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Myristoylation of viral and bacterial proteins
Review
TRENDS in Microbiology
Vol.12 No.4 April 2004
Myristoylation of viral and bacterial proteins Sebastian Maurer-Stroh and Frank Eisenhaber IMP – Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria
Myristoylation, the N-terminal attachment of a myristoyl lipid anchor to a glycine residue, can reversibly direct protein –membrane and protein –protein interactions. Apart from two entomopoxviruses, viruses and bacteria usually lack the enzyme N-myristoyltransferase (NMT) that is required for this modification, and their proteins are consequently processed by NMTs of their eukaryotic hosts. This review gives an overview of the multiplicity of viral and the few known bacterial proteins that can undergo glycine myristoylation. In particular, we discuss the role of the myristoyl anchor in viral entry into host cells, in retroviral proteins and in the envelopment of large DNA viruses. We review evidence for myristoylation in arena- and flaviviruses as well as bacterial proteins after their secretion into eukaryotic host cells. Recent evidence shows that myristoylation of noneukaryotic proteins is far more than just a curiosity. From the time that the myristoyl lipid modification of viral proteins was discovered in the early eighties, considerable new insight has been accumulated that has increased the number of known myristoylated proteins in viruses (Table 1). This progress is now aided by the availability of reliable prediction methods for the occurrence of this modification [1,2] and a comprehensive collection of the resulting data at sites such as MYRbase (http://mendel. imp.univie.ac.at/myristate/myrbase/)p. Recently, it was discovered that some bacterial proteins can also undergo lipid modification after injection into the host when using a type III secretion system (TTSS) [3]. Typically, the attachment of a 14-carbon saturated fatty acid (myristate) to N-terminal glycine residues of substrate proteins is executed by N-myristoyltransferases (NMTs) [4] of the eukaryotic host, but there are indications for NMTs in two viral species [5]. Several lower eukaryotic parasites also have their own NMTs for protein myristoylation [5]. Owing to mechanistic reasons, lipid modification by NMT strictly occurs solely on N-terminal glycines, which can become N-terminal after proteolytic cleavage [6]. In eukaryotic cells, the largest families of proteins with a conserved myristoylation motif (e.g. Ga subunits, ADP-ribosylation factors, serine/threonine and tyrosine kinases, and EF-hand calcium-binding proteins) are Corresponding author: Sebastian Maurer-Stroh ([email protected]). * See also the recent publication by Maurer-Stroh, S. et al. (2004) MYRbase: analysis of genome-wide glycine myristoylation enlarges the functional spectrum of eukaryotic myristoylated proteins. Genome Biology 5, R21
primarily involved in signaling processes. However, recent studies [7] have identified a series of smaller families that increase the functional spectrum of myristoylated proteins. The limited hydrophobicity of the myristoyl anchor permits reversible membrane interactions and requires additional membrane attachment factors (MAFs) for enhancing membrane targeting. Typical MAFs include the subsequent palmitoylation of nearby cysteine residues, clusters of positive charges, phospholipid-binding domains (e.g. for PIP2 interactions), transmembrane regions or direct protein –protein interactions. Both targeting specificity and membrane affinity are achieved through the concerted effects of a combination of these MAFs. For example, dual acylation (myristoylation and palmitoylation) often directs proteins to lipid rafts [8]. In viruses in particular, the role of the myristoyl moiety includes not only targeting to diverse membranes and membrane subcompartments, but it can also be involved in direct interactions with other proteins [9] or conformational switches that affect tertiary and quaternary structure [10]. This review aims to cover the existing knowledge surrounding myristoylated viral and bacterial proteins and summarizes the involvement of the lipid anchor in various steps of the life cycle of viruses, including several of enormous medical importance. Involvement of myristoylated proteins in viral entry into host cells Besides roles in assembly, structure, budding and intracellular host interactions, myristoylated viral proteins are increasingly implicated in viral entry. Typically, hydrophobic patches of proteins in both enveloped and nonenveloped viruses are exposed as a result of conformational changes that can be induced by changes in pH (e.g. acidification following internalization into endosomes), physiological temperature, cleavage and receptor-binding. Membrane fusion [11] and pore formation [12] are essential in the invasion process of enveloped and non-enveloped viruses, respectively. Although myristoyl lipid anchors usually enter membranes from the cytoplasmic or viral matrix side, a completely different topology can be found for the large surface antigen of human and duck hepatitis B viruses (HBV). C-terminal transmembrane regions anchor the protein to the envelope membrane and the myristoylated N-terminus appears on the outside [13]. Furthermore, the lipid modification is not required for viral assembly or protein localization, but for infectivity [14]. HBV receptor
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Review
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Table 1. Viral proteins with experimental evidence for glycine myristoylation Protein(s)
Total number Viruses in MYRbase
Refs
Negative factor (nef) Gag Total P17 P15 (matrix protein)
2498 1583 1444 85
[64]
P10 (matrix protein)
29
P19 (component of the inner protein layer of the viral nucleocapsid) VP4 coat protein
25 566
Large surface antigen L1R (VV) envelope protein
415 31
v-src A16L (VV) late protein VP2 coat protein G9R/F1 (VV) Tegument protein UL11
26 20 19 14 13
UL99, U71, BBLF1
13
Major outer capsid protein m1 E7R (VV) P220 Membrane fusion protein p15
9 5 1 1
Lentiviruses (HIV, SIV) Retroviruses, Poxviruses Lentiviruses (HIV, SIV, FIV) Gammaretroviruses (e.g. murine and feline leukemia viruses, rat and avian sarcoma virus, avian spleen necrosis virus, baboon and porcine endogenous retroviruses, reticuloendotheliosis virus), human endogenous retrovirus S71, Poxviridae (Fowl pox) Betaretroviruses (mouse mammary tumor virus, Mason-Pfizer monkey virus, simian retrovirus 2, ovine pulmonary adenocarcinoma virus), wooly monkey sarcoma virus, human endogenous retrovirus K, hamster intracisternal Aparticle and python endogenous retrovirus Deltaretroviruses (human/simian T-cell lymphotropic virus, bovine leukemia virus) Picornaviridae [rhino-, cardio-, aphtho-, entero- (echo-, coxsackie-, polio-) viruses] Hepadnaviridae (hepatitis B virus) Poxviridae [Chordopoxvirinae (e.g. variola, vaccinia) and Enteropoxvirinae ], Asfiviridae, Iridoviridae (lymphocysti-, rana-, iridovirus), Ascoviridae (Ascovirus) Rous sarcoma virus Poxviridae [Chordopoxvirinae (e.g. variola, vaccinia) and Enteropoxvirinae ] Polyomaviruses Poxviridae [Chordopoxvirinae (e.g. variola, vaccinia)] Alphaherpesvirinae (simplex-, varicello-, Marek’s disease-like and infectious laryngotracheitis-like viruses) Betaherpesvirinae (roseolo-, cytomegalo-, muromegalovirus), Gammaherpesvirinae (e.g. lymphocryptovirus), Alphaherpesvirinae Ortho- and aquareoviruses Orthopoxviruses Asfivirus Orthoreovirus
recognition occurs over an N-terminal QLDPAF sequence in the vicinity of the myristoylation motif [13]. It remains to be established whether the lipid moiety is involved in direct receptor interaction [15], if it induces conformational constraints on the receptor-binding region, inserts directly into host membranes or plays a role in later fusion events. Among the non-enveloped viruses, involvement of the myristoyl anchor in viral entry has recently been established for the structural protein m1 of reoviruses [10]. Autocatalytic cleavage of this protein into m1N and m1C, and proteolytic removal of s3 (which shields m1 at the virion surface), allow exposure of the myristoyl moiety in m1N and insertion of the anchor into the host membrane. Interestingly, a non-structural reoviral protein (p15 from the S4 genome segment of Baboon reovirus) has been shown to be myristoylated in the host cell where it induces cell – cell fusion as an integral membrane protein [16]. Similar to the myristoyl switch of m1, the structural protein VP4 of picornaviruses undergoes conformational changes following cleavage, resulting in exposure and host cell membrane insertion of the lipid anchor [17]. A model for involvement in viral entry has also been proposed for the myristoylated VP2 protein of polyomaviruses found on the virion surface [18]. The entry of some plant viruses can take plant-specific shortcuts and, as we suggest, involve myristoylated proteins. For example, C4 proteins of several geminiviruses are predicted to be modified with this lipid anchor (Table 2) [19]. These proteins localize to the plasmodesmata (channels interconnecting plant cells) and are implicated in cell to cell movement of the respective viruses [20]. www.sciencedirect.com
