Virus Research 54 (1998) 117 – 122
Conference report
Strategies in virus–host relationships An international symposium on virus – host relationships was held in Lyon, France, on the 16 – 18 February 1998, under the sponsorship of the Marcel Merieux Foundation. The theme of the symposium was to compare the strategies of different viruses (including those of vertebrates, bacteria, plants and insects) and especially in relation to the infections they establish. Held at the Ecole Normale Superieure de Lyon, the Symposium was organised in five sessions with invited speakers and submitted papers, the latter mostly given in the form of poster presentations. The meeting started with welcoming addresses by the renowned Dr Charles Merieux (son of Marcel Merieux) and by Jean-Francois Mornex on behalf of the organising committee.
1. Session I This began with talks on what is understood by the term viruses and ‘virus species’, particularly in relation to how one can categorise viruses that continually vary through accumulated mutations and genetic recombination. The Chairman (JeanLuc Darlix) began with a brief overview of the subject and this was followed by Marc van Regenmortel who discussed the unique position viruses hold in biology. Firstly, they are not organisms, but rather, viruses are obligate, intracellular parasites that exploit the lives of organisms. Secondly, like other biological forms, they can be categorised into groups (Orders, Families, Gen-
era) and species. The broad definition: ‘‘a virus species is a polythetic class of viruses that constitutes a replicating lineage and occupies a particular ecological niche’’ has been endorsed by the International Committee for the Taxonomy of Viruses. However van Regenmortel pointed out that in genetic, and other ways, viruses have hazy boundaries and are therefore ‘fuzzy sets’, changing with passage and time, not like sharply defined chemicals, or stable genetic entities. Esteban Domingo continued the theme of what constitutes a virus species and the value and otherwise of the terms ‘quasi-species’ and ‘replicative mutant swarms’. He referred to the fact that a derived virus sequence is only a statistical average when the population is sequenced in toto, or a single clone when an individual is sequenced. He noted, also, that in biological terms, within virus populations members vary with, for example, individuals that are less, or better, fit to survive challenge (e.g. antibody escape mutants, etc.), including some with altered propensities for infection—such as foot-and-mouth disease viruses (Picornaviridae) that can be obtained with altered RGD receptor binding motifs. Claude Fanquet then discussed his recent studies on the ssDNA genome geminiviruses (Geminiviridae) using a method he has developed termed ‘quantitative taxonomy’ that involves the processive comparison of aligned sequences to delineate virus individuals into species and strains, as well as recombinant entities. By such analyses he has
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evidence from studies of recent epidemics of cassava in Uganda, cotton in Pakistan and tomato in several places in the world, for the presence of recombinant geminiviruses between individuals of different species of the same genus, or between individuals from different genera. He has been able to conclude that virus recombination is a very frequent event, sometimes in association with the introduction of new plants, or new insect vectors, or new viruses to new ecological niches. Session I finished with a presentation by Duncan McGeoch on the genetic structure of the large DNA herpesviruses (Herpesviridae, that have some 70–200 viral genes, depending on the species). He presented evidence for the existence of a common subset of 30 – 40 genes shared by many mammalian/avian herpesviruses, some of which can be used to compare the respective viruses, their mutation rates and genetic relationships. His analyses have revealed that the divergence of these herpesviruses correlates well with the historical divergence of their hosts (i.e. suggesting co-speciation), arguably involving an extended time-scale (millions of years). He also referred to another feature about herpesviruses concerning their host (cell/tissue) preferences, which may involve lifelong associations of both low-activity (latent/persistent) and productive states.
