Cell-associated Enveloped Virus Research Articles

Characterisation of an Anti-Vaccinia Virus F13 Single Chain Fragment Variable from a Human Anti-Vaccinia Virus-Specific Recombinant Immunoglobulin Library.

Vaccinia virus (VACV) belongs to the genus Orthopoxvirus of the family Poxviridae. There are four different forms of infectious virus particles: intracellular mature virus (IMV), intracellular en-veloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). The F13 protein occupies the inner side of the CEV- and EEV-membranes and the outer side of the IEV-membranes. It plays an important role in wrapping progress and EEV production. We constructed a human single-chain fragment variable (scFv) library with a diversity of ≥4 × 108 independent colonies using peripheral blood from four vaccinated donors. One anti-F13 scFv was isolated and characterised after three rounds of panning. In Western blotting assays, the scFv 3E2 reacted with the recombinant F13VACV protein with a reduction of binding under denatured and reduced conditions. Two antigenic binding sites (139-GSIHTIKTLGVYSDY-153 and 169-AFNSAKNSWLNL-188) of scFv 3E2 were mapped using a cellulose membrane encompassing 372 15-mere peptides with 12 overlaps covering the whole F13 protein. No neutralisation capa-bilities were observed either in the presence or absence of complement. In conclusion, the con-struction of recombinant immunoglobulin libraries is a promising strategy to isolate specific scFvs to enable the study of the host-pathogen interaction.

Vaccinia virus dissemination requires p21-activated kinase 1.

The orthopoxvirus vaccinia virus (VACV) interacts with both actin and microtubule cytoskeletons in order to generate and spread progeny virions. Here, we present evidence demonstrating the involvement of PAK1 (p21-activated kinase 1) in the dissemination of VACV. Although PAK1 activation has previously been associated with optimal VACV entry via macropinocytosis, its absence does not affect the production of intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs). Our data demonstrate that low-multiplicity infection of PAK1(-/-) MEFs leads to a reduction in plaque size followed by decreased production of both IMVs and EEVs, strongly suggesting that virus spread was impaired in the absence of PAK1. Confocal and scanning electron microscopy showed a substantial reduction in the amount of VACV-induced actin tails in PAK1(-/-) MEFs, but no significant alteration in the total amount of cell-associated enveloped virions (CEVs). Furthermore, the decreased VACV dissemination in PAK1(-/-) cells was correlated with the absence of phosphorylated ARPC1 (Thr21), a downstream target of PAK1 and a key regulatory subunit of the ARP2/3 complex, which is necessary for the formation of actin tails and viral spread. We conclude that PAK1, besides its role in virus entry, also plays a relevant role in VACV dissemination.

Development and comparison of a quantitative TaqMan-MGB real-time PCR assay to three other methods of quantifying vaccinia virions

Plaque assays are a widely used method to quantify stocks of viruses. Although this method is well established for titrating viral stocks, it is time consuming and can take several days to complete. In this study, the creation and validation of a quantitative real-time PCR (qPCR) assay for enumerating virions of vaccinia virus is reported. PCR primers and a minor groove-binding probe were designed to hybridize to the DNA polymerase gene (E9L) from a number of different orthopoxviruses. The number of viral genomes determined using qPCR was approximately similar to results obtained using OD260 measurements and a direct count of fluorescent virions by microscopy indicating that all three methods are comparable in their ability to quantify virions from a purified stock. In addition, this report describes methodologies to harvest and quantify, using the qPCR assay, three of the four types of vaccinia virions produced during morphogenesis: intracellular mature virions, cell-associated enveloped virions, and extracellular enveloped virions. Using these procedures a particle to plaque forming unit of 61:1, 14:1 and 6:1 was calculated for IMV, CEV and EEV, respectively. These results show that qPCR can be used as a fast and accurate assay to quantify stocks of vaccinia virus over several orders of magnitude from both purified and unpurified stocks and should be applicable to other members of the orthopoxvirus genera.

