New concepts in measles virus replication: Getting in and out in vivo and modulating the host cell environment

Virus Research 162 (2011) 47–62

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Review

New concepts in measles virus replication: Getting in and out in vivo and modulating the host cell environment Bert K. Rima a,∗ , W. Paul Duprex a,b a b

School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast BT9 7BL, Northern Ireland, UK Department of Microbiology, Boston University School of Medicine, 72 East Concord Street, Boston, MA 02043, USA

a r t i c l e

i n f o

Article history: Available online 6 October 2011 Keywords: Measles virus Receptors Pathogenesis Host factors Innate immunity

a b s t r a c t This review focuses on new concepts important for the understanding of the pathogenesis of measles virus. First the requirement for specific entry receptors restricts the cell types that measles can enter during the initial stages of infection in the human host. Recently, the paradigm for measles has shifted from an epithelial infection similar to that caused in the respiratory tract by other members of the paramyxoviruses to one which displays more similarity to the infection of the immune system by HIV-1, though the route of infection is different. Secondly we review the role of host proteins that support viral replication as well as those that modify the cellular environment in order to promote measles virus replication. The role of specific virus proteins in the anti-antiviral response is also reviewed. Measles virus counteracts all pathways known to induce interferon synthesis as well as signalling by interferons, exemplifying the importance of these in the virulence/attenuation of the virus. We conclude that only studies in relevant animal model systems or humans or in vitro or ex vivo studies of relevant cell types and tissues will bring us closer to an understanding of the pathogenesis of the virus, factors that have often been overlooked in past studies. © 2011 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Getting in and out . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Attenuation and virulence; the importance of the cellular environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Dependence on the cellular environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Modifying the cellular environment to favour virus replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Combating cellular antiviral responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. The role of the viral V and P proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. The role of the C protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3. A role for the N and M protein? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

47 49 51 52 53 54 54 55 57 57 58 58

1. Introduction Many excellent and recent reviews have been published about measles virus (MV) molecular biology (Gerlier and Valentin, 2009; Rima and Duprex, 2009; Griffin and Oldstone, 2009a,b), immunosuppression (Griffin, 2010; Schneider-Schaulies and SchneiderSchaulies, 2009), persistence and neurologicaldisease (Rima and Duprex, 2005) but few deal with the pathogenesis induced by the

∗ Corresponding author. Tel.: +44 289097 5858; fax: +44 289097 2124. E-mail address: [email protected] (B.K. Rima). 0168-1702/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2011.09.021

virus in a comprehensive way. Hence in this review we will concentrate on two aspects of the viral life-cycle that have seen significant new developments in the past few years through research which fundamentally changes our perception of MV from a respiratory virus to one that predominantly affects the immune system in a manner similar to human immunodeficiency virus type 1 (HIV1). These primarily involve interactions of the virus with cellular receptors and they have altered our view of how the virus gets in and out of cells both in vitro and in vivo. Secondly, we will review new studies, dealing with the interaction of the virus with

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components of the host cell as these appear to be prime mediators of attenuation and virulence. As reviewed elsewhere, MV is a spherical enveloped virus with a diameter of between 100 and 300 nm (Griffin, 2007). The lipid bilayer envelope is derived from the host cell. The virion is surrounded by a fringe of spikes, which in contrast to influenza viruses, appear to be uniform in size, 5 nm in diameter and up to 15 nm in length. These spikes probably consist of complexes of the fusion (F) and hemagglutinin (H) glycoproteins, two key viral proteins which unite to form the biologically active fusion complex. Their function and interaction with cellular receptors and the importance in pathogenesis is described in Section 2. Inside the virion is a negative sense (−) ribonucleo-protein complex (RNP), the (−)RNP, which is the basic unit of infectivity (Rozenblatt et al., 1979) in the sense that both protein and RNA are required for the successful propagation of the virus (Lamb, 1993). It consists of the viral genome, which is 15,894 nucleotides in length, and at least three viral proteins. When observed by electron microscopy (EM) it appears as a helical structure which is 1 ␮m in length and 18–21 nm in diameter, with a central core, which is 5 nm in diameter. Its major function, following uncoating and deposition into the cytoplasm, is to act as the template for primary transcription by the virus RNA dependent RNA polymerase (vRdRp) which is comprised of the of the large (L) and phospho-(P)-proteins. After negative staining it displays the herringbone structure characteristic of all paramyxovirus RNPs and is indistinguishable from the (+)RNP which serves as an intermediate in genome replication, again being produced by the vRdRp. Analysis of “Y” forms of the RNP that are replicative intermediates (Thorne and Dermott, 1977) indicates that the herringbone “Points” to the 3 end of the genome. There are thirteen nucleocapsid (N) protein molecules per helical turn (Desfosses et al., 2011; Bhella et al., 2002). The genome must consist of a multiple of six nucleotides (6 × 2649). A suggested explanation for the rule of six (Calain and Roux, 1993) is that each N protein may associate with exactly six nucleotides of the genomic RNA and that the phasing of the nucleotides with respect to the helical protein coat is important in transcription and editing of the viral mRNAs. This would mean that there are approximately 204 helical turns in each RNP, a number which has been validated by direct observation (Lund et al., 1984). It also means that residues 78 (6 × 13) nucleotides apart would be almost adjacent to each other. This fits well with the alignment of the so called A and B boxes, which are present at the genome termini and in the 5 untranslated region (UTR) of the N and the 3 UTR of the L mRNAs which are key conserved promoter sequences essential for RNA synthesis. How the RNA and protein are arranged in the RNP is not yet clear, but three-dimensional reconstructions, based on negative stained cryo-EM, indicate that the RNP is very flexible (Bhella et al., 2002). By analogy to other non-segmented negative strand (NNS) RNA viruses it is likely that the RNA is located on the outside of the RNP and thus available to the vRdRp, its cofactors and substrates. This arrangement also implies a specific topological requirement for the processes of transcription and replication as it is likely that the vRdRp and the nascent RNA molecules and replicative intermediates are stationary in the cell whilst the template rotates as it is being copied. The alternative would be energetically too costly and would involve dragging large RNA molecules or RNP structures through the cytoplasm as the vRdRp “circles” the template to avoid entanglement. The MV genome contains six transcription units (genes), which are separated from each other by trinucleotide intergenic sequences. These six transcription units are preceded by the leader (Le) region at the 3 -terminus encoding a short Le RNA of 56 nucleotides and are followed at the 5 -terminus by a trailer (Tr) region comprised of 40 nucleotides. The sequence of the first five intergenic regions in the genome is 3 -GAA-5 , whilst the last which

separates the H and L genes is 3 -GCA-5 . The sizes of the various transcription units, the coding regions, and viral proteins are given in the annotated genome (Table 1). The entire sequence has been determined for many MV strains including the prototype Edmonston strain, which was isolated in 1954 as well as many vaccine, laboratory-adapted and wild-type strains. Nucleotide sequencing has led to the recognition of 23 different genotypes of measles virus which are distributed over eight clades (Rota et al., 2011). The MV genome encodes at least eight proteins, six of which are components of the virion (Table 1). The major structural protein of the RNP is the N protein and minor components are the P and L proteins, which comprise the vRdRp. The virus glycoprotein spikes consist of the trimeric F (Zhu et al., 2003) and the H glycoproteins (Hashiguchi et al., 2007), which are integral type 1 and type 2 membrane proteins, respectively. The H protein is a dimer or a dimer of dimers (Hashiguchi et al., 2007). The F0 glycoprotein precursor is biologically activated by proteolytic cleavage in the Golgi or post-Golgi involving furin-like proteases (Navaratnarajah et al., 2011; Watanabe et al., 1995). These cleave the glycoprotein into an F1 –F2 complex, which is held together by a disulfide bridge between two cysteine residues which are conserved throughout the Paramyxovirinae. Cleavage takes place at the amino terminus of a very hydrophobic stretch of amino acids (aa), which are essential for virus-to-cell fusion. The furin proteases, which perform the cleavage are ubiquitously expressed in a large number of cell types meaning that biological activation of the F0 glycoprotein does not constitute a limiting factor in MV egress, cell-to-cell spread and transmission. This is in contrast to other enveloped RNA viruses, such as Sendai and influenza viruses, which productively infect and spread in many fewer cell types. The H and F glycoproteins interact laterally but it is not known exactly how. The interactions are likely to lead to significant changes in protein structures to form the fusion complexes that allow entry of the viral RNP into the cytoplasm following interaction with the cellular receptors (Hashiguchi et al., 2011). Simple co-expression of the H and F glycoproteins in transiently transfected cells leads to cell-to-cell fusion and the production of multinucleated syncytia, which are indistinguishable from those produced upon infection. The inside of the virion membrane is coated with the matrix (M) protein, which interacts with the cytoplasmic tails of the F and H glycoproteins forming the “bridge” between these and the (−)RNP. A recombinant virus has been generated, which does not express the M protein and this virus is highly cell-associated and grows to very low titres only in vitro (Cathomen et al., 1998). At least two additional proteins are generated from the gene encoding the P protein, which are nonstructural in the sense that they are not detected in the virion. The C protein is translated from the P mRNA from an overlapping open reading frame (ORF) (Bellini et al., 1985). The initiation codons for the P and C proteins are not in the optimal context meaning that cap-dependent ribosome scanning allows translation initiation at the start of the P ORF (position 60) in the mRNA or that of the C ORF (position 83). The V protein is generated by co-transcriptional editing of 30–50% of the P/V/C gene transcripts by the non-templated addition of a single guanosine residue at a slippery sequence in the gene, the so-called editing site (Cattaneo et al., 1989; Bankamp et al., 2008). These edited mRNAs encode the V protein which shares the first amino terminal 231 aa with the P protein until the additional residue leads to a switch in reading frame resulting in a completely different 68 aa long cysteine rich carboxyl terminus. The role of the viral proteins and especially the P, C and V proteins in modifying the host environment is described in Section 3. The tremendous progress that MV research has made in the last 15 years is largely based on the achievement of the development of the original reverse genetics system (Spielhofer, 1995; Radecke et al., 1995) and the resulting recombinant (r) MVs have greatly facilitated our understanding of virus molecular biology.