2. Session II This developed further the theme of virus genome variability (or constancy) specifically in relation to host relationships and viral pathogenicity. The chairman (Tim Greenland) initially discussed evidence for genetic exchange between viruses and their hosts, and the selection and maintenance of virus variants that are produced. He alluded to viruses which exhibit a considerable degree of variability but are nevertheless highly host-specific, and others (e.g. morbilliviruses) in which viruses are less variable, but swap hosts. Stephen Goff then discussed evidence for the interactions of retroviruses (Retroviridae) with host proteins (e.g. cyclophilin A in the case of the gag protein of HIV-1, and other host env-interacting proteins for murine leukemia virus), including
describing their effects on virus infectivities and infection processes. Daniel Kolakofsky discussed another strategy of viruses found during virus infections, one involving the differential expression of a nested set of genes (the C proteins of Sendai virus, Paramyxoviridae) that are translated from alternative initiation sites. The studies from his laboratory have illustrated the translational gymnastics that can occur in gene product expression. Jim Strauss also presented data on virus strategies involving gene product translational, drawing on the insect-transmitted alphaviruses (Togaviridae) and the processes and consequences of post-translational processing. He, too, presented evidence for the involvement of host proteins in virus replication (e.g. the La protein), and in relation to the recognition of viral promoters within mammalian and insect hosts. Just Vlak in his talk discussed his work on cassettes of genes that are rearranged among different insect baculoviruses (Baculoviridae, including the silkworm baculovirus that in previous times was a scurge of the French silk industry around Lyon, and elsewhere), and likened baculovirus genetic variation as akin to shuffling a deck of playing cards to produce the gene rearrangements that he has observed. Further, he described the course of baculovirus infections within their natural hosts and the accumulating evidence for baculovirus genes that may have been acquired from their hosts, or other species (e.g. bacterial chitinase genes). Finally, in submitted papers, Pascal Leblanc reported recent studies on characterising an integrated invertebrate retrovirus (Retroviridae, ZAM) identified from Drosophila melanogaster that is present in multiple copies in the insect genome, depending on the strain. Also, Sacha Lucchini described the genetic variation observed among clinical specimens of human rotaviruses (Reoviridae), including evidence for cross-species evolution, and in a separate study, the movement of genes among Streptococcus thermophilus bacteriophages (Siphoviridae). Finally, Tomas Bergstrom talked about evidence for human herpes simplex virus-2 (Herpesviridae) epitope conservation among clinical specimens of the virus.
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3. Session III This session had as its subject viral structures, phenotypes and host cell interactions. David Bishop noted in his introductory remarks that research themes in virology follow fashions, and that a recent very rewarding fashion in virology is to understand the atomic structures of viruses (and their gene products), and to use such information to investigate protein organisation and function. Dennis Bamford took up this theme by describing the structures and functions of the Pseudomonas ¥6 bacteriophage (Cystoviridae), a multisegmented dsRNA virus. Drawing upon information about the structure and infection course of this phage, he concluded that in many aspects it is similar to the mammalian (or arthropod, or fish, or plant) multisegmented dsRNA reoviruses (Reoviridae), although in other aspects it is different (e.g. the coding strategy of ¥6 involves polycistronic transcription and translational, and the virus has a unique mechanism of entry into bacteria). Polly Roy then discussed her studies on the structure of Bluetongue virus (BTV, Reoviridae), a gnat-transmitted orbivirus that can cause lethal infections in sheep, and the functions of the BTV-encoded gene products. She described what is presently understood from cryoEM and atomic analyses of the virus, and the locations and arrangements of the four major structural proteins. She also reported recent fine analysis experiments from her laboratory on the functions of the three minor structural proteins that are involved in mRNA transcription of the ten genomic dsRNA species in virus-derived cores (i.e. the viral RNA polymerase, guanylyl transferase, helicase). Later, B.V.V. Prasad discussed his studies on the similarly-structured rotaviruses (Reoviridae; viruses that cause diarrhoea in humans and other species), also drawing on cryoEM and other analyses. He described evidence for the location and arrangement of the three minor transcriptase proteins in rotavirus virions, and how the viral RNA is organised underneath the outer capsid proteins. He showed how nascent mRNA transcripts leave virus cores at the 5-fold vertices of the particles and how these exit pores can be effectively blocked using a monoclonal