Variola and Monkeypox Viruses Utilize Conserved Mechanisms of Virion Motility and Release That Depend on Abl and Src Family Tyrosine Kinases

Vaccinia virus (VacV) enters mammalian cells, replicates extranuclearly, and produces virions that move to the cell surface along microtubules, fuse with the plasma membrane, and move from infected cells toward apposing cells on actin-filled membranous protrusions or actin tails. To form actin tails, cell-associated enveloped virions (CEV) require Abl and Src family tyrosine kinases. Furthermore, release of CEV from the cell requires Abl but not Src family tyrosine kinases and is blocked by imatinib mesylate (STI-571; Gleevec), an Abl family kinase inhibitor used to treat chronic myelogenous leukemia in humans. Here we demonstrate that the Poxviridae family members monkeypox virus (MPX) and variola virus (VarV) use conserved mechanisms for actin motility and extracellular enveloped virion (EEV) release. Furthermore, we show that imatinib mesylate is effective in a mouse model of infection with VacV, whether delivered prophylactically or postinfection, and restricts spread of virions from the site of inoculation. While inhibitors of both Src and Abl family kinases, such as dasatinib (BMS-354825; Sprycel), are effective in limiting dissemination of VacV, VarV, and MPX in vitro, members of this class of drugs appear to have immunosuppressive effects in vivo that preclude their use as anti-infectives. Together, these data suggest a possible utility for imatinib mesylate in treating smallpox or MPX infections or complications associated with vaccination.

A trans -Golgi Network Resident Protein, golgin-97, Accumulates in Viral Factories and Incorporates into Virions during Poxvirus Infection

Poxviruses are the only DNA viruses known to replicate and assemble in the cytoplasm of infected cells. Poxvirus morphogenesis is a complicated process in which four distinct infectious forms of the virus are produced: intracellular mature virus, intracellular enveloped virus, cell-associated enveloped virus, and extracellular enveloped virus. The source of primary membrane wrapping the intracellular mature virus, the first infectious form, is still unknown. Although the membrane was suggested to originate from the endoplasmic reticulum-Golgi intermediate compartment, none of the marker proteins from this or any other cell compartments has been found in the intracellular mature virus. Thus, it was hypothesized that the membrane is either extensively modified by the virus or synthesized de novo. In the work described here, we demonstrate that a host cell protein residing in the trans-Golgi network membrane, golgin-97, is transported to the sites of virus replication and assembly and becomes incorporated into the virions during poxvirus infection. Inside the virion, golgin-97 is associated with the insoluble core protein fraction. Being able to adopt a long rod-like structure, the protein apparently extends through the virion envelope and protrudes from its surface. Here we discuss the potential role and functions of golgin-97 in poxvirus replication and propose two working models.

The Envelope G3L Protein Is Essential for Entry of Vaccinia Virus into Host Cells

The vaccinia virus G3L/WR079 gene encodes a conserved protein with a predicted transmembrane domain. Our proteomic analyses of vaccinia virus revealed that G3L protein is incorporated into intracellular mature virus; however, the function of G3L protein in the vaccinia virus life cycle has not been investigated. In this study, a recombinant vaccinia virus, viG3L, expressing G3L protein under IPTG (isopropyl-beta-d-thiogalactopyranoside) regulation was constructed. Under permissive conditions when G3L protein was expressed, the vaccinia virus life cycle proceeded normally, resulting in plaque formation in BSC40 cells. In contrast, under nonpermissive conditions when G3L protein expression was repressed, no plaques were formed, showing that G3L protein is essential for vaccinia virus growth in cell cultures. In infected cells when G3L protein was not expressed, the formation of intracellular mature virus (IMV) and cell-associated enveloped virus occurred normally, showing that G3L protein is not required for virion morphogenesis. IMV particles containing (G3L(+)) or lacking (G3L(-)) G3L protein were purified and were found to be indistinguishable on microscopic examination. Both G3L(+) and G3L(-) IMV bound to HeLa cells; however, G3L(-) IMV failed to enter the cells, showing that G3L protein is required for IMV penetration into cells. Finally, G3L protein was required for fusion of the infected cells under low-pH treatment. Thus, our results provide direct evidence that G3L is an essential component of the vaccinia virus fusion complex, in addition to the previously reported A28, H2, L5, A21, and A16 proteins.

