Journal of General Virology (2002),
83, 2915-2931.
© 2002 Society for General Microbiology
The formation and function of extracellular enveloped vaccinia virus
Geoffrey L. Smith1,
Alain Vanderplasschenb,1 and
Mansun Law1
Department of Virology, Room 333, The Wright–Fleming Institute, Faculty of Medicine, Imperial College of Science, Technology & Medicine, St Mary’s Campus, Norfolk Place, London W2 1PG, UK1
Author for correspondence: Geoffrey L. Smith. Fax +44 207 594 3973. e-mail glsmith{at}ic.ac.uk
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Abstract
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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.
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Introduction
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Vaccinia virus (VV) is a large DNA virus that replicates in the cytoplasm and is a member of the genus Orthopoxvirus of the family Poxviridae (Moss, 2001
). Other orthopoxviruses include variola virus (VAR), the cause of smallpox, cowpox virus (CPV), monkeypox virus (MPV), ectromelia virus (ECT), camelpox virus (CMPV), raccoonpox virus and taterapoxvirus (Fenner et al., 1989 ). The genomes of VV (Goebel et al., 1990 ), VAR (Massung et al., 1994 ), MPV (Shchelkunov et al., 2001 ), CMPV (Afonso et al., 2002 ; Gubser & Smith, 2002 ), ECT (www.sanger.ac.uk), and CPV (AF482758; Shchelkunov et al., 1998 ) have been sequenced. These viruses are all morphologically indistinguishable and antigenically related, such that prior infection with any one provides some protection against each member of the genus (Fenner et al., 1989 ). This was the basis for CPV (Jenner, 1798 ) and later VV (Downie, 1939 ) being effective vaccines against smallpox. VV has been developed as an expression vector for foreign genes and as a live recombinant vaccine for infectious diseases and cancer (Mackett et al., 1982 ; Panicali & Paoletti, 1982 ).
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An overview of morphogenesis
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An overview of VV morphogenesis is shown in Fig. 1. Intracellular mature virus (IMV) particles are formed within cytoplasmic factories from non-infectious precursors called crescents and immature virus (IV). IMV represent the majority of infectious progeny and mostly remain within the cell until lysis. However, some IMV leave the factory on microtubules and become wrapped by a double layer of intracellular membrane derived from the early endosomes or trans-Golgi network (TGN) to form intracellular enveloped virus (IEV). IEV then move to the cell surface (again requiring microtubules) where the outer membrane fuses with the plasma membrane exposing an enveloped virion on the cell surface. Particles retained on the cell surface are termed cell-associated enveloped virus (CEV) and those released are called extracellular enveloped virus (EEV). CEV and EEV are physically indistinguishable and contain one fewer membrane than IEV and one more membrane than IMV. CEV induce the formation of actin tails that drive the virions away from the cell and are important for cell-to-cell spread. EEV mediate long-range dissemination of virus.
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Fig. 1. Overview of VV morphogenesis. IMV are made in a virus factory and move on microtubules (MT) to the wrapping membranes derived from the trans-Golgi network or early endosomes. Here IMV are wrapped by a double membrane to form IEV that move to the cell surface on microtubules. At the cell surface the outermost IEV membrane fuses with the plasma membrane to form CEV that induce actin tail formation to drive the virion away from the cell. CEV may also be released to form EEV.