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49

Table 1 Annotation of the measles genome. Nucleotide numbersb

mRNA

ORF lengtha

Number of codons

Protein

1–52 56–1744

Leader N

– 108–1682 (N)

– 525

1748–3402

P/V/C

1807–3327 (P) 1807–2702 + 1G (V)

507 231 + 69 = 300

1829–2386 (C)

186

– Nucleocapsid protein: phosphorylated protein, which encapsidates the RNA Phosphoprotein associated with RNP in polymerase complex V protein: prevents interferon-induced transcriptional responses and signalling C protein: prevents interferon-induced transcriptional responses; acts as an infectivity factor and regulates replication/transcription Matrix protein: hydrophobic protein on inner leaflet of membrane/regulates transcription Fusion glycoprotein: cleaved to a disulfide linked F2–F1 complex Hemagglutinin glycoprotein: attachment glycoprotein Large protein: enzymatic activity of the RNA polymerase complex –

3406–4872

M

3438–4442 (M)

335

4876–7247 7251–9208 9212–15,854 15,858–15,894

F H L Trailer?

5458–7107 (F) 7271–9121 (H) 9234–15,782 (L) –

550 617 2183 –

a b

ORF, open reading frame. Nucleotides are numbered 1–15,894 in positive sense.

However, this laboratory-adapted Edtag-based system has probably been superceded by vaccine and wild-type based systems which have been produced directly from vaccine stocks or from clinical isolates which have been passaged in disease-relevant cell lines which express the appropriate cellular receptor (Takeda et al., 2000; Combredet et al., 2003; Devaux et al., 2007; Lemon et al., 2011). Going forward, these will be more useful for the study of attenuation and pathogenesis and the development of vectored vaccines and gene therapeutic agents. The development of reverse genetics based on the generation of the viral N, P and L proteins in the cell by transient expression from plasmids and the generation of RNA molecules which contain the sequences required for transcription and replication as well as those of reporter genes – so called mini-genomes, has also allowed direct assessment of the effect of host and viral factors and specific mutations on viral replication and transcription. Probably one of the most useful approaches developed has been to insert an additional transcription unit into the promoter proximal position (position 1) of the genome. These additional genes typically contain an ORF encoding a fluorescent protein such as enhanced green fluorescent protein (EGFP) or DsRed (Spielhofer, 1995; Duprex et al., 1999, 2000; Naim et al., 2000; Rager et al., 2002; Hashimoto et al., 2002; Ludlow et al., 2005; Lemon et al., 2011). Such rMVs have helped to redefine primary MV pathogenesis and allow infected cells to be identified even in the absence of the syncytium formation, a defining feature of MV-infected cells. Thus infection in epithelial cells can be visualized even in the absence of syncytium formation (Ludlow et al., 2010). How rMVs, especially those which express fluorescent reporter proteins, have begun to assist our understanding of MV pathogenesis and attenuation in natural models of disease is described in Section 2.

2. Getting in and out Unlike other members of the Paramyxoviridae, which utilize sialic acid molecules present on glycoproteins and glycolipids to enter cells, morbilliviruses use specific cellular receptors. Thus rather than a hemagglutinin-neuraminidase (HN) glycoprotein, which is needed to cleave sialic acid moieties from the cell surface during egress, or a simple glycoprotein (G), which lacks both neuraminidase and hemagglutination activities, the MV receptor binding protein is simply an H glycoprotein which agglutinates macaque erythrocytes but not human red blood cells. The H glycoprotein is comprised of 617 aa, has an ectodomain of 559 aa, a transmembrane domain of 24 aa and a cytoplasmic tail of 34 aa (Table 1). Given its critical role in cell entry and by extension in vivo

pathogenesis, it is therefore not surprising that significant efforts have been expended to determine and model the three dimensional structure of the glycoprotein. Such a goal is not without its challenges as glycoproteins are notoriously problematic to express, purify and crystallize in sufficient quantities for their structures to be resolved at atomic resolution. Thus the successful resolution of the soluble head domain of the H glycoprotein from both a laboratory-adapted and a wild-type strain of the virus which is known to be pathogenic in the macaque, is a significant achievement (Hashiguchi et al., 2007). The in vitro expressed ectodomain fragment (resides 149–617) crystallized as a disulfide-linked dimer at 2.6 A˚ resolution and the head of the molecule is a cuboidal ␤-propeller structure comprised of six, four-stranded antiparallel ␤-sheets (␤1–␤6). Two asparagine residues on the globular head, aa 200 and 215, are glycosylated and these shield the surface and skew the dimers in opposite directions. The impact of glycosylation of the H glycoprotein on disease outcome is most apparent in a small animal model of acute encephalitis where two functionally linked residues, one being aa 200, have been identified as key molecular determinants of neurovirulence (Moeller et al., 2001; Moeller-Ehrlich et al., 2007). Reverse genetics was used to demonstrate that ablation of the glycosylation site at position 200 alongside modification of aa 195, from arginine to serine, were jointly critical for neurovirulence and neither one mutation nor the other in isolation caused disease in this small animal model. It is tempting to speculate that loss of the glycosylation site unmasks a novel rodent-brain receptor binding region of the head domain of the H glycoprotein, although at present there is no evidence to that effect. Determination of the atomic structure of the H glycoprotein alongside mapping of the known receptor binding sites adds weight to this suggestion in that these sites lie outside of the regions masked by the sugars (Hashiguchi et al., 2007). Whether this rodent receptor might shed any light on mechanisms of neurovirulence in the human remains to be determined although it may be interesting to determine the three dimensional structure of this mutated H glycoprotein and see if in addition to the local changes in structure loss of the glycosylation site leads to more profound changes in the spatial orientation of the head domain. Neutralizing antibodies, which bind exposed epitopes and aa essential for receptor are located in these unmasked regions of the H glycoprotein (see below). The fact that dimerisation tilts the globular domains emphasizes that the H glycoprotein should not be considered in isolation and critical homotypic (H/H) and heterotypic (H/F) interactions between the viral glycoproteins in the same membrane alongside heterotypic H/receptor interactions across opposing membranes are important in understanding the biological properties of the glycoprotein (Fournier et al., 1997; Plemper et al., 2001; Navaratnarajah et al.,

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2008; Hashiguchi et al., 2011). Therefore knowing the atomic structure of the H glycoprotein represents only one aspect of what is required to understand virus-to-cell fusion during cell entry, tropism and viral pathogenesis in the broader context. Movement of the globular heads during receptor binding and transmission of the signal to the F glycoprotein within the fusion complex is critical for controlling fusogenicity (Mühlebach et al., 2008). Previous reviews tend to introduce MV receptors in the chronological order in which they were identified. However, with the recent identification of the elusive epithelial receptor (Noyce et al., 2011), we will focus on the receptors in terms of their importance in understanding MV pathogenesis. Signalling lymphocyte activation molecule (SLAM), more correctly designated as CD150, is the primary MV receptor which was originally identified by Tatsuo et al. (2000) being confirmed as a receptor by others the following year using a variety of approaches (Tatsuo et al., 2001; Hsu et al., 2001; Erlenhöfer et al., 2001). Its distribution on activated Tcells, B-cells, macrophages, mature dendritic cells (DCs), platelets, and thymocytes is consistent with the highly lymphotropic nature of MV (Sidorenko and Clark, 1993; Cocks et al., 1995; Kiel et al., 2005). Clinical isolates and viruses passaged in disease-relevant cell lines, such as the often used marmoset B95a cells (Kobune et al., 1990) or human B-LCL cells (van Binnendijk et al., 1989) which express CD150, use this molecule exclusively (Ono et al., 2001). Indeed every naturally occurring MV uses CD150 as a receptor (Erlenhöfer et al., 2002). Three residues on the V domain of CD150 (I60, H61 and V63) are required for binding to the MV H glycoprotein (Ohno et al., 2003). Ten residues (I194, D505, D507, Y529, D530, T531, R533, H538, Y553 and P554) have been identified as important for CD150-dependent fusion with I194 being critical for receptor binding (Masse et al., 2004; Vongpunsawad et al., 2004; Navaratnarajah et al., 2008). Recently the structure of the marmoset receptor/glycoprotein complex has been resolved at 3.15 A˚ resolution and interestingly rather than binding at the top of the cuboidal domain a ␤-sheet on the side of the ␤-propeller is the key site of interaction (Hashiguchi et al., 2011). Four components of a 1050 A˚ 2 region (sites 1–4) have been defined and hydrophobic interactions, salt bridges, and intermolecular ␤-sheets are involved in H/CD150 binding. An rMV based on a wild-type has been generated which binds CD150 about 40 times less efficiently (Leonard et al., 2010). Although termed “SLAM-blind”, somewhat of a misnomer, the inability to fully engage with the receptor led to the single-step attenuation of the wild-type virus. This reduction in virulence could be due to fewer CD150-positive cells being infected, a restriction in cell-to-cell spread or due the virus having to bind to another receptor initiating the infection in a non-canonical manner. Textbook descriptions of MV pathogenesis (Griffin, 2007; Navaratnarajah et al., 2009) should be re-examined based on the discovery that neither epithelial cells in the upper respiratory tract nor CD150-expressing cells in the tonsils are the first cells targeted by wild-type virus (Lemon et al., 2011). A wild-type reverse genetics system, based on a consensus sequence obtained from an unpassaged clinical sample obtained from Khartoum, Sudan (KS), was used to recover rMVKS EGFP(1), a rMV which expresses EGFP from the promoter proximal position of the genome, position 1 (1). Macaques, which display many of the symptoms of the human infection and become systemically infected following infection via the respiratory route, were infected with rMVKS EGFP(1), which resulted in the observation that alveolar macrophages and DCs in the lower respiratory tract were the primary cells targeted early in the infection. How the virus formally enters these cells remains to be determined but it is clear they are the “vehicles” by which the infection is seeded in the deep lung. Shortly after infection of these cells MV replicates in localised areas of bronchial associated lymphoid tissue (BALT) where the close spatial proximity of CD150 positive T-, B- and myeloid cells amplify the infection. Thereafter