Vaccinia virus intracellular enveloped virions move to the cell periphery on microtubules in the absence of the A36R protein

Vaccinia virus (VACV) intracellular enveloped virus (IEV) particles are transported to the cell periphery on microtubules where they fuse with the plasma membrane to form cell-associated enveloped virus (CEV). Two IEV-specific proteins, F12L and A36R, are implicated in mediating transport of IEV. Without F12L, virus morphogenesis halts after formation of IEV, and CEV is not formed, whereas without A36R, IEV was reported not to be transported, yet CEV was formed, To address the roles of A36R and F12L in IEV transport, viruses with deletions of either F12L (vdeltaF12L) or A36R (vdeltaA36R) were labelled with enhanced green fluorescent protein (EGFP) fused to the core protein A5L, and used to follow CEV production with time. Without F12L, CEV production was inhibited by >99 %, whereas without A36R, CEV were produced at approximately 60 % of wild-type levels at 24 h post-infection. Depolymerization of microtubules, but not actin, inhibited CEV formation in vdeltaA36R-infected cells. Moreover, vdeltaA36R IEV labelled with EGFP fused to the B5R protein co-localized with microtubules, showing that the A36R protein is not required for the interaction of IEV with microtubules. Time-lapse confocal microscopy confirmed that both wild-type and vdeltaA36R IEV moved in a stop-start manner at speeds consistent with microtubular movement, although the mean length of vdeltaA36R IEV movement was shorter. These data demonstrate that VACV IEV is transported to the cell surface using microtubules in the absence of A36R, and therefore IEV must attach to microtubule motors using at least one protein other than A36R.

Disabling poxvirus pathogenesis by inhibition of Abl-family tyrosine kinases

The Poxviridae family members vaccinia and variola virus enter mammalian cells, replicate outside the nucleus and produce virions that travel to the cell surface along microtubules, fuse with the plasma membrane and egress from infected cells toward apposing cells on actin-filled membranous protrusions. We show that cell-associated enveloped virions (CEV) use Abl- and Src-family tyrosine kinases for actin motility, and that these kinases act in a redundant fashion, perhaps permitting motility in a greater range of cell types. Additionally, release of CEV from the cell requires Abl- but not Src-family tyrosine kinases, and is blocked by STI-571 (Gleevec), an Abl-family kinase inhibitor used to treat chronic myelogenous leukemia in humans. Finally, we show that STI-571 reduces viral dissemination by five orders of magnitude and promotes survival in infected mice, suggesting possible use for this drug in treating smallpox or complications associated with vaccination. This therapeutic approach may prove generally efficacious in treating microbial infections that rely on host tyrosine kinases, and, because the drug targets host but not viral molecules, this strategy is much less likely to engender resistance compared to conventional antimicrobial therapies.

The exit of Vaccinia virus from infected cells

Vaccinia virus (VACV) is the prototypic member of the Poxviridae a group of large DNA viruses that replicate in the cell cytoplasm. The entry and exit of VACV are complicated by the existence of two distinct forms of virus, intracellular mature virus (IMV) and extracellular enveloped virus (EEV), that are surrounded by different numbers of lipid membranes and have different surface proteins. Here the mechanisms used by these different forms of VACV to leave the infected cell are reviewed. Whereas some enveloped viruses complete virus assembly by budding through the plasma membrane, infectious poxvirus particles (IMV) are produced within the cytoplasm. These particles are either further enveloped by intracellular membranes to form intracellular enveloped virus (IEV) that are transported to the cell surface on microtubules and exposed on the cell surface by exocytosis, or are released after cell lysis. If the enveloped virion remains attached to the cell surface it is called cell-associated enveloped virus (CEV) and is propelled into surrounding cells by growing actin tails beneath the plasma membrane. Alternatively, the surface virion may be released as EEV.