[65] [66]
[67]
[68] [69] [70] [71]
[72] [46] [73] [46] [74] [41] [75] [46] [76] [16]
Prominent myristoylation in retroviruses The Nef protein found in the lentivirus subgroup of retroviruses, which includes human immunodeficiency virus type 1 (HIV-1), HIV-2 and simian immunodeficiency virus (SIV) among its members, contains a series of signals that act together in adjusting host cells to optimal viral replication conditions, one of these signals is the myristoylation motif. Typically, the lipid anchor directs the protein to membranes and co-localizes it with putative interaction partners that are recognized over additional binding determinants (e.g. the di-leucine motif and proline-rich SH3-binding motif). These interactions involve the endocytic machinery [compromising the host’s immune response by down- or upregulation of specific membrane protein abundances (e.g. down: CD4, CD28 and MHC class I molecules; up: AP-1, AP-3, TNF, LIGHT and DC-SIGN)] on the one hand and interference with signaling pathways on the other [affecting T-cell activation and apoptosis (e.g. interaction with Hck, Lck, Fyn, Lyn, PAK, PKC theta, Vav, MAPK, p53, Raf 1, PI3K and TcR chain zeta)]. Details on the multiplicity of the molecular functions of Nef can be found, for example, in Ref. [21]. The structural precursor polyprotein Gag is conserved among retroviruses and is essential for viral assembly [22]. Its N-terminal matrix (MA) domain is myristoylated and the lipid anchor, in addition to a cluster of positive charges, facilitates targeting to lipid rafts in the plasma membrane where Gag multimerization, viral assembly and budding occur [23]. Proteolytic processing by a viral protease results in a conformational switch involving the myristoyl anchor and
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TRENDS in Microbiology
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Table 2. Viral proteins predicted for glycine myristoylation Protein(s)
Total number in MYRbase
Representative Gis in MYRbase (number in cluster)
Viruses
C4
105
28380577 (75), 20177150 (21), 22901050 (8), 9187889 (1) 23452258 (22) new world, 75456 (10) old world, 23452268 (1) new world
Geminiviridae (Begomoviruses)
Glycoprotein GPC
33
RING finger protein Z
5
23343513 (5)
Immediate-early R1.5 circ protein Non-structural protein NSP1.orf1 Early protein EP0 (transcription activator) Major envelope antigen p43K Orf 15L of multigene family 100 K5L (VV) Unknown non-globular protein VP4-related FPV229, FPV221 vaccinia A47L homologue E66 homologue V0 K10
5
319990 (3), 420432 (1), 12084887 (1) MDL
4
1360868 (3), 4106794 (1)
Arenaviruses [old world (e.g. lymphocytic choriomeningitis, lassa, mopeia,…) and new world (e.g. junin, pichinde, tacaribe, latino, flexal, allpahuayo, bear canyon,…) viruses] Old world arenaviruses (e.g. lymphocytic choriomeningitis, lassa,…} Alphaherpesvirinae (varicello-, Marek’s disease-like viruses) Rotaviruses
3
93140 (3)
Alphaherpesvirinae (varicellovirus)
3 2 2 2 2 2
7444364 (3) 9628255 (2) 93481 (2) 9629594 (2) 22960707 (2) 9634899 (1), 9634891 (1)
Chordopoxvirinae (molluscipoxvirus) Asfiviruses Orthopoxviruses Gammaherpesvirinae Reoviridae (coltiviruses) Chordopoxvirinae (avipoxvirus)
2 2 2
9631098 (2) 76992 (2) 18846026 (2)
Baculoviridae (nucleopolyhedrovirus) Geminiviridae (mastrevirus) Gammaherpesvirinae (rhadinovirus)
leads to membrane detachment of the MA domain [24]. The role for the lipid anchor in the MA domain-mediated transport of viral components to and from the nucleus is unclear [25]. There also exists an MA domain variant in Rous sarcoma virus gag protein where a cluster of positive charges without the myristoyl anchor is sufficient for membrane targeting and the role in viral assembly. Interestingly, there are several examples of retroviral oncogenes containing Gag MA domains that are myristoylated [26]. The transcription factor v-Fos (viral Fos) [27], the GTPase v-Ras [28] and the kinases v-Abl [29], v-Akt [30], v-Fgr [31] and v-Mos [32] belong to this group. It remains to be established whether the myristoyl anchor, when combined with a cluster of positive charges alone or with the complete MA domain, contributes to the oncogenic potential of each example. In the case of myristoylated v-Src, its counterpart c-Src (host cellular Src) already includes a myristoylation motif and a positive charge cluster, which is retained in the viral oncogene [33] but without a MA domain. In v-Abl and c-Abl, a myristoyl anchor is also conserved, and in v-Abl it is part of an N-terminal MA domain. In c-Abl, the lipid anchor functions as a reversible membrane switch [34]. In several of these cases the myristoylated MA domain might result in the strengthening of membrane attachment that is usually weak or reversible. Common myristoylation-dependent mechanisms for envelopment in large DNA viruses Among enveloped viruses, several large DNA viruses are wrapped by a second lipid layer that is derived from ER-trans-Golgi membranes [35]. In the wellstudied Vaccinia poxvirus, a myristoylated transmembrane protein L1R is located within the first envelope, and in addition to other functions is involved in the www.sciencedirect.com
acquisition of the second envelope [36]. Whether the lipid anchor is required for trafficking and specific targeting or for direct membrane lipid recruiting remains to be established. Interestingly, database searches have identified a superfamily of pox-viral sequences that are related to L1R but also have homologues in non-poxviruses. In addition to similarities at the sequence level, all proteins have a conserved architecture, including an N-terminal myristoylation motif (except for the poxviral F9L subfamily), a globular domain and two transmembrane regions with C-terminal extensions; some of the members also have an additional transmembrane region (Figure 1). In poxviruses, close relatives of L1R share six conserved cysteine residues that form intramolecular disulfide bridges in a recently established pathway [37]. In the alignment of the superfamily, only two of the cysteines remain conserved (Figure 1). This suggests spatial vicinity of the respective residues in the unresolved structure of the globular L1R domain. The superfamily includes proteins from pox-, asco-, asfi- and iridoviruses, which are all large DNA viruses with similar membrane arrangements [35], possibly pointing to a conserved envelopment mechanism in agreement with proposed evolutionary relationships [38]. The myristoylated proteins UL11 and UL99 in herpesviruses, another group of large DNA viruses with secondary envelopment from Golgi cisternae [39], do not share sequence similarities with the L1R family. However, they are also targeted to the compartments where envelopment takes place [40,41]. Other herpesvirus proteins with completely different functions, which are predicted to be myristoylated, include the immediate-early R1.5 circ protein [42] and the early transcription activator EP0 [43] (Table 2).
Review
TRENDS in Microbiology
oPox
250
ePox
246
Asfi
248
Asco2
241
Asco1
441
lIrido
40
459
rIrido
70
523
iIrido1
80
515
iIrido2
70
495
uIrido
60
485 TRENDS in Microbiology
Figure 1. Schematic conserved architecture of a superfamily of myristoylated proteins related to Vaccinia L1R. Green, myristoylation motif; dark blue, transmembrane regions predicted with DAS-TMfilter [63] using conservative thresholds; light blue, transmembrane regions in agreement with hydrophobicity patterns in the alignment; yellow, two conserved cysteines forming a disulfide bridge; numbers in the end give the total protein length. Accession numbers and virus details: oPox, NP_042117.1 Variola virus; ePox, NP_064999.1 Amsacta moorei entomopoxvirus; Asfi, NP_042824.1 African swine fever virus; Asco2, CAC84467.1 Spodoptera frugiperda ascovirus 1; Asco1, CAC19148.1 Ascovirus DpAV4; lIrido, NP_078665.1 Lymphocystis disease virus 1; rIrido, AAK54492.1 Regina ranavirus; iIrido1, T03070 Chilo iridescent virus; iIrido2, NP_149921.1 Invertebrate iridescent virus 6; uIrido, NP_612229.1 infectious spleen and kidney necrosis virus.