the virus spreads first to the tracheobronchial lymph node prior to dissemination throughout the body. However, even though these studies reemphasized the lymphotropic nature of MV in this, and a previous study (de Swart et al., 2007), infected ciliated epithelial cells, which do not express CD150 are readily detectable at the peak of infection in the lower and upper respiratory tract, confirming what others have reported in other experimentally infected non-human primate models (Sakaguchi et al., 1986) and humans (Lightwood and Nolan, 1970; Kimura et al., 1975; Olding-Stenkvist and Bjorvatn, 1976; Moench et al., 1988). These in vivo pathological observations are mirrored by an in vitro study which shows that it is not possible to infect differentiated ciliated human bronchial epithelial cells with wild-type MV strains from the apical surface and extremely difficult to infect ex vivo-cultured human corneal rims, other than at side of the tissue where the integrity was compromised during surgical removal (Ludlow et al., 2010). However, the virus efficiently entered and replicated in the differentiated ciliated human bronchial epithelial cells when applied basolaterally or if the cell sheets where mechanically disrupted, which might be consistent with entry using a specific epithelial receptor, although this remains to be proven. Other in vitro model epithelial cell systems can be apically infected (Takeda et al., 2007; Tahara et al., 2008) and non-transformed small airway epithelial cells (SAEC) cells can be infected with well-defined wild-type viruses, although only when pre-treated with fetal calf serum in a CD150independent manner (Takeuchi et al., 2003b). Combined, these studies suggested that epithelial cells can be infected by wild-type viruses and that the unknown receptor was expressed temporally and possibly spatially on epithelial cells and cell lines. Identification of mutations in the H glycoprotein which abolished binding to the receptor, generation of rMVs expressing the modified glycoprotein and infection of macaques demonstrated that the wild-type, non-epithelial receptor binding virus could still cause a systemic infection but was suggested no longer to be shed from the respiratory epithelium (Leonard et al., 2008). This suggests that this receptor ranks second to CD150 in terms of pathological importance being critical for exit of the virus and therefore vital for MV transmission in the absence of its identification it has been termed “EpR”. Five residues in the H glycoprotein (L482, F483, P497, Y541 and Y543) have been identified as important for EpR-dependent cell-to-cell fusion (Leonard et al., 2008; Tahara et al., 2008). Mapping of these residues onto the atomic structure shows they lie in a region which is distinct from the CD150 binding site and in a region which is also not shielded by sugars. Uncharged polar and nonpolar interactions between these residues in blades 4 and 5 of the ␤-propeller make up the binding site, although interestingly no single residue is more that 28% exposed in the structure (Leonard et al., 2008; Navaratnarajah et al., 2009). Recent identification of nectin-4, which is present in the adherent junctions of epithelial cells below the tight junctions, as the epithelial receptor finally completes the MV pathogenesis jigsaw puzzle (Noyce et al., 2011). Understanding how the wild-type virus uses this molecule to gain entry into epithelial cells and the subsequent steps in assembly, budding and transmission will be of great interest both to those developing MV-vectored vaccines and retargeted, replicating viruses for gene therapy and those who are studying primary pathogenesis. Although not a formal entry receptor like CD150 and nectin-4 the C-type lectin DC-SIGN facilitates MV entry into DCs (de Witte et al., 2006). Inhibitors block attachment and infection although DC-SIGN expressing Chinese hamster ovary (CHO) cells cannot be productively infected. This is generally accepted as standard proof that a molecule acts as a true MV receptor and therefore DC-SIGN is considered to facilitate attachment to the cell rather than to permit delivery of the RNP into the cytoplasm. Moreover, no mutations in specific aa which ablate MV H glycoprotein binding to DC-SIGN have been reported. Given that alveolar macrophages and DCs in

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the deep lung are the primary target cells infected by wild-type viruses it is likely to have some pathological relevance (Lemon et al., 2011). DC-SIGN mediated trans-infection of lymphocytes by DCs (de Witte et al., 2008) and cell-to-cell spread in general (Duprex et al., 1999) is an important means by which MV, as a highly cellassociated virus, initiates and perpetuates the infection in vivo. Moreover, understanding the early events in the infection using more tractable ex vivo and in vitro models will be important in ongoing studies which are examining the aerosol delivery of MV vaccine (Lin et al., 2011; Simon et al., 2011; Wong-Chew et al., 2011). It is critical to understand if the route of MV immunization affects immunogenicity and protection. The first, and in hindsight arguably the least pathologically relevant MV receptor identified, is CD46 (Dörig et al., 1993; Naniche et al., 1993). Expressed on nearly all nucleated human cells the molecule is responsible for protection from complement-mediated attack and regulation of immune responses (Seya et al., 1988; Cole et al., 1985). It is a type 1 glycoprotein containing four short consensus repeats (SCRs), a serine/threonine/proline region and one of two differentially spliced cytoplasmic tails (Liszewski and Atkinson, 1992; Kemper and Atkinson, 2009). SCR1 and 2 of CD46 (residues 37–59) are required for binding to the MV H glycoprotein (Buchholz et al., 1996; Casasnovas et al., 1999; Devaux et al., 1997; Manchester et al., 1997). Six residues (F431, V451, Y481, P486, I487 and G546) have been identified as important for CD46-dependent fusion, with residues 451, 481 and 546 being most important (Bartz et al., 1996; Rima et al., 1997; Lecouturier et al., 1996; Tahara et al., 2007; Shibahara et al., 1994; Li and Qi, 2002). Again these aa are located in a distinct region of the atomic structure from those which interact with CD150 and nectin-4, once again in an area to the side of the cuboidal head domain separate from the glycosylation sites at blade 4 of the ␤-propeller (Hashiguchi et al., 2007; Navaratnarajah et al., 2009). No “true” wild-type MV uses CD46 to enter cells in vitro and its in vivo relevance is also questionable for vaccine viruses (Yanagi et al., 2009). When a CD150/CD46 utilizing laboratory-adapted Edmonston-based rMV expressing EGFP was administered virus by the respiratory route the virus predominantly infects CD150-positive cells (de Vries et al., 2010). Moreover, when post-mortem tissue samples are examined from a human vaccine-associated case, caused following the vaccination of a child who had an unidentified genetic immunodeficiency, viral antigen is found exclusively in SLAM positive cells (Allen et al., submitted). These in vivo data clearly demonstrate that the CD46 use is most likely an in vitro artefact and illustrates that thought should be given before in vivo CD46 transgenic models are used to address questions relating to vaccinology, gene therapy and pathogenesis. This is especially the case in transgenic animals which lack the interferon ␣/␤ receptors (Horvat et al., 1996; Rall et al., 1997; Mrkic et al., 1998; Oldstone et al., 1999; Marie et al., 2002; Kemper et al., 2005; Shingai et al., 2005). This is not to say that the molecule has no importance and it is clearly used during the propagation of vaccine and laboratory-adapted viruses in vitro. CD150 and nectin-4 account for the lymphotropic and epitheliotropic properties of natural MV. However, the virus is also endotheliotropic and neurotropic, indeed sub-acute sclerosing panencephalitis (SSPE) represents a paradigm for the long term persistent infection of an RNA virus in humans (reviewed in Rima and Duprex, 2005). Whether nectin-4 is used to infect endothelial cells and neurons remains to be determined. It has been suggested that neurokinin-1 (NK-1) may act a receptor in neurons although the F glycoprotein rather than the H glycoprotein is bound by NK-1 (Makhortova et al., 2007). Whether this receptor or another facilitates trans-synaptic spread, which has been suggested as a means by which the virus spreads (Duprex et al., 2000; Lawrence et al., 2000; Rima and Duprex, 2005), also remains to be determined. Non-specific and possibly non-biologically relevant entry has been

51

seen in some cell lines in vitro which is CD150 and CD46 independent (Hashimoto et al., 2002). At best such entry is mediated by low-affinity receptors and possibly even macropinocytosis as subsequent cell-to-cell spread in highly restricted.