Vaccinia virus cores are transported on microtubules.

Infection with Vaccinia virus (VV) produces several distinct virions called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). In this report, we have investigated how incoming virus cores derived from IMV are transported within the cell. To do this, recombinant VVs (vA5L-EGFP-N and vA5L-EGFP-C) were generated in which the A5L virus core protein was fused with the enhanced green fluorescent protein (EGFP) at the N or C terminus. These viruses were viable, induced formation of actin tails and had a plaque size similar to wild-type. Immunoblotting showed the A5L-EGFP fusion protein was present in IMV particles and immunoelectron microscopy showed that the fusion protein was incorporated into VV cores. IMV made by vA5L-EGFP-N were used to follow the location and movement of cores after infection of PtK(2) cells. Confocal microscopy showed that virus cores were stained with anti-core antibody only after they had entered the cell and, once intracellular, were negative for the IMV surface protein D8L. These cores co-localized with microtubules and moved in a stop-start manner with an average speed of 51.8 (+/-3.9) microm min(-1), consistent with microtubular movement. Treatment of cells with nocodazole or colchicine inhibited core movement, but addition of cytochalasin D did not. These data show that VV cores derived from IMV use microtubules for intracellular transport after entry.

The formation and function of extracellular enveloped vaccinia virus

Vaccinia virus produces four different types of virion from each infected cell called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). These virions have different abundance, structure, location and roles in the virus life-cycle. Here, the formation and function of these virions are considered with emphasis on the EEV form and its precursors, IEV and CEV. IMV is the most abundant form of virus and is retained in cells until lysis; it is a robust, stable virion and is well suited to transmit infection between hosts. IEV is formed by wrapping of IMV with intracellular membranes, and is an intermediate between IMV and CEV/EEV that enables efficient virus dissemination to the cell surface on microtubules. CEV induces the formation of actin tails that drive CEV particles away from the cell and is important for cell-to-cell spread. Lastly, EEV mediates the long-range dissemination of virus in cell culture and, probably, in vivo. Seven virus-encoded proteins have been identified that are components of IEV, and five of them are present in CEV or EEV. The roles of these proteins in virus morphogenesis and dissemination, and as targets for neutralizing antibody are reviewed. The production of several different virus particles in the VV replication cycle represents a coordinated strategy to exploit cell biology to promote virus spread and to aid virus evasion of antibody and complement.

An investigation of incorporation of cellular antigens into vaccinia virus particles.

Vaccinia virus (VV) infection produces several types of virus particle called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). Some cellular antigens are associated with EEV and these vary with the cell type used to grow the virus. To investigate if specific cell antigens are associated with VV particles, and to address the origin of membranes used to envelope IMV and IEV/CEV/EEV, we have studied whether cell antigens and foreign antigens expressed by recombinant VVs are incorporated into VV particles. Membrane proteins that are incorporated into the endoplasmic reticulum (ER), intermediate compartment (IC), cis/medial-Golgi, trans-Golgi network (TGN) or plasma membrane were not detected in purified IMV particles. In contrast, proteins present in the TGN or membrane compartments further downstream in the exocytic pathway co-purify with EEV particles when analysed by immunoblotting. Immunoelectron microscopy found only low levels of these proteins in IEV, CEV/EEV. The incorporation of foreign antigens into VV particles was not affected by loss of individual IEV or EEV-specific proteins or by redirection of B5R to the ER. These data suggest that (i) host cell antigens are excluded from the lipid envelope surrounding the IMV particle and (ii) membranes of the ER, IC and cis/medial-Golgi are not used to wrap IMV particles to form IEV. Lastly, the VV haemagglutinin was absent from one-third of IEV and CEV/EEV particles, whereas other EEV antigens were present in all these virions.