By contrast, the lipid modification status of Vaccinia poxvirus proteins (not only myristoylation, but also palmitoylation, which is technically more difficult to detect) is experimentally well established [44], with the exception of A14L. The reported myristoylation of Vaccinia A14L protein at glycine residues that are not N-terminal [45] is mechanistically impossible and might represent a different acylation (such as palmitoylation or the rare lysine myristoylation) at other sites instead [44]. However, this would leave a myristoylated protein represented by a 14 – 15 kDa band in gel shift experiments of earlier work unassigned [46]. The only remaining Vaccinia protein in concordance with this mass that contains a myristoylation motif would be encoded by the open reading frame K5L. Other proteins exist in related but distinct poxviruses
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whose myristoylation status still requires experimental verification (Table 2). For example, p43K of the human tumorigenic molluscipoxvirus [47] is a clear homologue of Vaccinia major envelope antigen p37K (F13L). Although p37K lacks a myristoylation motif, it has already been shown to be lipid modified with palmitoyl anchors instead, which are required for the proper localization to the transGolgi network membranes [48]. Owing to similar physical properties of the lipid anchors (16- instead of 14-carbon saturated alkyl chains), palmitoylation is expected to result in similar targeting as myristoylation. Furthermore, whether several entomopoxviral proteins with an N-terminal glycine residue and a degenerated myristoylation motif are modified by the host or a putative viral NMT remains elusive [5]. Myristoylation in arena- and flaviviruses Despite the lack of experimental reports about myristoyl lipid modifications in proteins of arenaviruses, inhibiting the myristoylating enzyme NMT in infected cells has proven effective in inhibiting replication of the Junin virus [49], suggesting that myristoylated arenaviral proteins play a central role in replication. A possible candidate that has a conserved motif for glycine myristoylation is a glycoprotein from both old world and new world arenaviruses (Table 2). As host NMT inhibition did not abolish glycoprotein surface expression [49], it could be concluded that the lipid anchor is not important in its processing and localization. However, in analogy to other examples of myristoylated viral surface proteins, such as the hepatitis B virus large surface antigen, it could be involved in host cell recognition and viral entry. Interestingly, the RING finger protein Z of old world arenaviruses also carries a myristoylation motif (Table 2). However, truncation experiments have shown that its N-terminus is not required for its function in inhibiting viral RNA synthesis [50]. The medically important flaviviruses also lack examples of experimentally verified myristoylated proteins despite the existence of possible candidates. For example, mature NS5 is produced from a polyprotein in the host cytosol [51] and contains a valid N-myristoylation motif that is visible after cleavage (Table 3). The glycine that becomes N-terminal after
Table 3. Flaviviral proteins predicted for glycine myristoylation after proteolytic cleavagea Protein ID (flavivirus)
Cleavage motifb
N-terminus after cleavage (position of G in sequence)
59339 (Yellow fever virus)* 11528014 (West Nile virus)* 22474436 (Montana myotis leukoencephalitis virus)* 7939634 (Apoi virus)* 20177456 (Modoc virus)* 464427 (Dengue virus type 1) 266813 (Dengue virus type 2) 130437 (Dengue virus type 3) 130438 (Dengue virus type 4) 130520 (Tick-borne encephalitis virus) 730353 (Tick-borne powassan virus)
TGRR GLKR PGRR ENRR SNRR GGRR NARR TGKR TPRR GSRR GARR
(2507) GSANGK-TLGEVWKRELNLLDKRQFELYKR (2526) GGAKGR-TLGEVWKERLNHMTKEEFTRYRK (2478) GLSLSHLTLGEDWKLKLNKMTKSDFLEYRT (2475) GVSSSYITYGEQWKRELNKLNAQAFFLYKS (2477) GICSSAPTLGEIWKRKLNQLDAKEFMAYRR (2493) GTGAKGKHWERNGKDRLNQLSKSEFNTYKR (2492) GTGNIGETLGEKWKSRLNALGKSEFQIYKK (2491) GTGSQGETLGEKWKKKLNQLSRKEFDLYKK (2487) GTGTTGETLGEKWKRQLNSLDRKEFEEYKR (2511) GGSE-GDTLGDLWKRKLNGCTKEEFFAYRR (2513) GGAE-GSTLGDIWKQRLNSCTKEEFFAYRR
a
Available prediction methods [1] only predict potential myristoylation for NS5 from some of the many different flaviviruses (marked with asterisks). However, the remaining sequence fragments fail the prediction limit because of the dominant negative profile term that can be biased towards the learning set, but several of them appear to be in principal agreement with the physical properties required for productive N-myristoyltransferase (NMT) interaction. b Color code: red, myristoylatable glycines; yellow, palmitoylatable cysteines (additional lipid anchor can strengthen membrane attachment); blue, positive charges (can strengthen membrane attachment). www.sciencedirect.com
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Table 4. Myristoylated type III secreted bacterial proteins with different levels of experimental verification status Protein
Organism
Position
Evidence for type III secretion and myristoylation AvRpm1 Pseudomonas 2 syringae
Sequence motifa
Myristoylation
Type III secretion
GenInfo numbers
MGCVSSTSRSTGYYSGYENHE
[3H]-myrb, G2Ac [3]
[3]
[3H]-myr, G2A [3]
[3]
420847, 420848, 2664216 280002
G2A [3]
[3]
249817
G2A [77]
[78]
28871145, 479718
Predicted [56]
[56]
19071483
Predicted [58]
[57]
28868774
Predicted
[56]
19071488
Predicted [58]
[58]
28867732
Predicted (twilight) [56,58]
[56]
29171493
Predicted (twilight)
[79]
22036282, 22036280
Predicted
21231082
Predicted
By similarity to HopPmaB By similarity to HopPmaB By similarity to avrB By similarity to avrB/avrC By similarity to HopPmaD Predicted
14905926
Predicted
Predicted
21243956
Predicted (twilight)
Predicted
25139439
Predicted (twilight)
Predicted
17548434
Predicted (twilight)
Predicted
17548525
Predicted
Predictedd
20560111
Predicted (twilight)
Predicted
d
28870724
Predicted (twilight)
Predictedd
23472108
Predicted (twilight)
Predictedd
21243721
AvrB
Pseudomonas 2 MGCVSSKSTTVLSPQTSFNEA syringae AvrPphb Pseudomonas 63 GCASSSGVSLEDDSHTQVS syringae AvrPto Pseudomonas 2 MGNICVGGSRMAHQVNSPDRV syringae Evidence for type III secretion and predicted myristoylation HopPmaB Pseudomonas 2 MGLCVSKGSTASSPQHYAVRY syringae HopPtoJ Pseudomonas 2 MGLCISKHSGSSYSYSDSDRW syringae HopPmaD Pseudomonas 2 MGNICIGGPRMSQQVYSPERA syringae HopPtoF Pseudomonas 2 MGNICGTSGSRHVYSPSHTQR syringae HopPtoS1/ Pseudomonas 2 MGNICGTSGSNHVYSPPISPQ HopPtoO syringae IpaB Shigella sonnei 2 MGNVSAATTGLSLAKILASTE Predicted type III secretion and predicted myristoylation HopPmaB Xanthomonas 2 MGLCVSKPSVAGSPDHYATHA homolog campestris HopPmaB Xanthomonas 2 MGLCVSRPATSGSSVAASPEQ homologue axonopodis AvrC Pseudomonas 2 MGNVCFRPSRSHVSQEFSQSE syringae AvrB homologue Pseudomonas 2 MGCVSSKASVISSDSFRASYT syringae AvrPpiG1 Pseudomonas 2 MGICVSKPSVRHDYNEDYGRN syringae Outer protein J Xanthomonas 2 MGLCVSKPSVAGSPEHYAAHV campestris Outer protein J Xanthomonas 2 MGLCTSKPSVVGSPVAGSPEH homologue axonopodis PthG Pantoea 2 MGCFNVTGASGRANNYVVEHH agglomerans N-terminus Ralstonia 2 MGCFNVTGTSGTASNYVAREH similar to PthG solanacearum AvrPphD-related Ralstonia 2 MGNLQIKASSAPYALLSDVSP solanacearum Wzz-like Pseudomonas 2 MGCIVTNGIGSRKQSKDYEID aeruginosa Gluconokinase Pseudomonas 2 MGVSSCGKSAVGAEIARNSGG syringae RNA helicase Pseudomonas 2 MGLCGCTRGRPMTQEIGGFAA syringae Metal-dependent Xanthomonas 2 MGQLGAAARPPARQTSSQPGP hydrolase axonopodis
Predicted Predicted [3] Predicted Predicted
21241060 280003, 501130 8037787 11322463
a Color code: red, myristoylatable glycines; yellow, palmitoylatable cysteines (additional lipid anchor can strengthen membrane attachment); blue, positive charges (can strengthen membrane attachment). b Incorporates radioactive myristate dependent on N-terminal glycine. c G2A mutation abolishes membrane localization. d Fulfills basic N-terminal amino acid requirements but genetic context for capability of type III secretion is not clear.