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antibody that binds to an adjacent structural protein (VP6), thereby inhibiting mRNA transcription. Continuing the theme of virus structures, Jack Johnson described elegant molecular switches of a simple icosahedral RNA nodavirus (Nodaviridae) and the effects of mutating viral genetic information to eliminate sequences that are needed for proper virus assembly. He showed that the ability to package alternate RNAs could be altered by deletions in the capsid protein gene involving only ten amino acids. Such mutations abrogated proper virus assembly and resulted in an heterogeneous mixture of particles in which the particle sizes were proportional to the amounts of RNA packaged. Thus, with a large enough RNA, a T= 3 particle class was formed, but with smaller-sized RNAs other forms of particle were produced. Johnson also described the maturation autoproteolysis of these insect nodaviruses and proposed how RNA was associated with the capsids. The other talks in this session concerned descriptions and analyses of the structures and functions of certain surface glycoproteins of two groups of lipid-enveloped RNA viruses. Franz Heinz described the assembly and atomic structure of the envelope (E) glycoprotein of tick-borne encephalitis virus (TBEV, Flaviviridae). In assembly (and the processes involving virus egress and release from an infected cell) the fusion domain of the TBEV dimeric E protein is protected by a conserved glycan side-chain and by interaction with a second envelope protein, PrM. Subsequently, PrM is cleaved by furin during exocytosis. Later, during infection, and following receptor-mediated endocytosis, the E protein undergoes an irreversible conformational change at acid pH to provide the trimeric and fusogenic form of E necessary for membrane penetration. John Skehel likewise described the current understanding of the structures and functions of influenza virus (Orthomyxoviridae) haemagglutinin (HA) proteins. HA is a sialic-acid-receptor binding and membrane fusion glycoprotein. It is made as a precursor protein that is subsequently proteolytically cleaved to HA1 and HA2 in order to activate its membrane fusion potential. Skehel described what is now known about the HA1–
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HA2 cleavage event and the likely rearrangements in the structure that follow cleavage (i.e. burying the derived fusogenic HA2 amino terminus). From analyses of the HA1 – HA2 crystallographic structure (in complexes with receptor analogues, as well as by mutational analyses), both the binding residues in HA1 and the interactions involved in receptor binding have been elucidated. Skehel further described what occurs to the HA1 – HA2 complex when exposed to endosomal (acid) pH and involving the further rearrangements of HA2 to achieve the close apposition of virus and cellular membranes that eventually results in fusion.
4. Session IV This session developed further the theme of virus –host relationships and the aspects of interchange between viral and host genes (and gene products) and how these affect immune responses and the persistence, virulence and pathogenesis of the virus. The Chairman (Robin Weiss), introduced the subject by referring to the obligatory integration of transforming retroviral DNA proviral genomes into host chromosomal DNA which is similar to the integration into bacterial genomes of lysogenic, temperate bacteriophage, and also comparable to the integration of genes of small oncogenic DNA viruses such as papovaviruses, adenoviruses and hepadnaviruses and which occur as rare consequences of virus infection. He noted that, conversely, viral genomes may acquire host genes, as first described with certain acutely transforming retroviruses. Further, from sequencing data of many viruses (e.g. herpesviruses and poxviruses) it is now apparent that sets of host genes have been acquired by viruses, genes that help them replicate by, for example, inhibiting apoptosis, or down-modulating the host immune attack. He mentioned, also, that different viruses interact, and may depend on each other in the same host cells (e.g. the adeno-associated parvoviruses, the hepatitis d-agent), and that integrated avian retroviral long terminal repeats are found in fowlpox and Marek’s disease viral genomes. Following this introduction, Paul Klenerman reviewed earlier and recent information