Replacing the SCR domains of vaccinia virus protein B5R with EGFP causes a reduction in plaque size and actin tail formation but enveloped virions are still transported to the cell surface.

A vaccinia virus (VV) recombinant is described in which the outer envelope of extracellular enveloped virus (EEV), cell-associated enveloped virus (CEV) and intracellular enveloped virus (IEV) is labelled with the enhanced green fluorescent protein (EGFP) derived from Aequorea victoria. To construct this virus, EGFP was fused to the VV B5R protein from which the four short consensus repeats (SCRs) of the extracellular domain had been deleted. Cells infected with the recombinant virus expressed a B5R-EGFP fusion protein of 40 kDa that was present on IEV, CEV and EEV, but was absent from IMV. The recombinant virus produced 2- and 3-fold reduced levels of IMV and EEV, respectively. Analysis of infected cells by confocal microscopy showed that actin tail formation by the mutant virus was reduced by 86% compared to wild-type (WT). The virus formed a small plaque compared to WT, consistent with a role for actin tails in promoting cell-to-cell spread of virus. However, the enveloped virions were still transported to the cell surface, confirming that this process is independent of actin tail formation. Lastly, we compared the mutant virus with a recombinant VV in which the B5R SCR domains were deleted and show that, contrary to a previous report, the plaque size of the latter virus was reduced compared to WT. This observation reconciles an inconsistency in the field and confirms that viruses deficient in formation of actin tails form small plaques.

The vaccinia virus F12L protein is associated with intracellular enveloped virus particles and is required for their egress to the cell surface.

The vaccinia virus (VV) F12L gene encodes a 65 kDa protein that is expressed late during infection and is important for plaque formation, EEV production and virulence. Here we have used a recombinant virus (vF12LHA) in which the F12L protein is tagged at the C terminus with an epitope recognized by a monoclonal antibody to determine the location of F12L in infected cells and whether it associates with virions. Using confocal and electron microscopy we show that the F12L protein is located on intracellular enveloped virus (IEV) particles, but is absent from immature virions (IV), intracellular mature virus (IMV) and cell-associated enveloped virus (CEV). In addition, F12L shows co-localization with endosomal compartments and microtubules. F12L did not co-localize with virions attached to actin tails, providing further evidence that actin tails are associated with CEV but not IEV particles. In vDeltaF12L-infected cells, virus morphogenesis was arrested after the formation of IEV particles, so that the movement of these virions to the cell surface was inhibited and CEV particles were not found. Previously, virus mutants lacking IEV- or EEV-specific proteins were either unable to make IEV particles (vDeltaF13L and vDeltaB5R), or were unable to form actin tails after formation of CEV particles (vDeltaA36R, vDeltaA33R, vDeltaA34R). The F12L deletion mutant therefore defines a new stage in the morphogenic pathway and the F12L protein is implicated as necessary for microtubule-mediated egress of IEV particles to the cell surface.

Antibody-sensitive and antibody-resistant cell-to-cell spread by vaccinia virus: role of the A33R protein in antibody-resistant spread.

The roles of vaccinia virus (VV) intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV) and their associated proteins in virus spread were investigated. The plaques made by VV mutants lacking individual IEV- or EEV-specific proteins (vDeltaA33R, vDeltaA34R, vDeltaA36R, vDeltaA56R, vDeltaB5R, vDeltaF12L and vDeltaF13L) were compared in the presence of IMV- or EEV-neutralizing antibodies (Ab). Data presented show that for long-range spread, the comet-shaped plaques of VV were caused by the unidirectional spread of EEV probably by convection currents, and for cell-to-cell spread, VV uses a combination of Ab-resistant and Ab-sensitive pathways. Actin tails play a major role in the Ab-resistant pathway, but mutants such as vDeltaA34R and vDeltaA36R that do not make actin tails still spread from cell to cell in the presence of Ab. Most strikingly, the Ab-resistant pathway was abolished when the A33R gene was deleted. This effect was not due to alterations in the efficiency of neutralization of EEV made by this mutant, nor due to a deficiency in IMV wrapping to form IEV, which was indispensable for EEV formation by vDeltaA33R and vDeltaA34R. We suggest a role for A33R in promoting Ab-resistant cell-to-cell spread of virus. The roles of the different virus forms in the VV life-cycle are discussed.