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Recognition of intracellular and extracellular virus
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Immunization against smallpox with live VV caused some vaccine-related complications that varied with the strain of VV used (Fenner et al., 1988 ). Although safer strains of virus, such as Lister and New York City Board of Health (Wyeth), were used during the latter part of the smallpox eradication campaign, the complications prompted inactivated (killed) virus vaccine preparations or more attenuated strains, such as LC16m8 (in Japan) or modified vaccinia virus Ankara (in Germany), to be considered as alternatives. Killed vaccines were prepared from virus released by disruption of infected cells, predominantly IMV, that was inactivated by ultra-violet light (Collier et al., 1955 ), formaldehyde (Amies, 1961 ) or heat (Madeley, 1968 ; Turner et al., 1970 ) and were largely ineffective in preventing orthopoxvirus infection in man (Kaplan, 1962 ; Kaplan et al., 1962 , 1965 ) and in animal models (Madeley, 1968 ; Boulter et al., 1971 ; Turner & Squires, 1971 ; Appleyard & Andrews, 1974 ). For instance, 18 out of 20 infants immunized with killed vaccine developed high levels of antibody to VV but had typical primary ‘takes’ when subsequently inoculated with live vaccine (Kaplan et al., 1965 ). Similarly, rabbits immunized with killed IMV vaccine had high levels of antibody but still developed lesions at the site of intradermal challenge (Boulter, 1969 ; Boulter et al., 1971 ; Turner & Squires, 1971 ). Such antibody did not prevent secondary plaque formation by virus growing in cell monolayers and did not neutralize virus released into the supernatants of infected cells (Boulter, 1969 ; Appleyard et al., 1971 ; Turner & Squires, 1971 ; Appleyard & Andrews, 1974 ). Evidently, antibody resulting from immunization with inactivated IMV lacked some component(s) that was (were) essential for protecting cells from infection in vitro and in vivo and this led to the proposal that extracellular virus (EEV) differed antigenically from the virus retained in cells (Boulter, 1969 ). This proposal was supported by several observations: (i) antibody raised against inactivated intracellular virus failed to neutralize extracellular virus (Madeley, 1968 ; Boulter, 1969 ; Appleyard et al., 1971 ; Turner & Squires, 1971 ); (ii) the proteins immunoprecipitated with anti-IMV and anti-EEV antibody were different (Turner & Squires, 1971 ); and (iii) electron microscopy showed that EEV contained an additional lipid envelope (Appleyard et al., 1971 ; Ichihashi et al., 1971 ). Consistent with the extra lipid envelope, EEV had a lower buoyant density (1·23–1·24 g/ml) in caesium chloride density gradients than IMV (1·27–1·28 g/ml) (Boulter & Appleyard, 1973 ). Later, Payne (1978 , 1979 ) identified 10 proteins in EEV that were absent from IMV. These early studies demonstrated the existence of antigenically and structurally distinct forms of infectious virions and showed that antibody to the EEV was important for protection against live virus challenge. Although antibody that neutralizes IMV can have benefit in protecting against challenge with IMV (Hooper et al., 2000 ; Ramírez et al., 2002 ), where the roles of antibody to IMV and EEV have been compared side-by-side, the anti-EEV antibody has much greater benefit, and in particular this benefit extends to after virus challenge, when the virus infection is spread by EEV against which the anti-IMV antibody is ineffective (Boulter et al., 1971 ; Boulter & Appleyard, 1973 ).
An important conclusion from these studies, and one pertinent to the current development of second generation smallpox vaccines, was that tests to measure neutralizing antibody that are relevant to immunological protection should utilize EEV rather than IMV (Boulter & Appleyard, 1973 ; Appleyard & Andrews, 1974 ). The EEV neutralization test is difficult because of the presence of contaminating IMV in EEV preparations and the fragility of the EEV outer envelope (Boulter & Appleyard, 1973 ). However, Appleyard et al. (1971) described two methods for measuring antibody to EEV: (i) the anti-comet test; and (ii) the modified neutralization test using EEV pretreated with antibody against inactivated IMV. Some strains of VV [such as rabbitpox and International Health Department (IHD)-J] release high levels of EEV and if these viruses are allowed to grow on cell monolayers they give rise to characteristic comet-shaped plaques in which the head of the comet represents the primary plaque and the comet tails represents secondary plaques caused by unidirectional spread of EEV by convection currents (Law et al., 2002 ). The formation of comets is inhibited by antibody to EEV but not IMV.