cleavage could be subject to myristoylation by host NMTs, as occurs in other proteolytically processed viral proteins. The cell biology surrounding flaviviral NS5 appears compatible with myristoylation as a signal for partial or dynamic membrane-targeting of these proteins. NS5 is a multidomain protein involved in the viral replication cycle, which is suggested to take place in specialized membrane compartments [52]. It contains an N-terminal (nucleoside20 -O)-methyltransferase domain that is involved in RNA www.sciencedirect.com
capping and also a C-terminal RNA-dependent RNA polymerase domain. Interestingly, a cluster of positive charges (mainly comprised in an amphipathic helix) is in spatial vicinity to the flexible N-terminus in the resolved structure of the Dengue virus NS5 N-terminal domain [53]. This cluster might serve as an additional membrane attachment factor. Although a C-terminally His-tagged NS5 shows full polymerase activity, this activity is reduced when the His-tag is attached to the N-terminus [54], which
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would also abolish potential myristoylation among many other effects.
several more examples for glycine myristoylation of pathogenic bacterial proteins in the future.
Myristoylation of bacterial proteins after secretion into eukaryotic hosts Originally, glycine myristoylation was believed to be a unique posttranslational modification of proteins from eukaryotes and their viruses. However, Nimchuk et al. [3] corrected this view by demonstrating that proteins from pathogenic bacteria can also be myristoylated after secretion into eukaryotic host cells. Concomitantly, considerable improvements were achieved in understanding the TTSS (reviewed in Ref. [55]), which also resulted in the formulation of sequence requirements for predicting TTSS translocations [56 –58]. Table 4 summarizes the recently accumulated knowledge on myristoylated bacterial proteins with differing levels of experimental or computationally derived evidence for injection into the host and modification by the host NMT. Notably, most of the examples are from plant pathogens. Many of these proteins have N-terminal cysteine residues that could be subject to subsequent palmitoylation, which further strengthens attachment to membranes. It is at this cellular localization that the bacterial virulence or effector proteins are suspected to act on their targets, however, their efficiency can be reduced if the host carries specific resistance genes [59]. In this context, it is important that recent data has suggested that several plant disease resistance proteins are myristoylated [7], which could protect the effector targets by interfering with pathogenic proteins through myristoylation-dependent co-localization in specific membrane compartments. It is possible that some of these myristoylated plant disease resistance proteins could also be involved in host-specific innate immune responses against geminiviruses by inhibiting viral cell to cell translocation through co-localization with C4 proteins (see also viral entry section).
Acknowledgements
Concluding remarks: medical issues The number of myristoylated proteins identified in viruses is constantly increasing. Nevertheless, substantial additional work will be required to shed light on the multiple functions that could be executed by the respective proteins as well as their involvement in the viral life cycle and elucidation of the role of the lipid anchor. Inhibition of NMT or replacing myristic acid by analogs in infected cells is known to affect numerous viruses of medical importance, for example, HIV [60], hepatitis B virus [61], herpesviruses [62] and arenaviruses [49]. This approach might be generally suitable for influencing infections by viruses that take advantage of the eukaryotic myristoylation machinery. However as a side effect, inhibition of host NMTs affects myristoylation of vital cellular proteins (mainly involved in signal transduction pathways) that are processed by the same enzyme. Characterization of bacterial proteins that are secreted into eukaryotic host cells, including those that use translocation machineries that differ from the TTSS, is still in its infancy. Hence, it would not be surprising to find www.sciencedirect.com
We are grateful for continuous support from Boehringer Ingelheim. This project was partly funded by the Fonds zur Fo¨rderung der wissenschaf¨ sterreichs (FWF grant P15037), the Austrian tlichen Forschung O ¨ sterreichische Nationalbank) and the Austrian National Bank (OeNB - O Gen-AU bioinformatics integration network (BIN) sponsored by BM-BWK.
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49 Cordo, S.M. et al. (1999) Myristic acid analogs are inhibitors of Junin virus replication. Microbes Infect. 1, 609– 614 50 Cornu, T.I. and de la Torre, J.C. (2002) Characterization of the arenavirus RING finger Z protein regions required for Z-mediated inhibition of viral RNA synthesis. J. Virol. 76, 6678 – 6688 51 Zhang, L. et al. (1992) Processing and localization of Dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5. J. Virol. 66, 7549– 7554 52 Uchil, P.D. and Satchidanandam, V. (2003) Architecture of the flaviviral replication complex: protease, nuclease and detergents reveal encasement within double-layered membrane compartments. J. Biol. Chem. 278, 24388 – 24398 53 Egloff, M.P. et al. (2002) An RNA cap (nucleoside-20 -O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization. EMBO J. 21, 2757 – 2768 54 Guyatt, K.J. et al. (2001) Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin. J. Virol. Methods 92, 37 – 44 55 Thomas, N.A. and Brett, F.B. (2003) Establishing order for type III secretion substrates – a hierarchical process. Trends Microbiol. 11, 398– 403 56 Guttman, D.S. et al. (2002) A functional screen for the type III (Hrp) secretome of the plant pathogen Pseudomonas syringae. Science 295, 1722– 1726 57 Petnicki-Ocwieja, T. et al. (2002) Genome wide identification of proteins secreted by the Hrp type III protein secretion system of Pseudomonas syringae pv. tomato DC3000. Proc. Natl. Acad. Sci. U. S. A. 99, 7652– 7657 58 Collmer, A. et al. (2002) Genomic mining type III secretion system effectors in Pseudomonas syringae yields new picks for all TTSS prospectors. Trends Microbiol. 10, 462 – 469 59 McGuinness, D.H. et al. (2003) Pattern recognition molecules and innate immunity to parasites. Trends Parasitol. 19, 312 – 319 60 Bryant, M.L. et al. (1989) Replication of human immunodeficiency virus 1 and Moloney murine leukemia virus is inhibited by different heteroatom-containing analogs of myristic acid. Proc. Natl. Acad. Sci. U. S. A. 86, 8655– 8659 61 Parang, K. et al. (1997) In vitro antiviral activities of myristic acid analogs against human immunodeficiency and hepatitis B viruses. Antiviral Res. 34, 75– 90 62 Harper, D.R. et al. (1993) Inhibition of varicella – zoster virus replication by an inhibitor of protein myristoylation. J. Gen. Virol. 74, 1181 – 1184 63 Cserzo, M. et al. (2002) On filtering false positive transmembrane protein predictions. Protein Eng. 15, 745– 752 64 Yu, G. and Felsted, R.L. (1992) Effect of myristoylation on p27 nef subcellular distribution and suppression of HIV-LTR transcription. Virology 187, 46 – 55 65 Ono, A. and Freed, E.O. (1999) Binding of human immunodeficiency virus type 1 Gag to membrane: role of the matrix amino terminus. J. Virol. 73, 4136– 4144 66 Rein, A. et al. (1986) Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus. Proc. Natl. Acad. Sci. U. S. A. 83, 7246 – 7250 67 Hizi, A. et al. (1989) Analysis of gag proteins from mouse mammary tumor virus. J. Virol. 63, 2543 – 2549 68 Shoji, S. et al. (1989) Antibodies to an NH2-terminal myristoyl glycine moiety can detect NH2-terminal myristoylated proteins in the retrovirus-infected cells. Biochem. Biophys. Res. Commun. 162, 724 – 732 69 Moscufo, N. et al. (1991) Myristoylation is important at multiple stages in poliovirus assembly. J. Virol. 65, 2372– 2380 70 Gripon, P. et al. (1995) Myristylation of the hepatitis B virus large surface protein is essential for viral infectivity. Virology 213, 292 – 299 71 Ravanello, M.P. et al. (1993) An NH2-terminal peptide from the vaccinia virus L1R protein directs the myristylation and virion envelope localization of a heterologous fusion protein. J. Biol. Chem. 268, 7585– 7593 72 Resh, M.D. (1994) Myristylation and palmitylation of Src family members: the fats of the matter. Cell 76, 411 – 413 73 Streuli, C.H. and Griffin, B.E. (1987) Myristic acid is coupled to a structural protein of polyoma virus and SV40. Nature 326, 619 – 622 74 MacLean, C.A. et al. (1992) The myristylated virion proteins of herpes
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simplex virus type 1: investigation of their role in the virus life cycle. J. Gen. Virol. 73, 539 – 547 75 Tillotson, L. and Shatkin, A.J. (1992) Reovirus polypeptide sigma 3 and N-terminal myristoylation of polypeptide mu 1 are required for site-specific cleavage to mu 1C in transfected cells. J. Virol. 66, 2180 – 2186 76 Simon-Mateo, C. et al. (1993) Polyprotein processing in African swine fever virus: a novel gene expression strategy for a DNA virus. EMBO J. 12, 2977 – 2987 77 Shan, L. et al. (2000) The pseudomonas AvrPto protein is differentially