Movements of vaccinia virus intracellular enveloped virions with GFP tagged to the F13L envelope protein.

Vaccinia virus produces several forms of infectious virions. Intracellular mature virions (IMV) assemble in areas close to the cell nucleus. Some IMV acquire an envelope from intracellular membranes derived from the trans-Golgi network, producing enveloped forms found in the cytosol (intracellular enveloped virus; IEV), on the cell surface (cell-associated enveloped virus) or free in the medium (extracellular enveloped virus; EEV). Blockage of IMV envelopment inhibits transport of virions to the cell surface, indicating that enveloped virus forms are required for virion movement from the Golgi area. To date, the induction of actin tails that propel IEV is the only well-characterized mechanism for enveloped virus transport. However, enveloped virus transport and release occur under conditions where actin tails are not formed. In order to study these events, recombinant vaccinia viruses were constructed with GFP fused to the most abundant protein in the EEV envelope, P37 (F13L). The P37-GFP fusion, like normal P37, accumulated in the Golgi area and was incorporated efficiently into enveloped virions. These recombinants allowed the monitoring of enveloped virus movements in vivo. In addition to a variety of relatively slow movements (<0.4 microm/s), faster, saltatory movements both towards and away from the Golgi area were observed. These movements were different from those dependent on actin tails and were inhibited by the microtubule-disrupting drug nocodazole, but not by the myosin inhibitor 2,3-butanedione monoxime. Video microscopy (5 frames per s) revealed that saltatory movements had speeds of up to, and occasionally more than, 3 microm/s. These results suggest that a second, microtubule-dependent mechanism exists for intracellular transport of enveloped vaccinia virions.

Vaccinia virus utilizes microtubules for movement to the cell surface

Vaccinia virus (VV) egress has been studied using confocal, video, and electron microscopy. Previously, intracellular-enveloped virus (IEV) particles were proposed to induce the polymerization of actin tails, which propel IEV particles to the cell surface. However, data presented support an alternative model in which microtubules transport virions to the cell surface and actin tails form beneath cell-associated enveloped virus (CEV) particles at the cell surface. Thus, VV is unique in using both microtubules and actin filaments for egress. The following data support this proposal. (a) Microscopy detected actin tails at the surface but not the center of cells. (b) VV mutants lacking the A33R, A34R, or A36R proteins are unable to induce actin tail formation but produce CEV and extracellular-enveloped virus. (c) CEV formation is inhibited by nocodazole but not cytochalasin D or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP1). (d) IEV particles tagged with the enhanced green fluorescent protein fused to the VV B5R protein moved inside cells at 60 μm/min. This movement was stop-start, was along defined pathways, and was inhibited reversibly by nocodazole. This velocity was 20-fold greater than VV movement on actin tails and consonant with the rate of movement of organelles along microtubules.

Vaccinia virus F12L protein is required for actin tail formation, normal plaque size, and virulence.