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IMV formation
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Investigations of VV morphogenesis by electron microscopy reported that the initial stages of virion formation take place in cytoplasmic sites called virus factories from which cellular organelles are largely excluded (Dales & Siminovitch, 1961 ). Within these factories the first visible viral structures were crescents composed of lipid and virus-encoded protein. These crescents grow to form ovals, called IV, which then mature by proteolytic cleavage of several capsid proteins (Moss & Rosenblum, 1973 ) and condensation of the virus core to form IMV.
The nature and origin of the crescents are disputed. Early investigators proposed that these were formed from a single lipid bilayer that was synthesized de novo and lacked continuity with cellular membranes (Dales, 1963 ; Dales & Mosbach, 1968 ). Subsequently, it was proposed that the crescent was composed of a pair of tightly apposed membranes that were derived from and were continuous with cell membranes of the intermediate compartment (IC) between the endoplasmic reticulum (ER) and the Golgi stack (Sodeik et al., 1993 ). Another study reported no continuity between virus and cellular membranes and only a single lipid bilayer (Hollinshead et al., 1999 ). Recently, additional reports claimed IMV has two (Risco et al., 2002 ) or more membranes (Griffiths et al., 2001 ). The de novo model of membrane biosynthesis contradicts dogma stating that membranes grow from existing membranes. However, a single membrane around the outside of IMV simplifies the virus re-entry mechanism (see below). In contrast, the double membrane model fits with our knowledge of cell biology, but creates a topological difficulty during virus re-entry: namely, how the multiple membranes surrounding the virus are shed to release the core into the cytosol. The issue is fundamental to aspects of virus morphogenesis and re-entry and additional study is needed. This review considers events after IMV formation and builds on an earlier review (Smith & Vanderplasschen, 1998 ).
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Egress of IMV from factories
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After their formation IMV particles may move to sites where they become wrapped by a double layer of membrane to form an IEV. Alternatively, IMV may remain in the cell until cell lysis or, with some orthopoxviruses, become occluded in A-type inclusion (ATI) bodies (see below). Late in infection some IMV bud through the plasma membrane (Ichihashi et al., 1971 ; Tsutsui, 1983 ).
The movement of IMV particles from the factory to the wrapping membranes requires microtubules and the A27L protein (Sanderson et al., 2000 ), which is present on the IMV surface and is a target for antibodies that neutralize IMV infectivity (Rodriguez et al., 1985 ). Repression of A27L gene expression caused a deficiency in IEV formation, a small plaque size and 20-fold reduced EEV production (Rodriguez & Smith, 1990 ). The A27L protein is required for both transport and wrapping since loss of A27L prevented IMV transport and an Ala-25 to Asp substitution permitted transport but wrapping was inhibited (Sanderson et al., 2000 ). This multi-functional protein also forms a complex with two other IMV proteins (A17L and A14L) (Rodriguez et al., 1993 ) and promotes cell-to-cell fusion (Rodriguez et al., 1987 ).
In another study virus particles were found to accumulate near the microtubule organizing centre (MTOC) and this accumulation was prevented by disruption of microtubules by nocodazole or by expressing dominant negative mutants of p50/dynamitin, which disrupts the function of dynein–dynactin (Ploubidou et al., 2000 ). These observations supported the requirement for microtubules for IMV transport. Later during infection the MTOC was disrupted (Ploubidou et al., 2000 ).
Late during infection, a greater proportion of IMV particles remains unwrapped and may either stay in the cytosol until cell lysis, bud through the plasma membrane or become occluded in ATIs. ATIs are proteinaceous bodies that appear late in infection (Ichihashi et al., 1971 ) and are composed predominantly of a single polypeptide (160 kDa in cowpox virus) (Patel et al., 1986 ). The majority of orthopoxviruses, including VV, do not make ATIs because the gene encoding the 160 kDa protein is disrupted. However, several strains of cowpox virus and raccoonpox virus make ATIs (Ichihashi et al., 1971 ; Patel et al., 1986 ). The ATI enhances IMV stability after cell death and aids virus transmission between hosts.