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recognized by tomato and tobacco and is localized to the plant plasma membrane. Plant Cell 12, 2323 – 2338 78 van Dijk, K. et al. (1999) The Avr (effector) proteins HrmA (HopPsyA) and AvrPto are secreted in culture from Pseudomonas syringae pathovars via the Hrp (type III) protein secretion system in a temperature- and pH-sensitive manner. J. Bacteriol. 181, 4790 – 4797 79 Menard, R. et al. (1994) The secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD. EMBO J. 13, 5293– 5302
Microbiology websites The Microbiology information portal http://www.microbes.info/ A useful website providing a variety of microbiology links, including links to feature articles and microbiology-related news stories. All the virology on the WWW http://www.tulane.edu/~dmsander/garryfavweb.html A comprehensive website on virology, including great pictures, virology jobs, a bookshop and links to other useful sites. The Picornavirus homepage http://www.iah.bbsrc.ac.uk/virus/Picornaviridae/ A website dedicated to the family of the Picornaviridae. Includes important up-to-date news features and articles, ongoing research at the Institute for Animal Health, comprehensive virus classification, links to other useful websites and details of future EUROPIC conferences. The WWW Virtual Library: Mycology http://biodiversity.bio.uno.edu/~fungi/ A useful website that provides extensive links to other related sites. The World Wide Web Virtual Library: Parasitology http://www.diplectanum.dsl.pipex.com/purls/ A website that provides extensive links to other related sites. Parasitology links http://www.galenica.cl/club/rec_parasitologia.html The world of parasites http://martin.parasitology.mcgill.ca/JIMSPAGE/WORLDOF.HTM Find out what parasites live with you in your country! The aspergillus website http://www.aspergillus.man.ac.uk/ This site includes laboratory protocols, treatment information, DNA sequence data, a comprehensive bibliographic database, an image library and discussion groups. The E. coli index http://web.bham.ac.uk/bcm4ght6/res.html A comprehensive guide to information relating to the model organism Escherichia coli. Bacterial infections and mycoses http://www.mic.ki.se/Diseases/c1.html Lots of useful bacterial links. www.sciencedirect.com
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Myristoylation of viral and bacterial proteins Sebastian Maurer-Stroh and Frank Eisenhaber IMP – Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria
Myristoylation, the N-terminal attachment of a myristoyl lipid anchor to a glycine residue, can reversibly direct protein –membrane and protein –protein interactions. Apart from two entomopoxviruses, viruses and bacteria usually lack the enzyme N-myristoyltransferase (NMT) that is required for this modification, and their proteins are consequently processed by NMTs of their eukaryotic hosts. This review gives an overview of the multiplicity of viral and the few known bacterial proteins that can undergo glycine myristoylation. In particular, we discuss the role of the myristoyl anchor in viral entry into host cells, in retroviral proteins and in the envelopment of large DNA viruses. We review evidence for myristoylation in arena- and flaviviruses as well as bacterial proteins after their secretion into eukaryotic host cells. Recent evidence shows that myristoylation of noneukaryotic proteins is far more than just a curiosity. From the time that the myristoyl lipid modification of viral proteins was discovered in the early eighties, considerable new insight has been accumulated that has increased the number of known myristoylated proteins in viruses (Table 1). This progress is now aided by the availability of reliable prediction methods for the occurrence of this modification [1,2] and a comprehensive collection of the resulting data at sites such as MYRbase (http://mendel. imp.univie.ac.at/myristate/myrbase/)p. Recently, it was discovered that some bacterial proteins can also undergo lipid modification after injection into the host when using a type III secretion system (TTSS) [3]. Typically, the attachment of a 14-carbon saturated fatty acid (myristate) to N-terminal glycine residues of substrate proteins is executed by N-myristoyltransferases (NMTs) [4] of the eukaryotic host, but there are indications for NMTs in two viral species [5]. Several lower eukaryotic parasites also have their own NMTs for protein myristoylation [5]. Owing to mechanistic reasons, lipid modification by NMT strictly occurs solely on N-terminal glycines, which can become N-terminal after proteolytic cleavage [6]. In eukaryotic cells, the largest families of proteins with a conserved myristoylation motif (e.g. Ga subunits, ADP-ribosylation factors, serine/threonine and tyrosine kinases, and EF-hand calcium-binding proteins) are Corresponding author: Sebastian Maurer-Stroh ([email protected]). * See also the recent publication by Maurer-Stroh, S. et al. (2004) MYRbase: analysis of genome-wide glycine myristoylation enlarges the functional spectrum of eukaryotic myristoylated proteins. Genome Biology 5, R21
primarily involved in signaling processes. However, recent studies [7] have identified a series of smaller families that increase the functional spectrum of myristoylated proteins. The limited hydrophobicity of the myristoyl anchor permits reversible membrane interactions and requires additional membrane attachment factors (MAFs) for enhancing membrane targeting. Typical MAFs include the subsequent palmitoylation of nearby cysteine residues, clusters of positive charges, phospholipid-binding domains (e.g. for PIP2 interactions), transmembrane regions or direct protein –protein interactions. Both targeting specificity and membrane affinity are achieved through the concerted effects of a combination of these MAFs. For example, dual acylation (myristoylation and palmitoylation) often directs proteins to lipid rafts [8]. In viruses in particular, the role of the myristoyl moiety includes not only targeting to diverse membranes and membrane subcompartments, but it can also be involved in direct interactions with other proteins [9] or conformational switches that affect tertiary and quaternary structure [10]. This review aims to cover the existing knowledge surrounding myristoylated viral and bacterial proteins and summarizes the involvement of the lipid anchor in various steps of the life cycle of viruses, including several of enormous medical importance. Involvement of myristoylated proteins in viral entry into host cells Besides roles in assembly, structure, budding and intracellular host interactions, myristoylated viral proteins are increasingly implicated in viral entry. Typically, hydrophobic patches of proteins in both enveloped and nonenveloped viruses are exposed as a result of conformational changes that can be induced by changes in pH (e.g. acidification following internalization into endosomes), physiological temperature, cleavage and receptor-binding. Membrane fusion [11] and pore formation [12] are essential in the invasion process of enveloped and non-enveloped viruses, respectively. Although myristoyl lipid anchors usually enter membranes from the cytoplasmic or viral matrix side, a completely different topology can be found for the large surface antigen of human and duck hepatitis B viruses (HBV). C-terminal transmembrane regions anchor the protein to the envelope membrane and the myristoylated N-terminus appears on the outside [13]. Furthermore, the lipid modification is not required for viral assembly or protein localization, but for infectivity [14]. HBV receptor
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Table 1. Viral proteins with experimental evidence for glycine myristoylation Protein(s)
Total number Viruses in MYRbase
Refs
Negative factor (nef) Gag Total P17 P15 (matrix protein)
2498 1583 1444 85
[64]
P10 (matrix protein)
29
P19 (component of the inner protein layer of the viral nucleocapsid) VP4 coat protein
25 566
Large surface antigen L1R (VV) envelope protein
415 31
v-src A16L (VV) late protein VP2 coat protein G9R/F1 (VV) Tegument protein UL11
26 20 19 14 13
UL99, U71, BBLF1
13
Major outer capsid protein m1 E7R (VV) P220 Membrane fusion protein p15
9 5 1 1
Lentiviruses (HIV, SIV) Retroviruses, Poxviruses Lentiviruses (HIV, SIV, FIV) Gammaretroviruses (e.g. murine and feline leukemia viruses, rat and avian sarcoma virus, avian spleen necrosis virus, baboon and porcine endogenous retroviruses, reticuloendotheliosis virus), human endogenous retrovirus S71, Poxviridae (Fowl pox) Betaretroviruses (mouse mammary tumor virus, Mason-Pfizer monkey virus, simian retrovirus 2, ovine pulmonary adenocarcinoma virus), wooly monkey sarcoma virus, human endogenous retrovirus K, hamster intracisternal Aparticle and python endogenous retrovirus Deltaretroviruses (human/simian T-cell lymphotropic virus, bovine leukemia virus) Picornaviridae [rhino-, cardio-, aphtho-, entero- (echo-, coxsackie-, polio-) viruses] Hepadnaviridae (hepatitis B virus) Poxviridae [Chordopoxvirinae (e.g. variola, vaccinia) and Enteropoxvirinae ], Asfiviridae, Iridoviridae (lymphocysti-, rana-, iridovirus), Ascoviridae (Ascovirus) Rous sarcoma virus Poxviridae [Chordopoxvirinae (e.g. variola, vaccinia) and Enteropoxvirinae ] Polyomaviruses Poxviridae [Chordopoxvirinae (e.g. variola, vaccinia)] Alphaherpesvirinae (simplex-, varicello-, Marek’s disease-like and infectious laryngotracheitis-like viruses) Betaherpesvirinae (roseolo-, cytomegalo-, muromegalovirus), Gammaherpesvirinae (e.g. lymphocryptovirus), Alphaherpesvirinae Ortho- and aquareoviruses Orthopoxviruses Asfivirus Orthoreovirus