3. Attenuation and virulence; the importance of the cellular environment. In the past decade it has become clear that there are very few instances in NSS RNA viruses where the virulence of a particular virus strain is determined by a single gene or genetic mutation. In almost all cases, virulence and attenuation, which actually represent two sides of the same coin, are multi-factorial because of the existing synergies between proteins and RNA sequences of the virus. These RNA viruses are highly optimised RNA/protein complexes in which sequence and structure of the RNA genome are completely attuned to the sequence and structures of the viral proteins by strong selective forces. These selective forces allow the successful propagation of a virus and provide an explanation for the relative stability of MV virus sequences in the field. In terms of fitness landscapes this means that the virus replicates on top of a very narrow fitness peak (Lauring and Andino, 2010) or may be better on top of a steep sided mountain on which there are a number of small tors representing the 22 circulating genotypes (Rota et al., 2011). Even though it appears that the RdRp functions with a substantial error rate and replicates the genomic RNA at the edge of error catastrophe, the sequence of the viral genotypes is relatively stable and nowhere near as variable as has been observed for other virus such as HIV and influenza A virus (Lauring and Andino, 2010). This may reflect the fact that MV has reached an accommodation with its human host after its introduction into the population. Using Bayesian statistical approaches to sequence comparisons, this introduction has recently been estimated to have occurred approximately 800 years ago (Furuse et al., 2010) but this is about 600 years later than its introduction in the human population in the sixth century suggested in its first description by Al Rhazes in the ninth century AD (Griffin, 2007). It has also become clear that the speed with which a virus replicates when invading its host is a primary determinant for its ultimate ability to be described as “virulent”. Adaptive immune responses to the virus are ultimately required for the host to control the virus and paradoxically these are generated successfully in the presence of the prolonged immunosuppression, which is a hallmark of morbillivirus infections (reviewed in Griffin, 2010; Schneider-Schaulies and Schneider-Schaulies, 2009). However, the initial determinant that establishes whether a virus can replicate successfully and causes clinical disease in the host appears to be strongly linked to its ability to deal with the innate immune system of the host as this is the primary line of host defence. Adaptive T-cell responses, especially those driven by cytotoxic T-cells are required for the ultimate control of viral infection but humoral ones tend to appear after substantial reductions in viral load have occurred (Schneider-Schaulies and Schneider-Schaulies, 2009). There is still controversy about the role of specific MV proteins in combating the host cell environment. It is also unlikely that our knowledge of the innate immune system is complete and that we understand all aspects of what the virus has to deal with when it enters a host cell. Hence it is difficult to know what precise cellular functions should be included as components of the innate immune system, particularly as this may well differ for each pathogen. Hence we use the terms such as modifying the host cell environment and prefer this to the term of combating innate immunity. Host factors that inhibit or are required for efficient viral replication are especially relevant to “cross-species jumps” by viruses and determine the height of the barriers that viruses need to overcome

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when they invade hosts of new species. The extent of the restriction depends most likely on the level of conservation and similarity between cellular proteins of the old and new host. A further point that needs to be made is that MV and other paramyxoviruses are able to establish long term persistent infections in cultured cells, but also, as is the case for MV, in vivo in the long term lethal neurological disease SSPE (reviewed in Rima and Duprex, 2005). In cultured cells these long term infections can be established by wild type viruses that have not mutated but have come to an equilibrium level of replication attuned to the host cell. These cells can be cured from their infection by treatment with antibody. Therefore modifications of the cellular environment must be tunable and even reversible. Hence gross modifications of, for example, the translation machinery that would favour exclusive translation of viral mRNAs or gross alterations to the cellular machinery that control apoptosis and other important cellular processes are unlikely to occur in these persistent MV infections as these would endanger the co-existence between the virus and the cell. Thus, whatever modulations of the cellular environment MV induces, they must be moderate and/or reversible and cells can be cured from their persistent MV infection. In SSPE it is not clear whether MV and neurones co-exist or whether this reflects an acute and progressive infection, but some patients survive for years to almost two decades (Rima and Duprex, 2005). Analogous to the situation with other paramyxoviruses, the MV P, C and V proteins have been investigated as the prime candidates that could play a role in combating the antiviral responses. However, in the flavi- and other viruses nucleocapsid proteins appear to suppress innate immune reactions. Hence there is no reason to exclude any intra-cytoplasmic proteins and protein domains (e.g. including the cytoplasmic tails of the H and F glycoproteins) as potential players. Furthermore, recently our understanding of conversion of information contained in RNA by the host’s translational machinery has developed such that we now know that this is not just governed by the simple concept of a 5 UTR structure, be it a cap or an internal ribosome entry site, an AUG start codon surrounded by a Kozak consensus sequence, a stop codon, a 3 UTR and a poly-A tail. Some of the variant forms of translation have been described in this virus group (Gould and Easton, 2007; Schneider-Schaulies et al., 1994; Powell, 2010; Schnorr et al., 1993) and hence small ORFs that are conserved in the sequence of the various strains of specific RNA viruses should all be considered potential players in overcoming host innate immunity. A number of these have been identified in MV and are actively investigated in our laboratories (Rennick et al., in preparation). The effects of MV on the host cell and host tissues will become clearer over the next few years when further studies using the various “omics” will be reported. So far only a small number of transcriptome analyses and one proteomic study have been reported for MV, but nevertheless it is clear that these technologies will have an impact on our understanding of the interactions with the host cell, though combined transcriptome and proteome studies will be required in order to really understand the effect of the virus on the cell as either one or the other taken in isolation may potentially give a misleading impression of the importance of any specific host factor. Transcriptome studies are particularly sensitive to potential artefacts due to the competition of viral RNA for the translation machinery of the cell and resulting destabilisation of cellular mRNAs. 3.1. Dependence on the cellular environment It is a truism to say that RNA viruses require the cell for their replication and that they need to avail cellular processes such as translation, movement of their components through the cytoplasm using the cytoskeleton and the glycoprotein synthesis, maturation

and export machinery. RNA viruses require the cellular translation machinery for successful replication. Potential modifications of the machinery by the virus that give viral mRNAs an advantage in the competition for ribosomes will be discussed below. Here we first deal with a number of host proteins that have been suggested to be required for viral replication. It is likely that the listings in Table 2 are far from comprehensive and further transcriptome and proteome studies may expand the list significantly (see above). Early in vitro RNA synthesis experiments in Sue Moyer’s laboratory identified tubulin as a cellular factor that was required for RNA synthesis (Horikami and Moyer, 1995) but this observation has not been confirmed. The potential role of this cytoskeletal protein does not necessarily indicate that the cytoskeleton is involved in MV replication because destruction of the cytoskeleton by cytochalasin B does not inhibit MV replication (Follett et al., 1976) and although the destruction of the actin cytoskeleton inhibits virus maturation it does not prevent efficient intracellular replication and syncytium formation (Berghall et al., 2004). It is also likely that casein kinase II which has been shown to play a role in the phosphorylation of serine residues (aa 86,151,180) of the P protein (Das et al., 1995) is required for MV replication but this has not been formerly proven. The same holds true for other unidentified kinases that phosphorylate residue 110 of the P protein (Devaux et al., 2007) and those that phosphorylated certain threonine and serine residues of the N protein (Robbins et al., 1980) and tyrosine residues in the N and P protein (Segev et al., 1995; Ofir et al., 1996). The phosphorylation of serine residues 479 and 510 in the N protein appears to activate viral transcription and replication in a mini-genome based expression system (Hagiwara et al., 2008). The Oglesbee laboratory has studied the role of thermoinducible and stress inducible heat shock protein 72 (HSP72) and demonstrated that it binds to two conserved sequence motifs in the carboxy terminal 150 aa variable part of the N protein (Taylor et al., 1991), described as box 2 (aa 489–506) and box 3 (aa 517–525) in the overall quite variable tail of the N protein (residues 400–525). Box 2 is highly conserved (Zhang et al., 2002), whilst in box 3 the change from aspartic acid to asparagine at position 522 changes the binding constant of HSP72 to the N protein tail (NTAIL ) (Zhang et al., 2002, 2005). This NTAIL is the same area where the XD domain of the P protein (aa 459–507) binds to the N protein (Bourhis et al., 2005) and it opens the way for a regulatory function of HSP72 in the interactions of the P and N proteins during replication and or transcription from the RNP. The effect of over-expression of HSP72 has been assessed in in vivo models in a HSP72 expressing transgenic mouse and after hyper-thermal preconditioning of mice, but the results are not clear (Carsillo et al., 2004, 2006). Recently another protein peroxiredoxin1 (Prdx1) has been identified as a critical component in MV replication (Watanabe et al., 2011). It was shown to bind to the same area of the N protein as the P protein did (box 2) and competed with P protein binding (Watanabe et al., 2011). A 50% reduction of Prdx1 expression by RNA silencing led to a substantial inhibition of viral gene expression (80–90%) and a tenfold reduction in production of infectious virus. Interestingly, a reduction in Prdx1 expression appears to make the MV transcription gradient steeper as it had less of an effect on N protein expression compared to L protein expression. Surface plasmon resonance analysis showed that the binding affinity of Prdx1 to NTAIL is approximately 40-fold lower than that of P protein to NTAIL , suggesting that Prdx1 may only play a role in MV RNA synthesis at the early stages of infection when the amount of cellular Prdx1 is much greater than that of the viral P protein (Watanabe et al., 2011). The XD domain of the P protein also appears to bind to PIRH2 (Chen et al., 2005), a p53-inducible E3 ligase containing the RING domain and this interaction stabilises its expression probably by preventing its (auto)-ubiquitination. The P protein alone or in combination with N protein also allows PIRH2 to accumulate

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Table 2 Established and proposed interactions between measles virus and host proteins. Process

Viral protein/RNA involved

Host proteins

Selected references

MV transcription/replication and protein synthesis

NTail and PXD

C, M and V (?) regulators L ? ?