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Proteins of IEV, CEV and EEV
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Before considering the next stages of morphogenesis the virus-encoded proteins that are associated with the IEV, CEV and EEV, but not IMV particles, are described. The properties and membrane topology of these proteins are illustrated in Fig. 2 and their locations on virions are illustrated in Fig. 3. The phenotypes of virus mutants lacking these proteins individually are shown in Table 1 and the stages at which morphogenesis is affected are shown in Fig. 4.
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Fig. 3. Virion localization of envelope proteins. IMV, IEV, CEV and EEV are shown together with the indicated proteins and topology. Note that A36R and F12L are present on the IEV outer envelope but are absent from CEV and EEV. A36R becomes concentrated beneath the CEV particle on the plasma membrane. A33R, A34R, A56R, and B5R are exposed on the outside of the CEV/EEV and F13L is located between the EEV outer envelope and the IMV surface.
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Fig. 4. The VV morphogenic pathway. Different stages of VV egress can be defined using inhibitors of IMV wrapping (IMCBH), actin (cytochalasin D) and microtubule (nocodazole) polymerization, and Src-family kinase (PP1). The A27L protein and microtubules are needed for the movement of IMV from the factory to site of wrapping. IMV wrapping to form IEV requires the B5R and F13L proteins. Movement of IEV to the cell surface requires microtubules and the F12L protein. Actin tail formation requires the A33R, A34R and A36R proteins and the phosphorylation of the A36R protein that can be inhibited by PP1. Release of CEV to form EEV is affected by the A33R, A34R and B5R proteins.
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Payne (1978 , 1979 ) identified ten proteins (210K, 110K, 89K, 42K, 37K, plus five proteins from 20K to 23K) that were present in purified EEV (strain IHD-J) and absent from IMV. All were glycosylated except 37K. In contrast, IMV contains only a single glycopeptide of 40 kDa (Holowczak, 1970 ; Garon & Moss, 1971 ). Monoclonal antibodies (mAbs) to several of these EEV proteins were reported (Shida, 1986a ; Payne, 1992 ; Schmelz et al., 1994 ) and the genes encoding most of these proteins have been identified. Gene A56R encodes the 89K virus haemagglutinin (HA) (Shida, 1986a ), B5R encodes the 42K protein (Engelstad et al., 1992 ; Isaacs et al., 1992 ), F13L encodes the 37K protein (Hirt et al., 1986 ), and genes A33R (Roper et al., 1996 ) and A34R (Duncan & Smith, 1992 ) encode the collection of smaller proteins with various degrees of glycosylation. Genes encoding the 210K and 110K proteins have not been identified and these may represent complexes (Payne, 1992 ) (Fig. 2
). The proteins encoded by F13L, A33R, A34R, A56R and B5R are present in IEV, CEV and EEV particles (Fig. 3), although approximately one-third of EEV particles lack A56R (Krauss et al., 2002 ).
In addition, genes A36R (Parkinson & Smith, 1994 ; van Eijl et al., 2000 ) and F12L (Zhang et al., 2000 ; van Eijl et al., 2002 ) encode proteins that are present on IEV. Although some A36R and F12L proteins co-purify with EEV preparations, immunoelectron microscopy showed that they are absent from CEV and EEV envelopes (van Eijl et al., 2000 , 2002 ). These proteins facilitate egress of IEV on microtubules (F12L) or CEV by actin polymerization (A36R) and therefore are termed transport proteins.
An interesting feature of the proteins encoded by these genes is that A33R, A36R, A56R, B5R and F13L proteins are palmitoylated (Grosenbach et al., 2000 ). Expression of most of these proteins individually by Semliki Forest virus vectors enabled the location of each protein to be studied in the absence of other VV proteins. The B5R, F13L and A34R proteins were present in intracellular vesicles, whereas the A33R and A56R proteins accumulated at the cell surface (Lorenzo et al., 2000 ).