recognition occurs over an N-terminal QLDPAF sequence in the vicinity of the myristoylation motif [13]. It remains to be established whether the lipid moiety is involved in direct receptor interaction [15], if it induces conformational constraints on the receptor-binding region, inserts directly into host membranes or plays a role in later fusion events. Among the non-enveloped viruses, involvement of the myristoyl anchor in viral entry has recently been established for the structural protein m1 of reoviruses [10]. Autocatalytic cleavage of this protein into m1N and m1C, and proteolytic removal of s3 (which shields m1 at the virion surface), allow exposure of the myristoyl moiety in m1N and insertion of the anchor into the host membrane. Interestingly, a non-structural reoviral protein (p15 from the S4 genome segment of Baboon reovirus) has been shown to be myristoylated in the host cell where it induces cell – cell fusion as an integral membrane protein [16]. Similar to the myristoyl switch of m1, the structural protein VP4 of picornaviruses undergoes conformational changes following cleavage, resulting in exposure and host cell membrane insertion of the lipid anchor [17]. A model for involvement in viral entry has also been proposed for the myristoylated VP2 protein of polyomaviruses found on the virion surface [18]. The entry of some plant viruses can take plant-specific shortcuts and, as we suggest, involve myristoylated proteins. For example, C4 proteins of several geminiviruses are predicted to be modified with this lipid anchor (Table 2) [19]. These proteins localize to the plasmodesmata (channels interconnecting plant cells) and are implicated in cell to cell movement of the respective viruses [20]. www.sciencedirect.com
[65] [66]
[67]
[68] [69] [70] [71]
[72] [46] [73] [46] [74] [41] [75] [46] [76] [16]
Prominent myristoylation in retroviruses The Nef protein found in the lentivirus subgroup of retroviruses, which includes human immunodeficiency virus type 1 (HIV-1), HIV-2 and simian immunodeficiency virus (SIV) among its members, contains a series of signals that act together in adjusting host cells to optimal viral replication conditions, one of these signals is the myristoylation motif. Typically, the lipid anchor directs the protein to membranes and co-localizes it with putative interaction partners that are recognized over additional binding determinants (e.g. the di-leucine motif and proline-rich SH3-binding motif). These interactions involve the endocytic machinery [compromising the host’s immune response by down- or upregulation of specific membrane protein abundances (e.g. down: CD4, CD28 and MHC class I molecules; up: AP-1, AP-3, TNF, LIGHT and DC-SIGN)] on the one hand and interference with signaling pathways on the other [affecting T-cell activation and apoptosis (e.g. interaction with Hck, Lck, Fyn, Lyn, PAK, PKC theta, Vav, MAPK, p53, Raf 1, PI3K and TcR chain zeta)]. Details on the multiplicity of the molecular functions of Nef can be found, for example, in Ref. [21]. The structural precursor polyprotein Gag is conserved among retroviruses and is essential for viral assembly [22]. Its N-terminal matrix (MA) domain is myristoylated and the lipid anchor, in addition to a cluster of positive charges, facilitates targeting to lipid rafts in the plasma membrane where Gag multimerization, viral assembly and budding occur [23]. Proteolytic processing by a viral protease results in a conformational switch involving the myristoyl anchor and
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Table 2. Viral proteins predicted for glycine myristoylation Protein(s)
Total number in MYRbase
Representative Gis in MYRbase (number in cluster)
Viruses
C4
105
28380577 (75), 20177150 (21), 22901050 (8), 9187889 (1) 23452258 (22) new world, 75456 (10) old world, 23452268 (1) new world
Geminiviridae (Begomoviruses)
Glycoprotein GPC
33
RING finger protein Z
5
23343513 (5)
Immediate-early R1.5 circ protein Non-structural protein NSP1.orf1 Early protein EP0 (transcription activator) Major envelope antigen p43K Orf 15L of multigene family 100 K5L (VV) Unknown non-globular protein VP4-related FPV229, FPV221 vaccinia A47L homologue E66 homologue V0 K10
5
319990 (3), 420432 (1), 12084887 (1) MDL
4
1360868 (3), 4106794 (1)
Arenaviruses [old world (e.g. lymphocytic choriomeningitis, lassa, mopeia,…) and new world (e.g. junin, pichinde, tacaribe, latino, flexal, allpahuayo, bear canyon,…) viruses] Old world arenaviruses (e.g. lymphocytic choriomeningitis, lassa,…} Alphaherpesvirinae (varicello-, Marek’s disease-like viruses) Rotaviruses
3
93140 (3)
Alphaherpesvirinae (varicellovirus)
3 2 2 2 2 2
7444364 (3) 9628255 (2) 93481 (2) 9629594 (2) 22960707 (2) 9634899 (1), 9634891 (1)
Chordopoxvirinae (molluscipoxvirus) Asfiviruses Orthopoxviruses Gammaherpesvirinae Reoviridae (coltiviruses) Chordopoxvirinae (avipoxvirus)
2 2 2
9631098 (2) 76992 (2) 18846026 (2)
Baculoviridae (nucleopolyhedrovirus) Geminiviridae (mastrevirus) Gammaherpesvirinae (rhadinovirus)
leads to membrane detachment of the MA domain [24]. The role for the lipid anchor in the MA domain-mediated transport of viral components to and from the nucleus is unclear [25]. There also exists an MA domain variant in Rous sarcoma virus gag protein where a cluster of positive charges without the myristoyl anchor is sufficient for membrane targeting and the role in viral assembly. Interestingly, there are several examples of retroviral oncogenes containing Gag MA domains that are myristoylated [26]. The transcription factor v-Fos (viral Fos) [27], the GTPase v-Ras [28] and the kinases v-Abl [29], v-Akt [30], v-Fgr [31] and v-Mos [32] belong to this group. It remains to be established whether the myristoyl anchor, when combined with a cluster of positive charges alone or with the complete MA domain, contributes to the oncogenic potential of each example. In the case of myristoylated v-Src, its counterpart c-Src (host cellular Src) already includes a myristoylation motif and a positive charge cluster, which is retained in the viral oncogene [33] but without a MA domain. In v-Abl and c-Abl, a myristoyl anchor is also conserved, and in v-Abl it is part of an N-terminal MA domain. In c-Abl, the lipid anchor functions as a reversible membrane switch [34]. In several of these cases the myristoylated MA domain might result in the strengthening of membrane attachment that is usually weak or reversible. Common myristoylation-dependent mechanisms for envelopment in large DNA viruses Among enveloped viruses, several large DNA viruses are wrapped by a second lipid layer that is derived from ER-trans-Golgi membranes [35]. In the wellstudied Vaccinia poxvirus, a myristoylated transmembrane protein L1R is located within the first envelope, and in addition to other functions is involved in the www.sciencedirect.com
acquisition of the second envelope [36]. Whether the lipid anchor is required for trafficking and specific targeting or for direct membrane lipid recruiting remains to be established. Interestingly, database searches have identified a superfamily of pox-viral sequences that are related to L1R but also have homologues in non-poxviruses. In addition to similarities at the sequence level, all proteins have a conserved architecture, including an N-terminal myristoylation motif (except for the poxviral F9L subfamily), a globular domain and two transmembrane regions with C-terminal extensions; some of the members also have an additional transmembrane region (Figure 1). In poxviruses, close relatives of L1R share six conserved cysteine residues that form intramolecular disulfide bridges in a recently established pathway [37]. In the alignment of the superfamily, only two of the cysteines remain conserved (Figure 1). This suggests spatial vicinity of the respective residues in the unresolved structure of the globular L1R domain. The superfamily includes proteins from pox-, asco-, asfi- and iridoviruses, which are all large DNA viruses with similar membrane arrangements [35], possibly pointing to a conserved envelopment mechanism in agreement with proposed evolutionary relationships [38]. The myristoylated proteins UL11 and UL99 in herpesviruses, another group of large DNA viruses with secondary envelopment from Golgi cisternae [39], do not share sequence similarities with the L1R family. However, they are also targeted to the compartments where envelopment takes place [40,41]. Other herpesvirus proteins with completely different functions, which are predicted to be myristoylated, include the immediate-early R1.5 circ protein [42] and the early transcription activator EP0 [43] (Table 2).