HSP72 Prdrx1 PIRH2 Tubulin (?) HSP90 (?) MxA ADAR1

Zhang et al. (2002) Watanabe et al. (2011) Chen et al. (2005) Horikami and Moyer (1995) Gerlier and Valentin (2009) Schneider-Schaulies et al. (1994) Ward et al. (2011)

Cellular translation

C N core

Inhibits eIF2␣ phosphorylation Interacts with p40 of eIF3

Nakatsu et al. (2006) Sato et al. (2007)

Cellular cell cycle, transcription and apoptosis

V P and N (?) N Soluble H

P53 and p73 SUMO-ylation A20 gene transcription Apoptosis induction Bystander apoptosis

Cruz et al. (2006) Yokota et al. (2008) tenOever et al. (2002) Iwasa et al. (2010)

Signalling through the JAK/STAT pathway

V (VNT) and P (PNT) V (VCT) C

JAK1 binding STAT1 and STAT2 binding Binds tyr-phospho-STAT1

Yokota et al. (2003) Caignard et al. (2009) Yokota et al. (2011)

Recognition of intracellular PAMPS

V (VCT) 5 Triphosphate at end of the leader RNA Ds RNA ? C

Sequesters MDA5 and LGP2 RIG-I (?) role unclear

Childs et al. (2009) Plumet et al. (2007)

RIG-I or MDA5 and LGP2? Reduces PKR phosphorylation

Killip et al. (2011) Toth et al. (2009a)

V

Pfaller and Conzelmann (2008)

V (VCT) H in wild type virus

Binding of IKK␣ blocks IRF7 phosphorylation Rel domain of p65 of NF␬B TLR2

N box 1

Fc␥RII

Tatsuo et al. (2001)

PAMPS and TLRs

B cell antibody production

in the cytoplasm within intracellular aggregates. The significance of this interaction is unknown. Clearly, the interplay between the XD domain of the P protein, HSP72, PIRH2 and Prdx1 and their binding partner the box 2 area of the NTAIL probably represents a very complex set of fine regulatory mechanisms for the control of transcription and replication, but the significance of this in the MV replication cycle is not clear. Furthermore, if the structure of the MV nucleocapsid indicates that the C terminal NTAIL domain is inside the helix (Desfosses et al., 2011), these interactions are most likely relevant to the regulation of the interaction of the P and N proteins in their pre-replication complex rather than in the interaction between P and N in the RNP. However others (reviewed in Longhi, 2009) locate the NTAIL on the outside of the helical RNP. The interaction of HSP90 as a chaperone of the L protein in other member viruses of the Mononegavirales has led Gerlier to speculate that it may play a role in the replication MV as well since the L proteins of this group of viruses show high levels of similarity (Gerlier and Valentin, 2009). 3.2. Modifying the cellular environment to favour virus replication RNA viruses depend on the mRNA translation machinery of the cell and many modify this to favour translation of their own mRNAs. However, such modifications are likely minor or reversible as explained above. This is most saliently indicated by the role of the C protein viz a viz the phosphorylation of the eukaryotic translation initiation factor eIF2␣. Many viruses induce phosphorylation of this initiation factor to suppress cellular mRNA translation. Others have proteases that cleave initiation factors (Lloyd, 2006). A rMV which failed to express the C protein (-C) virus allowed eIF2␣ phosphorylation to be demonstrated but in the corresponding wild type parent virus that was not observed (Nakatsu et al., 2006; Toth et al., 2009a). There is thus a balance between two activities of the virus: an unknown function or possibly the stress induced by

Schuhmann et al. (2011) Bieback et al. (2002)

infection itself, which induces eIF2␣ phosphorylation and a balancing inhibition of this function by the C protein. The MV N protein inhibits translation in vitro in rabbit reticulocyte lysates but the mechanism is unknown (Sato et al., 2007). The protein interacts with initiation factor eIF3-p40 via a binding site resides in the amino terminal part of the protein between aa residues 81 and 192. However, the amino terminal core of the N protein is also the area that binds to RNA. The N protein of MV can bind RNA in a non-sequence specific manner (Spehner et al., 1997). However the observation that the N protein does not inhibit translation of an IRES containing RNA argues against the inhibitory effect simply being a non-specific effect caused by the N protein sequestering RNA. Initial studies examining gene expression in MV infected cells based on analysis of the transcriptome (Bolt et al., 2002; Zilliox et al., 2006, 2007) and the proteome (van Diepen et al., 2010) have been reported. Apart from general increases in the proteins involved in the interferon responses, endoplasmic reticulum (ER) stress and apoptotic proteins it is difficult to draw general conclusions from these studies as the modulations in gene expression vary with the virus strain and its level of attenuation and are also cell type dependent (Sato et al., 2008). However again it is to be remembered that for a virus that can establish persistent infection, these effects must be moderate, reversible or being able to be counteracted and controlled by viral proteins. One example of this may be the binding of MV-V protein to the DNA binding domains of both p53 and p73 (Cruz et al., 2006). Inhibition of p73-dependent gene activation may lead to V acting as inhibitor of cell death by preventing the activation of PUMA, an apoptosis regulator (Cruz et al., 2006). This is another example of a viral protein that is not so much combating an antiviral effect but regulating a cellular (over)-reaction to infection. The question as to whether MV can induce apoptosis in cells has been debated largely in the context of the profound lymphopenia associated with immunosuppression in acute infection. Ito et al. (1996) demonstrated the induction of apoptosis by a wild

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type strain in the monocytic cell line THP1 and Auwaerter et al. (1996) demonstrated apoptosis in vivo in thymocytes in the SCIDHu mouse model but no linear relationship between viral burden and levels of apoptosis was visible in SSPE brain material (McQuaid et al., 1997). MV infection in T lymphocytes activated by some stimuli did give rise to apoptosis but stimulation with other stimuli lead to cell cycle arrest (Ito et al., 1997) which was also intimated in another report as potentially contributing to the lymphopenia (Schnorr et al., 1997). Bystander apoptosis (Okada et al., 2000; Vuorinen et al., 2003) was postulated to be a factor in lymphopenia in order to explain the remarkable difference between the extent of the lymphopenia and the levels of MV infected lymphocytes and TRAIL induction was demonstrated (Vidalain et al., 2000). Ectopic expression of the N protein of MV induces apoptosis (Bhaskar et al., 2011) and signals can be enhanced by syncytium formation (Scheller and Jassoy, 2001) and Iwasa et al. (2010) demonstrated the soluble form of the H molecule of MV can induce apoptosis in PBMCs. Whilst it appears reasonably clear that MV infection can induce apoptotic and cell growth inhibiting effects in PBMC, in DCs and in lymphocytes, it also appear that anti-apoptotic effects are induced by the virus (Cruz et al., 2006; van Diepen et al., 2010). Hence the balance between pro- and anti-apoptotic factors may depend on virus strain as well as cell type and further study is required. 3.3. Combating cellular antiviral responses The main antiviral response that has been long recognized as a factor in innate immunity is the interferon response. Hence it is important to address questions as to how does measles induce interferon, how does it counteract the cellular response of interferon synthesis and how does it combat the antiviral state induced by interferon by interfering with signalling in the cascades involved in innate immunity? The main players in this arena appear to be the viral P, C and V proteins. The latter two non-structural proteins have been described specifically as virulence factors (Patterson et al., 2000). 3.3.1. The role of the viral V and P proteins The V protein (Table 1) has two distinct domains: the amino terminus which is shared with the P protein representing 231 aa residues (VNT) and the carboxy terminus which is generated in MV by co-transcriptional editing of the transcripts from the P/V/C gene involving insertion of a single G residue that changes the reading frame to one which terminates after 68 aa (VCT) and generates a cysteine rich C terminal tail (Cattaneo et al., 1989) which forms a zinc finger, which has been shown to bind the metal (Liston and Briedis, 1994). The Zn finger structure is conserved in a large number of paramyxovirus “V” proteins (see above). The VNT thus has the same primary sequence as the N terminal part of the P protein (PNT) but may be folded differently as a peptide antibody raised against aa 65–87 of the shared amino terminus reacted with the P protein but not with the V protein (Wardrop and Briedis, 1991). Hence functions of PNT and VNT may not be the same though their primary sequence is. Variable levels of post-translational modification may of course further distinguish PNT from VNT. Whether V can bind N, P or L is not clear. Liston et al. (1995) demonstrated no interaction of V with other viral proteins whilst Tober et al. (1998) showed a complex interaction with N and P protein that may afford a regulatory role for the V protein in transcription and replication. Two reports (Witko et al., 2006; Parks et al., 2006) showed that V protein affected expression of reporter genes in a mini-genome based assay system but this has not been confirmed by others. Whether V can bind N is still an open question, which may be of relevance to the observed feature of the P protein which activates transcription of the ubiquitin-modifying enzyme A20 gene (Yokota et al.,