concerning the persistence of lymphocytic choriomeningitis virus (LCMV, Arenaviridae) and discussed his new findings on the occurrence of cDNA copies of LCMV sequences in certain cells. LCMV infection is initially controlled in its natural host, the mouse, by a vigorous cytotoxic T lymphocyte (CTL) response. As with many other viral antigens, the quality of the CTL response is determined by the distribution and kinetics of spread of the virus. However, after initial clearance, persistently activated CTLs are detected, suggesting the continued presence of viral antigens. During a search for persistence virus Klenerman has detected viral sequences present in the form of cDNA. These sequences were only found in situations of high levels of endogenous RT, possibly due to an endogenous retrovirus. Whether the viral DNA has any biological function or even is responsible for long term LCMV gene expression remains to be determined, but the potential for integration or recombination events may provide a source of antigen and contribute to a protective CTL memory. Jan Svoboda then discussed a range of pathogenicity and intracellular barriers to retrovirus infections. He described retrovirus oncogene rescue, as exemplified by vsrc in birds, and reviewed new findings of high viral load in ducks infected with avian leukosis virus (ALV, Retroviridae). Different aspects of the ability of reverse kanscriptase to produce cDNA copies were discussed using the example of v-src mRNA reverse transcription, integration and rescue. Retrovirus oncogenic activity, mediated by activation of proto-oncogenes, was described with the acutely transforming avian retrovirus PR2257. He also noted that oncogenic DNA-genome viruses employ other strategies, using either a proto-oncogene product for activation or producing specific proteins which inactivate cellular anti-oncogene products. In relation to the different diseases obtained following retrovirus infection and persistence, he presented data suggesting the formation of increased amyloid A deposits in ducks persistently infected by ALV-C. Also, that transmission of retroviruses to foreign species of cells can be controlled by the host by provirus DNA methylation and down-regulation of viral transcription. Delphine Sitterlin then dis-
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cussed how the hepatitis B (Hepadnaviridae) X regulatory protein interacts with a host cell protein (UV-damaged DNA binding protein, WDDB). She is presently addressing the question of the role of this interaction on the virus cycle in the woodchuck model, by screening mutants for altered interaction with UVDDB. Michele Bouloy subsequently described biochemical and genetic properties of the tri-segmented, RNA genome, Rift Valley Fever virus (RVFV, Bunyaviridae), and specifically in relation to its non-structural protein (NSs) as a potential virulence factor. Like other members of the Bunyaviridae family, the virus replicates in the host cytoplasm. However, during RVFV infections large amounts of the phosphorylated NSs protein form filaments in the nucleus although some is associated with the viral nucleocapsids found in the infected cell cytoplasm where it interacts with the viral nucleoprotein. However, viral nucleocapsids extracted from purified virus do not contain NSs protein. In another study, with RVFV clone C13 attenuated for mice and lacking much of the coding region of NSs, her group has shown, by genetic reassortment studies, that the RVFV C13 S RNA carries the attenuating properties, implicating either NSs (or the N protein that is also encoded by the S RNA) as the causative factor. Robin Weiss then reviewed the epidemiology and pathogenesis of Kaposi’s Sarcoma-associated herpesvirus (KSHV, Herpesviridae), also known as HHV-8. KSHV is a g-herpesvirus associated with Kaposi’s sarcoma and primary effusion lymphomas. These tumours occur at some 70000-fold higher frequencies in immunosuppressed persons (AIDS, or transplant patients). Interestingly, KSHV has captured a surprisingly large number of host genes that may play a role in KSHV latent and lytic phases of replication and in tumour development. Among these are apoptosis inhibitors, a cyclin D homologue that may promote cell proliferation, chemokines and chemokine receptors related to angiogenesis. In fact Karposi’s sarcoma is a type of angiosarcoma, although it remains controversial whether it represents a clonal malignancy or (more likely) a hyperplastic endothelial response to cellular and viral angiogenic factors. Weiss noted that Epstein-Barr virus (EBV), which is also
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an oncogenic g-herpesviruses, has an alternative strategy in terms of host modulation. Whereas KSHV has captured and incorporated several of these cellular genes into its genome, EBV proteins activate the expression of cellular genes for IL-6, bc1-2 and other regulatory factors. The great increase in KSHV-related tumours in immunosuppressed hosts implies that virus-infected cells are under the strong control of cell-mediated immunity in the immunocompetent individual. In ending the session, Mike Skinner discussed work on the fowlpox virus (FPV, Poxviridae) and the existence of cellular gene homologues in the virus genome, such as viral homologues of a mammalian type II membrane glycoprotein with exoenzyme activity, a protein involved in the secretory pathway and two predicted proteins of unknown function but found thus far only in the nematode Caenorhabditis elegans. Although these homologues are non-essential for virus replication in tissue culture they are conserved within fowlpox viruses (though apparently not in other avipoxviruses) and so may play a role in host interactions.