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oPox
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ePox
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Asfi
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Asco2
241
Asco1
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lIrido
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rIrido
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iIrido1
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iIrido2
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uIrido
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Figure 1. Schematic conserved architecture of a superfamily of myristoylated proteins related to Vaccinia L1R. Green, myristoylation motif; dark blue, transmembrane regions predicted with DAS-TMfilter [63] using conservative thresholds; light blue, transmembrane regions in agreement with hydrophobicity patterns in the alignment; yellow, two conserved cysteines forming a disulfide bridge; numbers in the end give the total protein length. Accession numbers and virus details: oPox, NP_042117.1 Variola virus; ePox, NP_064999.1 Amsacta moorei entomopoxvirus; Asfi, NP_042824.1 African swine fever virus; Asco2, CAC84467.1 Spodoptera frugiperda ascovirus 1; Asco1, CAC19148.1 Ascovirus DpAV4; lIrido, NP_078665.1 Lymphocystis disease virus 1; rIrido, AAK54492.1 Regina ranavirus; iIrido1, T03070 Chilo iridescent virus; iIrido2, NP_149921.1 Invertebrate iridescent virus 6; uIrido, NP_612229.1 infectious spleen and kidney necrosis virus.
By contrast, the lipid modification status of Vaccinia poxvirus proteins (not only myristoylation, but also palmitoylation, which is technically more difficult to detect) is experimentally well established [44], with the exception of A14L. The reported myristoylation of Vaccinia A14L protein at glycine residues that are not N-terminal [45] is mechanistically impossible and might represent a different acylation (such as palmitoylation or the rare lysine myristoylation) at other sites instead [44]. However, this would leave a myristoylated protein represented by a 14 – 15 kDa band in gel shift experiments of earlier work unassigned [46]. The only remaining Vaccinia protein in concordance with this mass that contains a myristoylation motif would be encoded by the open reading frame K5L. Other proteins exist in related but distinct poxviruses
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whose myristoylation status still requires experimental verification (Table 2). For example, p43K of the human tumorigenic molluscipoxvirus [47] is a clear homologue of Vaccinia major envelope antigen p37K (F13L). Although p37K lacks a myristoylation motif, it has already been shown to be lipid modified with palmitoyl anchors instead, which are required for the proper localization to the transGolgi network membranes [48]. Owing to similar physical properties of the lipid anchors (16- instead of 14-carbon saturated alkyl chains), palmitoylation is expected to result in similar targeting as myristoylation. Furthermore, whether several entomopoxviral proteins with an N-terminal glycine residue and a degenerated myristoylation motif are modified by the host or a putative viral NMT remains elusive [5]. Myristoylation in arena- and flaviviruses Despite the lack of experimental reports about myristoyl lipid modifications in proteins of arenaviruses, inhibiting the myristoylating enzyme NMT in infected cells has proven effective in inhibiting replication of the Junin virus [49], suggesting that myristoylated arenaviral proteins play a central role in replication. A possible candidate that has a conserved motif for glycine myristoylation is a glycoprotein from both old world and new world arenaviruses (Table 2). As host NMT inhibition did not abolish glycoprotein surface expression [49], it could be concluded that the lipid anchor is not important in its processing and localization. However, in analogy to other examples of myristoylated viral surface proteins, such as the hepatitis B virus large surface antigen, it could be involved in host cell recognition and viral entry. Interestingly, the RING finger protein Z of old world arenaviruses also carries a myristoylation motif (Table 2). However, truncation experiments have shown that its N-terminus is not required for its function in inhibiting viral RNA synthesis [50]. The medically important flaviviruses also lack examples of experimentally verified myristoylated proteins despite the existence of possible candidates. For example, mature NS5 is produced from a polyprotein in the host cytosol [51] and contains a valid N-myristoylation motif that is visible after cleavage (Table 3). The glycine that becomes N-terminal after
Table 3. Flaviviral proteins predicted for glycine myristoylation after proteolytic cleavagea Protein ID (flavivirus)
Cleavage motifb
N-terminus after cleavage (position of G in sequence)
59339 (Yellow fever virus)* 11528014 (West Nile virus)* 22474436 (Montana myotis leukoencephalitis virus)* 7939634 (Apoi virus)* 20177456 (Modoc virus)* 464427 (Dengue virus type 1) 266813 (Dengue virus type 2) 130437 (Dengue virus type 3) 130438 (Dengue virus type 4) 130520 (Tick-borne encephalitis virus) 730353 (Tick-borne powassan virus)
TGRR GLKR PGRR ENRR SNRR GGRR NARR TGKR TPRR GSRR GARR
(2507) GSANGK-TLGEVWKRELNLLDKRQFELYKR (2526) GGAKGR-TLGEVWKERLNHMTKEEFTRYRK (2478) GLSLSHLTLGEDWKLKLNKMTKSDFLEYRT (2475) GVSSSYITYGEQWKRELNKLNAQAFFLYKS (2477) GICSSAPTLGEIWKRKLNQLDAKEFMAYRR (2493) GTGAKGKHWERNGKDRLNQLSKSEFNTYKR (2492) GTGNIGETLGEKWKSRLNALGKSEFQIYKK (2491) GTGSQGETLGEKWKKKLNQLSRKEFDLYKK (2487) GTGTTGETLGEKWKRQLNSLDRKEFEEYKR (2511) GGSE-GDTLGDLWKRKLNGCTKEEFFAYRR (2513) GGAE-GSTLGDIWKQRLNSCTKEEFFAYRR
a
Available prediction methods [1] only predict potential myristoylation for NS5 from some of the many different flaviviruses (marked with asterisks). However, the remaining sequence fragments fail the prediction limit because of the dominant negative profile term that can be biased towards the learning set, but several of them appear to be in principal agreement with the physical properties required for productive N-myristoyltransferase (NMT) interaction. b Color code: red, myristoylatable glycines; yellow, palmitoylatable cysteines (additional lipid anchor can strengthen membrane attachment); blue, positive charges (can strengthen membrane attachment). www.sciencedirect.com
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Table 4. Myristoylated type III secreted bacterial proteins with different levels of experimental verification status Protein
Organism
Position
Evidence for type III secretion and myristoylation AvRpm1 Pseudomonas 2 syringae
Sequence motifa
Myristoylation
Type III secretion
GenInfo numbers
MGCVSSTSRSTGYYSGYENHE
[3H]-myrb, G2Ac [3]
[3]
[3H]-myr, G2A [3]
[3]
420847, 420848, 2664216 280002
G2A [3]
[3]
249817
G2A [77]
[78]
28871145, 479718
Predicted [56]
[56]
19071483
Predicted [58]
[57]
28868774
Predicted
[56]
19071488
Predicted [58]
[58]
28867732
Predicted (twilight) [56,58]
[56]
29171493
Predicted (twilight)
[79]
22036282, 22036280
Predicted
21231082
Predicted
By similarity to HopPmaB By similarity to HopPmaB By similarity to avrB By similarity to avrB/avrC By similarity to HopPmaD Predicted
14905926
Predicted
Predicted
21243956
Predicted (twilight)
Predicted
25139439
Predicted (twilight)
Predicted
17548434
Predicted (twilight)
Predicted
17548525
Predicted
Predictedd
20560111
Predicted (twilight)
Predicted
d
28870724
Predicted (twilight)
Predictedd
23472108
Predicted (twilight)
Predictedd
21243721
AvrB