2008). The P protein indirectly affects the promoter of this gene and enhances transcriptional activity of the gene whereas V does not. However, P is normally below detectable levels in the nucleus of the infected cell and hence how it would affect transcription and reaches the nucleus remains an enigma. In any case the difference between P and V may have more to do with the ability of P protein to bind to N and bring P into the nucleus whilst the ability of V to bind to N is controversial as discussed above. This effect of the P protein was also cell specific as it occurred in monocytic but not in epithelial cells (Yokota et al., 2008). The upregulation of A20 suppresses the activation of the NF␬B and AP1 in infected cells and the immunological silencing of the monocytes as TLR mediated responses are suppressed. This silencing may aid in the spread of MV in the infected host (Yokota et al., 2008). In order to discuss the role of viral protein combating antiviral responses it is necessary to briefly review how a cell detects infection by an RNA virus. Two main mechanisms are involved. The first involves the recognition of pathogen associated patterns (PAMPS) by trans-membrane TLR molecules (Kumar et al., 2011) and resulting signalling cascades that activate interferon ␣/␤ synthesis. In this context it is perhaps surprising that MV H protein of wild type but not vaccine strains is able to activate TLR2 (Bieback et al., 2002), whilst an expected response would be for the virus to counteract activation of TLRs. However, this TLR2 activation induces the expression of MV receptor CD 150 on the surface of the cell, which may allow spread of the virus to further macrophages and or dendritic cells (Bieback et al., 2002). In contrast to activation of a TLR signal, the Schwarz vaccine strain of MV was reported to block signalling from TLR7 and TLR9, but this was not universally observed in all MV strains (Druelle et al., 2008). Pfaller and Conzelmann (2008) have shown recently that this could be associated with the ability of the V protein to become a decoy substrate for IKK-␣, a kinase that phosphorylates IRF7. TLR9 recognizes CpG rich oligonucleotides and thus probably plays no role in anti RNA virus responses, but in this respect it is interesting to note that MV and many other RNA viruses display in their RNA genomes the same level of CpG suppression and bias against the most immunostimulatory motifs (Rima and McFerran, 1997) that have been identified in corresponding DNA oligonucleotides. Recently it was shown that CD14 and TLR7 (Baumann et al., 2010) are able to recognize ssRNA in acidic endosomes in cells infected with vesicular stomatitis virus and influenza virus (Iwasaki, 2007; Lee et al., 2007), thus potentially explaining the need for a virus to avoid these immunostimulatory motifs. There was no sequence specificity but the U content of RNAs appeared to be a crucial factor in determining the strength of the signal (Diebold et al., 2006). Incidentally the notable UpA suppression also observed in RNA virus genomes (Rima and McFerran, 1997) may reflect a reduction in the sensitivity of the virus to the 2–5A activated RNaseL (Malathi et al., 2007) which has been shown to prefer UpA and UpU dinucleotides as substrates (Han et al., 2004). A recent identification of a tryptophan rich motif and the recognition that the second tryptophan residue in a WX3WX9W motif is crucial in inhibiting TLR7 signalling in human parainfluenza virus type 2 infected cells (Kitagawa et al., 2011) indicates that the VCT of V protein of MV may do the same as the second and third tryptophan are strictly conserved in the VCT of all morbilliviruses. An important pathway that recognizes intracytosolic infection by RNA viruses is mediated by RIG-I (Hornung et al., 2006; Pichlmair et al., 2006). This leads ultimately to activation of transcription factors IRF3 and NF␬B and the generation of pro-inflammatory cytokines. RIG-I is activated as a result of the presence of a 5 triphosphate group on an RNA molecule. Generally cellular RNA synthesis leads to an absence of 5 triphosphates on RNAs but in MV infection the leader RNA probably has a 5 triphosphate group exposed to interact with RIG-I (Plumet et al., 2007). Whilst it is likely

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that the genome and antigenome RNA also carry this group, the encapsidation of the nascent RNA strand by N protein probably prevents these RNA molecules from being recognized by RIG-I (Plumet et al., 2007). It is not clear to what extent this interaction provides the main RIG-I mediated recognition of infection since defective interfering (DI) particles appear to play a major role in the induction of interferon and their removal almost completely abrogates the ability of MV to induce interferon (Rima, unpublished). Analogous to Sendai virus (Strahle et al., 2006), this may be explained by the fact that uncontrolled replication of DI RNA genomes does not contribute to the N protein pool in the cell but acts as a constant drain on this pool. The lack of N protein may lead to the accumulation of double-stranded (ds) RNA in the cell. These observations imply that the differences between strains observed in interactions of MV with the cellular environment are not only strain-dependent but also may depend on passage history and the presence of DI particles (Shingai et al., 2007). This component has often been overlooked in earlier studies, even in those using knockdown of RIG-I by siRNA (McAllister et al., 2010) and is particularly relevant to comparisons of the importance of the various aspects of IFN induction and other antiviral responses and the viral counter-reactions to changes in that environment, recently elegantly demonstrated in PIV5 infected cells (Killip et al., 2011). A second important intracytosolic recognition pathway relies on another set of helicase molecules such as MDA5 and a recently identified antiviral helicase LGP2 (Parisien et al., 2009). MV has developed a counteracting activity. The direct interaction of the V protein through the VCT with the helicase domain of MDA5 and LGP2 leads to an inhibition of their ATP hydrolysing activity and their consequential inactivation (Parisien et al., 2009). Childs et al. (2009) have suggested that binding of dsRNA to the helicase domain of these proteins is required for activation through the formation of homodimers. A vaccine strain associated mutation of residue 272 of the V protein (in VCT) has been shown to be important in the ability of MV to bind to MDA5 (Takaki et al., 2011). All the paramyxovirus V proteins appear to bind to MDA5 and prevent the activation by interfering with the homodimerisation. In contrast to MV, V proteins of other paramyxoviruses also block IFNAR signalling by acting as a “bridge” between STATs and E3 ligases that ubiquitinate the protein and so target it for proteasome mediated degradation (Randall and Goodbourn, 2008). The MV V VCT domain also binds directly in immunoprecipitation experiments to the rel domain of subunit p65 of NF␬B (Schuhmann et al., 2011) relocalising it thereby in the cytoplasm and rather than in the nucleus. This provides a further role for V in the inhibition of pro-inflammatory responses and provides an example of the redundancy that has been built in the inhibition of these pathways by the virus by acting at the beginning and the end of the signalling cascades. It is not clear from our current understanding of the MV replication cycle when and where dsRNA is generated and hence MDA5 activation should not occur. Goodbourn and Randall (Goodbourn and Randall, 2009) developed the concept of essential PAMPS i.e. those that are associated with its “normal” transcription and replication and “corrupted” PAMPS i.e. those associated with aberrant transcription. We probably do not have a complete understanding of all the RNA molecules induced by MV. For example, it important to remember the finding of a rather aberrant set of RNA molecules identified in MV infected cells that contain leader, mono or bicistronic N mRNA and a polyA tail (Castaneda and Wong, 1989). A similar argument about the general absence of dsRNA in the replicative cycle also applies to RIG-I mediated activation of IRF3 involving the kinase complex TBK1/IKK␧. In several paramyxoviruses V protein prevents IRF3 phosphorylation by becoming a decoy substrate (Goodbourn and Randall, 2009). This has not yet been shown to be the case for MV.

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Apart from silencing the cells ability to respond to extracellular or intracellular PAMPS it would also be advantageous for a virus to be able to interfere with intracellular signalling from the IFNAR receptor which involves the JAK/STAT pathway to block the effect of IFN. Both the P and V proteins (mediated by their shared PNT and VNT domains) prevent STAT1 phosphorylation (Takeuchi et al., 2003a; Ohno et al., 2004; Devaux et al., 2007; Caignard et al., 2007). The area surrounding tyrosine residue 110 is particularly important in this. Recently, Caignard et al. (2009) demonstrated that the 11 residue peptide representing the residues 110–120 in the PNT/VNT prevents STAT1 phosphorylation. A recombinant MV that cannot antagonise STAT1 phosphorylation is highly attenuated in vivo in a macaque model (Devaux et al., 2011). Furthermore the VCT domain is able to prevent the phosphorylation of STAT2 (Caignard et al., 2009). The same authors also demonstrated that these phosphorylation inhibiting activities interfered with signalling derived from IFN-␭ (Caignard et al., 2009). Yokota et al. (2003) suggested that the binding of MV V protein froze the IFNAR1 receptor with a scaffolding protein RACK1 and JAK into a complex that prevented signalling down the cascade. A further ability of the V protein to bind to the JH1 domain of JAK through its VNT domain stabilised by the VCT (Caignard et al., 2007) also appears to prevent STAT1 phosphorylation and so direct binding to the kinase (JAK) and its target (STAT) appears again to represent a redundancy but it also could suggest that other signalling pathways are still to be identified as involved in the antiviral response. 3.3.2. The role of the C protein The C protein (Table 1) consists of 186 aa and is translated from a second ORF in the mRNA transcribed from the P/V/C gene probably accessed by leaky scanning (see above). Known conserved sequence motifs are not evident in alignments of C proteins of the Paramyxovirinae. Approximately 22 aa (aa 104–125) are highly conserved across the morbillivirus genus (Fig. 1). These do not correspond to the previously identified nuclear localisation (aa 41–48) and nuclear export (aa 76–85) signals (Nishie et al., 2007), which led to the suggestion that the C protein cycles between the nucleus and the cytoplasm being located in the nucleus in the early phase of infection and in the cytoplasm at later time points. The 22 aa conserved motif in morbilliviruses cannot be linked to any other proteins in the data base by BLAST searches including the C proteins of other paramyxoviruses. Hence the significance of the conserved stretch remains unclear and it is impossible by bio-informatics to infer function. One explanation may be that the structure of the C protein is highly attuned to other viral components and has evolved to be optimal for interaction with the N, P, L and or M proteins rather than to interact with specific host components, which especially if they were to be part of the innate immune system, may be expected to provide conserved interacting surfaces. The precise role of the C protein in the MV life cycle is still not completely clear. A -C rMV is less infectious and it was proposed that the C protein is an infectivity factor (Shaffer et al., 2003; Takeuchi et al., 2005; Devaux and Cattaneo, 2004). Earlier studies showed that -C rMVs grow efficiently in cells in the absence of an interferon challenge such as the conditions of growth in Vero cells (Radecke and Billeter, 1996; Escoffier et al., 1999). These viruses grew differently in human thymus implants in SCID mice (Valsamakis et al., 1998) and in CD46 expressing transgenic mice (Patterson et al., 2000). Of note, all these studies used rMVs based on the original reverse genetic system, the so called Ed-tag backbone (Radecke et al., 1995). The full-length antigenomic plasmid clone p(+)MV, was derived from a strain that was highly adapted to growth in the laboratory and certainly does not represent the situation in wild type CD150 utilizing MVs and possibly not even a vaccine virus. However, later studies with wild-type virus in