5. Session V This session, introduced by Marian Horizinek, was devoted to a discussion of the relationships of viruses with their vertebrate hosts and insect vectors, vector habitat identification and virus emergence. Barry Beaty began by discussing his studies on La Crosse virus (LACV, Bunyaviridae), a mosquito-transmitted virus that in certain regions of the USA is sometimes responsible for fatal encephalitis in infants. LACV, like other bunyaviruses, uses host mRNA sequences to initiate mRNA synthesis (cap snatching). Since LACV is efficiently transmitted transovarially by its mosquito vectors he has examined the issue of viral gene expression during ovarian quiescence and embryo diapause. From such studies he has obtained evidence that viral mRNA synthesis is down-regulated, resurfacing when diapause is ended. Paul Sharp then discussed his views on the multiple emergence of HIV-1 and HIV-2 strains and their subsequent evolution as diverse clades in
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recent time. He also discussed the host preferences and time-scale of primate lentivirus evolution for simian lentiviruses (SIV, Retroviridae), specifically in relation to the issue of recombination. Paul Ewald, continuing the subject of sexually transmitted viruses, stated that according to theory and empirical evidence, sexually transmitted pathogens evolve to become more virulent when the potential for sexual transmission is high, and less damaging when the potential for sexual transmission is low. He claimed that data from HIV, human T-cell lymphotropic viruses, herpes simplex viruses, and the human papillomaviruses support this prediction. His view was that by reducing the potential for sexual transmission it should be possible to reverse the process of virus evolution so that sexually transmitted pathogens evolve towards a lower level of virulence. Brian Mahy began by summarising the recent outbreak of chicken influenza in Hong Kong and the transmission of this chicken virus to humans. He then reviewed the data on newly identified rodent hantaviruses in the Americas, some of which have caused fatal human pulmonary infections and in relation to the rodent hosts and their habitats. Scott Weaver continued the theme of habitat – vector identification in relation to Venezuelan equine encephalitis virus (VEE, Togaviridae). VEE is an important zoonotic arboviral disease that occurs in both enzootic and epizootic forms. The issue of what causes the epizootic forms of the virus to emerge from quiescent insect – small
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animal cycles to cause horse and human epizootics is a subject that is presently being developed taking advantage of remote sensing and habitat analyses using sentinel animals in Columbia and Western Venezuela. While the research is still at the formative stages, the combination of virological and ecological approaches offers exciting new opportunities for understanding and predicting zoonotic viral emergencies. In summary, the International Symposium on the Strategies in Virus–Host Relationships covered a wide range of subjects, issues and interests. Overall, the meeting reflected the shift in emphasis that is occurring elsewhere in molecular biology, i.e. away from genome-oriented reductionism to the more integrated approach of considering biological systems as a whole. For virology, this shift has meant that genome sequencing and identifying viral genes no longer holds centre stage, instead the cutting edge studies address the dynamic interplay between viruses and their hosts, including ecological considerations (vectors, habitats) and open up new opportunities for research and understanding in the future. Da6id H.L. Bishop Oxford University St. Cross College St. Giles Oxford OX1 3LZ England, UK