Vaccinia virus gene F12L is shown to encode a 65-kDa protein that is synthesized early and late during infection and that is not modified by glycosylation. Computational sequence comparison revealed that related proteins are encoded by all sequenced chordopoxviruses. A virus deletion mutant lacking the F12L gene (vDeltaF12L) and a revertant virus with the F12L gene reinserted into the deletion mutant (vF12L-rev) were constructed and analyzed. A version of the F12L gene with a C-terminal amino acid tag derived from the influenza virus hemagglutinin and that is recognized by a monoclonal antibody was also inserted into the F12L locus of vDeltaF12L. Loss of the F12L protein reduced the formation of IMV 2-fold, but there was a dramatic (99.5%) reduction in actin tail formation, and the levels of cell-associated enveloped virus and extracellular enveloped virus were reduced 8- to 11-fold and 7-fold, respectively. Consistent with the lack of actin tail formation, vDeltaF12L produced a very small plaque. The vDeltaF12L virus was severely attenuated in vivo, such that a dose of vDeltaF12L 10,000-fold greater than the dose of wild-type virus that induced severe disease was unable to induce disease in mice infected intranasally.

The Vaccinia Virus A36R Protein Is a Type Ib Membrane Protein Present on Intracellular but Not Extracellular Enveloped Virus Particles

Vaccinia virus gene A36R encodes a 45-kDa protein that is conserved in orthopoxviruses. A virus lacking the A36R protein formed a small plaque, was unable to induce the polymerization of actin tails, and was avirulent in vivo. Here we present a further characterization of the A36R protein by in vitro transcription and translation and analysis of infected cells by confocal microscopy and immunoelectron microscopy of cryosections using a monoclonal antibody raised against the C-terminal domain of the A36R protein. Translation of the A36R mRNA in vitro produced a protein of the same size whether or not the translation reaction was performed in the presence of canine pancreatic microsomes. However, the polypeptide synthesized in the presence of microsomes was associated integrally with the membrane and was sensitive to digestion by exogenous protease without permeabilization of the membrane with detergent, indicating that the majority of the protein is exposed on the outside of the vesicle. Consistent with this, immunofluorescent analysis of virus-infected cells demonstrated that the C-terminal domain of A36R was not exposed on the cell surface but was detected once the cell membrane was permeabilized. Immunoelectron microscopy of cryosections of infected cells showed that the protein was absent from IMV particles but present on intracellular enveloped virus (IEV) particles, predominantly on the cytosolic face of the IEV outer membrane. Where cell-associated enveloped virus (CEV) particles were attached to the cell surface, the A36R protein was detected only on the cytosolic surface of the plasma membrane where the virus particle remained attached to the cell and not elsewhere on the plasma membrane or on the CEV particle. A36R and actin copurified with EEV particles due to the association of fragments of cellular membranes with the EEV particles. Therefore, A36R represents the first example of a virus-encoded protein that is present on IEV but not CEV particles.

Roles of vaccinia virus EEV-specific proteins in intracellular actin tail formation and low pH-induced cell-cell fusion.

During vaccinia virus (VV) morphogenesis intracellular mature virus (IMV) is wrapped by two additional membranes to form intracellular enveloped virus (IEV). IEV particles can nucleate the formation of actin tails which aid movement of IEVs to the cell surface where the outer IEV membrane fuses with the plasma membrane forming cell-associated enveloped virus (CEV) which remains attached to the cell, or extracellular enveloped virus (EEV) which is shed from the cell. In this report, we have used a collection of VV mutants lacking individual EEV-specific proteins to compare the roles of these proteins in the formation of IEV and IEV-associated actin tails and fusion of infected cells after a low pH shock. Data presented here show that p45-50 (A36R) is not required for IEV formation or for acid-induced cell-cell fusion, but is required for formation of IEV-associated actin tails. In contrast, gp86 (A56R), the virus haemagglutinin, is not required for formation of either IEV or IEV-associated actin tails. Data presented also confirm that p37 (gene F13L), gp42 (B5R) and gp22-24 (A34R) are needed for formation of IEV-associated actin tails and for cell-cell fusion after low pH shock. The phenotypes of these mutants were not affected by the host cell type as similar results were obtained in a range of different cells. Lastly, comparisons of the phenotypes of VV strains Western Reserve, deltaA34R and deltaA36R demonstrate that actin tails are not required for low pH-induced cell-cell fusion.