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. . . . . . . MSKTDWNASGLSRPSPSAHWPSRKLWQHCQKYQTTQDRSEPPAG-KRRQAVRVSANHASQQLDQLKAVHLA ..TKA....R..G.D..TP.SL..PL..GSRPPKGKRLTVC.PTRPKQ-TI.I..S........A..AC.. ..IR.LSV.N..EKIRPMLSKL..PKLSEARPPAKNQARVITRTTPKK-TLLI.T...L.....KRTACYL ..TRG..V.SP.K.L.RIYPP.ETPLRAGERGSAPRAVQHRTLIRP.E-II.V.T...H..S..T.STC.L ..AKG....KP.ERILLTLRRFKRSAASET.PA.QAK.M..Q.CR...-TL.I.M..T...K..TMSAMYL ..VKG.S..RP.EKILLTWKRFKRSATSGIKPTSQAKKA..QVCKRKK-SL.I.M..TR..R..TVSAMYS . . . . . . . MV SAVRDLERAMTTLKLWESPQEISRHQALGYSVIMFMITAVKRLRESKMLTLSWFNQALMVIAPSQEETMN RPV VTIK...E.TAVMRS..HSLVTPQCI.PR..I...........................MVSK.G..MRN DMV VMIQ...HQV.S.MKESPS..T.ERRN.Q.D.T..........K..R...C...Q..V.MMQN.ET.MRA PPRV EIIS....SLATTVRLG.EESRGKDPT.K...T..IA.G.....D.R.......K.I.QLLTS.M..R.D CDV KII..V.N.ILR.WRRSG.L.RTSNGD.E.D...................V..YL...S..ED.R..KEA PDV KKI.EV..TILH.WRQKTVLKRIPK.D.Q.D...................V..YQ...Q..GD.K..REA . . . . MV LKTAMWILANLIPRDMLSLTGDLLPSLWGSGLLMLKLQKEGRSTSS 186 RPV .R............EV.P.........QQQEPP...Q 177 DMV .SR..VN..L...EEI.P.......G.RSRDRLT.R. 177 PPRV .T....T..QM..AEI.YM......AMMSL.PQ.S.N 177 CDV .MI.LR...KI..KE..H....I.SA.NRTEQ.. 174 PDV .MI.LK...KI..KE..H....I.LA.TQTEQ.. 174 MV RPV DMV PPRV CDV PDV

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140

MV sequence from the Edmonston strain; RPV (rinderpest virus) from the RBOK strain; DMV (dolphin morbillivirus) from our strain (Rima et al., 2005) ; PPRV (peste-des-petits ruminants virus) from the Nigeria 75/1 strain; CDV (canine distemper virus) from the Onderstepoort strain and PDV (phocine distemper virus) from the Ulster/88 strain. Fig. 1. Comparison of the c proteins of morbilliviruses. MV sequence from the Edmonston strain; RPV (rinderpest virus) from the RBOK strain; DMV (dolphin morbillivirus) from our strain (Rima et al., 2005); PPRV (peste-des-petits ruminants virus) from the Nigeria 75/1 strain; CDV (canine distemper virus) from the Onderstepoort strain and PDV (phocine distemper virus) from the Ulster/88 strain.

interferon competent HeLa-SLAM cells demonstrated that the C protein is required for efficient growth (Takeuchi et al., 2005). Furthermore a -C rMV failed to infect lymphoid tissue in a macaque model and replicated at least 3000 fold less efficiently in the tissue compared to wild-type virus (Takeuchi et al., 2005). One clearly identified function of the C protein is identified by its ability to suppress transcription and replication (Bankamp et al., 2005). This has been demonstrated in mini-genome expression assays (see above). A study of naturally occurring substitutions at aa positions 147 and 166, but not 88 and 186, were found to regulate MV C protein activity (Bankamp et al., 2005). Deletion of the carboxyl-terminal 19 aa (Miyajima et al., 2004) did not affect the polymerase-modulating activity, but aa changes in the MV C protein could not be linked to attenuation (Bankamp et al., 2005). This effect on the transcription and replication of the virus makes it difficult to distinguish the effects of the C protein on viral growth from the effects on interferon induction and signalling and no completely clear picture has emerged as yet (Nakatsu et al., 2008). Fontana et al. (2008) demonstrated that the C protein was less effective in blocking interferon signalling than V protein and Nakatsu et al. (2008) proposed that it has no ability to block signalling at all but that C protein inhibits IFN-␤ production as a result of its interference with the production of PKR. In contrast, Yokota et al. (2003) indicated a direct interaction of the C protein with the interferon receptor chain 1, STAT1 and the scaffolding protein RACK1). Though the mechanisms remains unknown, it is however clear from the induction of interferon in C rMV infected cells in the presence of a fully functional V protein that both viral C and V proteins are required for the virus to circumvent the interferon response of the cell (Nakatsu et al., 2008). A recent paper by Yokota et al. (2011) indicates that the C protein does affect the IFN-␥ signalling cascade by interacting with tyrosine phosphorylated STAT1 and suppressed the formation of the STAT1 dimers required for expression of IFN-␥ controlled genes. The virus induced IRF-1 expression which could result from IFN-␥ signalling and lead to growth arrest of host cells, is thereby counteracted by the C protein. This effect of the C protein in counteracting other viral responses appears to be similar to the balancing action of C in relation to the phosphorylation of eIF2␣ (Nakatsu et al., 2006). Recent studies show that the MV C protein suppresses PKR in the cell (Toth et al., 2009a; McAllister et al., 2010). PKR mediates IFN-␤ induction. By using -C and -V rMVs they show that the induction

of IFN-␤ depended on PKR and mitochondrial adapter protein IPS1, and correlated with the PKR mediated-enhancement of MAPK and NF␬B activation (McAllister et al., 2010). Suppression of PKR activity may also affect apoptosis (Zilliox et al., 2006), thereby linking C protein to suppression of apoptosis in MV by counteracting the pro-apoptotic and antiviral action of PKR (Toth et al., 2009a). Some of the effects of the C protein on host cell proteins might be explained if it activated protein phosphatases. There thus appears to be a multitude of functions which are influenced by the MV C protein. The fact that in some cases it may balance cellular responses to stress resulting from the activity of other viral proteins or RNAs or from infection itself as well as regulate overall levels of replication and transcription, makes it difficult to come to a coherent and simple understanding of the function(s) of this protein which is so little conserved even among the closely knit genus of the morbilliviruses (Fig. 1). Toth et al. (2009b) also described that activation of PKR kinase and IRF3 correlated with enhanced apoptosis in wild type MV infected cells in which in the long interferon inducible form of ADAR1 (George and Samuel, 1999) was knocked down by RNA silencing. These results suggested that ADAR1 is a proviral, anti-apoptotic host factor in the context of MV infection. However contrasting results have been reported for proor antiviral functions for ADAR1. ADAR has long been of interest to MV virologists as it was considered to be the enzyme that explained biased hypermutation of U to C resulting from deamination in the negative strands (Cattaneo and Billeter, 1992) and also A to G mutation (Baczko et al., 1993) in the MV genomes found in the brains of SSPE patients and in other viral RNAs (Bass, 2002). In human hepatitis virus infection, ADAR1 has been shown to target viral RNA and to suppress viral replication through dsRNA editing (Taylor et al., 2005) and have an antiviral effect. In contrast, a pro-viral effect of ADAR1 that enhances replication of vesicular stomatitis virus (VSV) through a mechanism independent of dsRNA editing has also been demonstrated (Nie et al., 2007). In this system ADAR1 interacts with dsRNA-activated protein kinase PKR, inhibits its kinase activity, and suppresses phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2␣). This effect of ADAR1 requires the amino-terminal domains but does not require the deaminase domain. These findings reveal a novel mechanism of ADAR1 that increases host susceptibility to VSV infection by inhibiting PKR activation (Nie et al., 2007). In MV

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infection the opposite has been found (Ward et al., 2011). Cells in which ADAR1 was knocked out but not WT MEF cells displayed extensive syncytium formation and cytopathic effect following infection with MV, consistent with an anti-MV role of the p150 isoform of ADAR1. MV titres were 3–4 log higher in p150(−/−) cells compared with WT cells and restoration of ADAR1 in p150(−/−) cells prevented MV cytopathology. In contrast to infection with MV, p150 disruption had no effect on VSV, reovirus or lymphocytic choriomeningitis virus replication but protected against CPE resulting from infection with Newcastle disease virus, Sendai virus and influenza A virus as well as another morbillivirus: canine distemper virus. Thus, ADAR1 appears as a restriction factor in the replication of these paramyxoviruses and orthomyxoviruses (Ward et al., 2011). Similar results have been obtained with another deaminase APOBECG3 which also inhibits the growth by orders of magnitude for MV, mumps virus and human respiratory syncytial virus (Fehrholz et al., submitted), again independent of the deaminase activity itself. However it is also clear that deaminase activity itself can inhibit the formation of functionally intact viral RNA progeny molecules in cells (Suspene et al., 2011). The deamination gives rise to RNA molecules with an enhanced GC content. The levels of these potentially non-functional RNA molecules in the cells is substantially underestimated by current RT-PCR protocols and they have shown to be present in MV vaccines (Suspene et al., 2011).