Pseudomonas 2 MGCVSSKSTTVLSPQTSFNEA syringae AvrPphb Pseudomonas 63 GCASSSGVSLEDDSHTQVS syringae AvrPto Pseudomonas 2 MGNICVGGSRMAHQVNSPDRV syringae Evidence for type III secretion and predicted myristoylation HopPmaB Pseudomonas 2 MGLCVSKGSTASSPQHYAVRY syringae HopPtoJ Pseudomonas 2 MGLCISKHSGSSYSYSDSDRW syringae HopPmaD Pseudomonas 2 MGNICIGGPRMSQQVYSPERA syringae HopPtoF Pseudomonas 2 MGNICGTSGSRHVYSPSHTQR syringae HopPtoS1/ Pseudomonas 2 MGNICGTSGSNHVYSPPISPQ HopPtoO syringae IpaB Shigella sonnei 2 MGNVSAATTGLSLAKILASTE Predicted type III secretion and predicted myristoylation HopPmaB Xanthomonas 2 MGLCVSKPSVAGSPDHYATHA homolog campestris HopPmaB Xanthomonas 2 MGLCVSRPATSGSSVAASPEQ homologue axonopodis AvrC Pseudomonas 2 MGNVCFRPSRSHVSQEFSQSE syringae AvrB homologue Pseudomonas 2 MGCVSSKASVISSDSFRASYT syringae AvrPpiG1 Pseudomonas 2 MGICVSKPSVRHDYNEDYGRN syringae Outer protein J Xanthomonas 2 MGLCVSKPSVAGSPEHYAAHV campestris Outer protein J Xanthomonas 2 MGLCTSKPSVVGSPVAGSPEH homologue axonopodis PthG Pantoea 2 MGCFNVTGASGRANNYVVEHH agglomerans N-terminus Ralstonia 2 MGCFNVTGTSGTASNYVAREH similar to PthG solanacearum AvrPphD-related Ralstonia 2 MGNLQIKASSAPYALLSDVSP solanacearum Wzz-like Pseudomonas 2 MGCIVTNGIGSRKQSKDYEID aeruginosa Gluconokinase Pseudomonas 2 MGVSSCGKSAVGAEIARNSGG syringae RNA helicase Pseudomonas 2 MGLCGCTRGRPMTQEIGGFAA syringae Metal-dependent Xanthomonas 2 MGQLGAAARPPARQTSSQPGP hydrolase axonopodis
Predicted Predicted [3] Predicted Predicted
21241060 280003, 501130 8037787 11322463
a Color code: red, myristoylatable glycines; yellow, palmitoylatable cysteines (additional lipid anchor can strengthen membrane attachment); blue, positive charges (can strengthen membrane attachment). b Incorporates radioactive myristate dependent on N-terminal glycine. c G2A mutation abolishes membrane localization. d Fulfills basic N-terminal amino acid requirements but genetic context for capability of type III secretion is not clear.
cleavage could be subject to myristoylation by host NMTs, as occurs in other proteolytically processed viral proteins. The cell biology surrounding flaviviral NS5 appears compatible with myristoylation as a signal for partial or dynamic membrane-targeting of these proteins. NS5 is a multidomain protein involved in the viral replication cycle, which is suggested to take place in specialized membrane compartments [52]. It contains an N-terminal (nucleoside20 -O)-methyltransferase domain that is involved in RNA www.sciencedirect.com
capping and also a C-terminal RNA-dependent RNA polymerase domain. Interestingly, a cluster of positive charges (mainly comprised in an amphipathic helix) is in spatial vicinity to the flexible N-terminus in the resolved structure of the Dengue virus NS5 N-terminal domain [53]. This cluster might serve as an additional membrane attachment factor. Although a C-terminally His-tagged NS5 shows full polymerase activity, this activity is reduced when the His-tag is attached to the N-terminus [54], which
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would also abolish potential myristoylation among many other effects.
several more examples for glycine myristoylation of pathogenic bacterial proteins in the future.
Myristoylation of bacterial proteins after secretion into eukaryotic hosts Originally, glycine myristoylation was believed to be a unique posttranslational modification of proteins from eukaryotes and their viruses. However, Nimchuk et al. [3] corrected this view by demonstrating that proteins from pathogenic bacteria can also be myristoylated after secretion into eukaryotic host cells. Concomitantly, considerable improvements were achieved in understanding the TTSS (reviewed in Ref. [55]), which also resulted in the formulation of sequence requirements for predicting TTSS translocations [56 –58]. Table 4 summarizes the recently accumulated knowledge on myristoylated bacterial proteins with differing levels of experimental or computationally derived evidence for injection into the host and modification by the host NMT. Notably, most of the examples are from plant pathogens. Many of these proteins have N-terminal cysteine residues that could be subject to subsequent palmitoylation, which further strengthens attachment to membranes. It is at this cellular localization that the bacterial virulence or effector proteins are suspected to act on their targets, however, their efficiency can be reduced if the host carries specific resistance genes [59]. In this context, it is important that recent data has suggested that several plant disease resistance proteins are myristoylated [7], which could protect the effector targets by interfering with pathogenic proteins through myristoylation-dependent co-localization in specific membrane compartments. It is possible that some of these myristoylated plant disease resistance proteins could also be involved in host-specific innate immune responses against geminiviruses by inhibiting viral cell to cell translocation through co-localization with C4 proteins (see also viral entry section).
Acknowledgements
Concluding remarks: medical issues The number of myristoylated proteins identified in viruses is constantly increasing. Nevertheless, substantial additional work will be required to shed light on the multiple functions that could be executed by the respective proteins as well as their involvement in the viral life cycle and elucidation of the role of the lipid anchor. Inhibition of NMT or replacing myristic acid by analogs in infected cells is known to affect numerous viruses of medical importance, for example, HIV [60], hepatitis B virus [61], herpesviruses [62] and arenaviruses [49]. This approach might be generally suitable for influencing infections by viruses that take advantage of the eukaryotic myristoylation machinery. However as a side effect, inhibition of host NMTs affects myristoylation of vital cellular proteins (mainly involved in signal transduction pathways) that are processed by the same enzyme. Characterization of bacterial proteins that are secreted into eukaryotic host cells, including those that use translocation machineries that differ from the TTSS, is still in its infancy. Hence, it would not be surprising to find www.sciencedirect.com
We are grateful for continuous support from Boehringer Ingelheim. This project was partly funded by the Fonds zur Fo¨rderung der wissenschaf¨ sterreichs (FWF grant P15037), the Austrian tlichen Forschung O ¨ sterreichische Nationalbank) and the Austrian National Bank (OeNB - O Gen-AU bioinformatics integration network (BIN) sponsored by BM-BWK.
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Microbiology websites The Microbiology information portal http://www.microbes.info/ A useful website providing a variety of microbiology links, including links to feature articles and microbiology-related news stories. All the virology on the WWW http://www.tulane.edu/~dmsander/garryfavweb.html A comprehensive website on virology, including great pictures, virology jobs, a bookshop and links to other useful sites. The Picornavirus homepage http://www.iah.bbsrc.ac.uk/virus/Picornaviridae/ A website dedicated to the family of the Picornaviridae. Includes important up-to-date news features and articles, ongoing research at the Institute for Animal Health, comprehensive virus classification, links to other useful websites and details of future EUROPIC conferences. The WWW Virtual Library: Mycology http://biodiversity.bio.uno.edu/~fungi/ A useful website that provides extensive links to other related sites. The World Wide Web Virtual Library: Parasitology http://www.diplectanum.dsl.pipex.com/purls/ A website that provides extensive links to other related sites. Parasitology links http://www.galenica.cl/club/rec_parasitologia.html The world of parasites http://martin.parasitology.mcgill.ca/JIMSPAGE/WORLDOF.HTM Find out what parasites live with you in your country! The aspergillus website http://www.aspergillus.man.ac.uk/ This site includes laboratory protocols, treatment information, DNA sequence data, a comprehensive bibliographic database, an image library and discussion groups. The E. coli index http://web.bham.ac.uk/bcm4ght6/res.html A comprehensive guide to information relating to the model organism Escherichia coli. Bacterial infections and mycoses http://www.mic.ki.se/Diseases/c1.html Lots of useful bacterial links. www.sciencedirect.com