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(Schneider-Schaulies et al., 1994; Schnorr et al., 1993). The MX proteins are GTPases involved in vesicle trafficking and the mouse equivalent MX proteins have been shown to inhibit early steps in the replication cycle of several RNA viruses, depending on the virus type affecting RNA synthesis or translation (Haller et al., 2007). MxA inhibition of MV occurs by an unknown mechanism and is cell type specific as it appears to affect transcription in glioblastoma cell line U87 (Schneider-Schaulies et al., 1994), but glycoprotein expression in monocytic cell line U937 (Schnorr et al., 1993). It appears that the virus has not developed a response to the inhibitory effects of MxA but at the same time the induction of MxA appears “survivable” for MV. Interestingly no role of the internal M protein in any of these processes has been observed or investigated. It has a clear structural role in budding but also acts as a regulatory factor for transcription in the cell (Suryanarayana et al., 1994; Iwasaki et al., 2009). One unique part of the morbillivirus genome and thus MV, which has been shown to have a functional role in replication and gene expression is the long combined 1 kb untranslated region between the ORFs for the M and F proteins (Takeda et al., 2005). Deletion mutants can grow but the effect of these mutations on changing the cellular environment has not been documented.

4. Conclusions and future directions 3.3.3. A role for the N and M protein? Apart from the aforementioned interaction of the N protein with eIF3-p40 and effects on translation, the N protein has been postulated to also play a role in immune-suppression by its ability to bind to extracellular receptors such as Fc␥RII (Ravanel et al., 1997), and an unidentified receptor NR expressed widely (Laine et al., 2003, 2005). The latter binding is through box 1 of the N protein (aa 401–420). The binding to these receptors induces apoptosis and cell proliferation and therefore has been suggested to play a role in the lymphopenia discussed earlier. It also reduced in vitro antibody production in B cells (Ravanel et al., 1997). The main question that this observation raises is how does the N protein end up on the outside of the cell able to bind cellular receptors? Marie et al. (2004) suggested that the N protein in combination with the Fc␥RII travels through the late endosomes to the cell membrane. The mechanism of transfer of the cytosolic N protein into the endosomal compartment remains unclear. Nevertheless, externalisation and recognition are implied from the remarkable early humoral immune reaction against the N protein which results in being that the N protein is the main antigen recognized in the infected patient early in infection. Thus antibody to the N protein is the complement fixing antibody and the most prevalent one early in infection (Graves et al., 1984). However, this does not fit well with the reported in vitro antibody inhibiting effect (Ravanel et al., 1997) of the binding of N protein to Fc␥RII. The RNP and the N protein alone, if transiently expressed, are able to activate IRF3 by phosphorylation of two critical serine residues in the carboxy terminal tail of this transcription factor (tenOever et al., 2002). This leads to the synthesis of proinflammatory cytokines but not IFN␤. Replication was shown to be required by using a rMV with a deletion in the L protein. Thus it is not the incoming RNP that activates an unidentified kinase. The binding site for IRF3 on the N protein was interestingly identified in NTAIL residues 415–523 (tenOever et al., 2002). Again the point has to be made that this is proposed to be on the inside of the helix (Desfosses et al., 2011) and hence it may be that activation of IRF3 requires the generation of new soluble N protein in quantities that depend on amplification by the incoming genomic RNP. A final molecule that has been described as part of the innate immune system (Haller et al., 2007) and that has been shown to interfere with MV replication is the IFN induced MxA protein

In this review we have striven to bring the reader up to date with the most important recent changes in measles virology. Although the original antigenomic clone and N, P and T7 RNA polymerase expressing cells lines are now used infrequently much of this knowledge depends on the reverse genetics system developed by Martin Billeter (Radecke et al., 1995; Billeter et al., 2009). Indeed all of the second and third generation rMV systems generated depend on this achievement. Our own studies using rMVs produced directly from clinical material have moved measles from an infection of the respiratory system to one which, like HIV-1, is highly lymphotropic to the extent that the first cells targeted by the virus are CD150 expressing cells. Based on these in vivo studies it seems futile to search for an entry receptor on epithelial cells. Nonetheless the virus is still transmitted by the aerosol route and understanding the optimal means of delivering the vaccine is now possible using a panel of vaccine-based rMVs which express EGFP. However, as the door closes on epithelial cells as important for MV entry at the primary infection site it opens to them as a potential key to understanding “exit” from the body and transmission to the next susceptible host. What will be learnt as we begin to unravel the part nectin-4 plays in pathogenesis may help to explain why MV is one of the most infectious human pathogens known. This is all the more important as MV eradication is considered (see Bellini and Rota, 2011). Although whether it would ever be safe or rational to suspend vaccination would require significant consideration, but eventual eradication may open up the way to combine continued MV vaccination with its already proven success as an expression vector for other antigens (Billeter et al., 2009). The intrinsic barriers to infection such as dependence on host cell factors and ability to deal with antiviral responses discussed in this review are undoubtedly important in maintaining species specificity of MV. However, the fact that all morbilliviruses use SLAM as a relatively conserved receptor lowers the barrier to cross-species jumps for this group of viruses significantly. A number of host proteins (Table 2) have been identified that interact with the NTAIL and the XD domain of P. The relevance of this and the mechanisms involved remain unclear but this interaction which are due to the importance of the N–P complex opens the way for a fine regulation of the replication of the viral RNPs. If as is indicated the NTAIL is indeed on the inside of the RNP and localised

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near the inner core then this interaction between host and viral proteins is less likely to play a role in transcription unless there is a large scale disruption of the RNP structure at the site of RNA synthesis by the RdRp. A virus such as MV which is able to establish persistent infections in vitro and in vivo must be able to tune antiviral responses and maintain a balance with the needs of the cell for continued survival and cellular growth and replication. This need is exemplified by the effect of infection on host translation which appears counteracted by the viral C protein that also promotes viral replication/transcription. Similar arguments apply to cellular apoptosis. There appears to be a regulated balance between pro-apoptotic effects of infection (mediated by the N protein and soluble H protein) and counteractions by the virus that restrict apoptosis. Though apoptosis is demonstrated in vitro to occur in certain cells and may play a role in lymphopenia, it is not a hallmark of MV infection. All three pathways that are involved in IFN responses are inhibited i.e. the JAK/STAT signalling is inhibited by what appears to be a mechanism that shows redundancy in acting at the intracellular beginning of the pathway (JAK) and the end (phosphorylated STAT). The role of the C protein in the inhibition of IFN is clear but the mechanism is not. The C protein clearly has a multitude of roles and only binding to tyrosine phosphorylated STAT1 and its role in suppression of PKR seem clear. The lack of conservation in the group of the paramyxoviruses and even in the morbilliviruses makes it difficult to understand how this relatively small protein can have so many functions. The number of binding partners of the V protein has grown over the years (Table 2) and clearly this provides substantial redundancy in the inhibition of signalling of intracytosolic infection. The binding of the V protein to MDA5 and LGP2 at the beginning of the pathways as well as to the p65 subunit component of NF␬B at the end and also direct binding to IKK␣ which phosphorylates IRF7 exemplifies this well. Binding to NF␬B and IKK␣ also inhibit signalling through activation of TLR7. The role of RIG-I is not clear and could be associated more with recognition of corrupted PAMPs such as DI RNA. However, in the end who knows what viral RNA molecules are still to be discovered in infected cells? In conclusion this review demonstrates two important lessons for the future. The first is that only studies in relevant animal models systems or humans can really bring us closer to an understanding of MV pathogenesis. The second is that studies in vitro or ex vivo are only useful if they are carried out with appropriately propagated wild type viruses in relevant cell types and tissues, a factor that has often been overlooked in the past. Acknowledgements We thank Chris Richardson for making his paper on the identification of the epithelial receptor available to us during the writing of this review. We also thank Linda Rennick, Ken Lemon and Ingrid Allen for sharing unpublished data with us and Martin Ludlow for critical comments on the manuscript. Work in our laboratories is supported by the Medical Research Council of the UK grant number G0801001 and FNIH grant number DUPREX09GCGH0. References Allen, I.V., McQuaid, S., Ludlow, M., Duprex, W.P., Rima, B.K. Macrophages and dendritic cells are the predominant cells infected in epithelia in measles in humans, submitted. Auwaerter, P.G., Kaneshima, H., McCune, J.M., Wiegand, G., Griffin, D.E., 1996. Measles virus infection of thymic epithelium in the SCID-hu mouse leads to thymocyte apoptosis. J. Virol. 70, 3734–3740. Baczko, K., Lampe, J., Liebert, U.G., Brinckmann, U., ter, M.V., Pardowitz, I., Budka, H., Cosby, S.L., Isserte, S., Rima, B.K., 1993. Clonal expansion of hypermutated measles virus in a SSPE brain. Virology 197, 188– 195.

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