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Wrapping of IMV to make IEV
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The origin of the membranes used to wrap IMV to form IEV is controversial. Some studies reported that the wrapping membranes were derived from early endosomes because fluid phase markers were incorporated into the lumen between the outer membranes of IEV particles (Tooze et al., 1993 ; van Eijl et al., 2002 ). Others reported that the membranes were from the Golgi (Ichihashi et al., 1971 ; Hiller & Weber, 1985 ) or TGN (Schmelz et al., 1994 ). There is increased traffic between these compartments late during VV infection (Tooze et al., 1993 ) and so these organelles might not be easily distinguished. Possibly both are used.
Early during infection the majority of IMV particles are wrapped to form IEV, whereas later during infection IMV predominate (Ulaeto et al., 1996 ), possibly due to depletion of wrapping membranes. The interaction of IMV with the wrapping membranes involves the cytosolic face of the wrapping membrane and the surface of IMV. The A27L protein on the IMV surface is implicated in this interaction (Sanderson et al., 2000 ); however, no direct physical evidence for this has been published. Glycoproteins A33R, A34R, A56R and B5R have the majority of their polypeptide chains positioned within the lumen of the wrapping membranes and have only a short tail (15–30 amino acids) in the cytosol (Fig. 3), whereas the non-glycosylated proteins A36R, F12L and F13L have the majority of their polypeptide chain in the cytosol and so are better placed to interact with IMV. Interestingly, proteins F12L and A36R are associated predominantly with the outer IEV membrane such that after fusion of IEV with the plasma membrane they are absent from CEV (van Eijl et al., 2000 , 2002 ). How they are excluded from the inner IEV wrapping membrane is unclear. One possibility is that during the progressive wrapping of IMV, A36R and F12L become displaced due to the bulk of their polypeptide chain being between the IMV surface and the wrapping membrane.
Analysis of virus mutants lacking individual genes has shown that the F13L (Blasco & Moss, 1991 ) and B5R (Engelstad & Smith, 1993 ; Wolffe et al., 1993 ) proteins are each required for efficient wrapping, whereas F12L, A33R, A34R, A36R and A56R are not (Table 1). Without A34R there is an increased production of EEV yet fewer IEV are seen (Duncan & Smith, 1992 ; Wolffe et al., 1997 ; Law et al., 2002 ).
Wrapping of IMV is inhibited by a drug, N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (IMCBH) (Kato et al., 1969 ; Payne & Kristensson, 1979 ; Hiller et al., 1981a ), that prevents targeting of the F13L protein to the wrapping membranes (Hiller et al., 1981a ). Passage of VV in the presence of IMCBH resulted in generation of drug-resistant virus containing an Asp to Tyr mutation within the F13L protein (Schmutz et al., 1991 ). The F13L protein is modified by acylation (palmitic and oleic acid) (Hiller & Weber, 1985 ; Child & Hruby, 1992 ; Payne, 1992 ). Mutation of Cys-185 and Cys-186 to serine prevented palmitoylation leaving the F13L protein soluble in the cytoplasm and preventing wrapping (Grosenbach et al., 1997 ; Grosenbach & Hruby, 1998 ; Grosenbach et al., 2000 ).
An interesting feature of the F13L protein is its limited amino acid similarity to phospholipase D (PLD) (Koonin, 1996 ; Ponting & Kerr, 1996 ). Although no PLD activity was detected in cells expressing F13L, mutagenesis of a motif conserved in PLDs disrupted F13L function and only tiny plaques were formed (Sung et al., 1997 ). Others reported that F13L is a broad specificity lipase with phospholipase C, phospholipase A and triacylglycerol lipase activity (Baek et al., 1997 ). Mammalian PLD1 is expressed in the Golgi membranes and regulates vesicular budding (Bednarek et al., 1996 ; Colley et al., 1997 ). A similar role has been suggested for F13L based on the observation that F13L expressed without other VV proteins localizes in the Golgi (Lorenzo et al., 2000 ) or post-Golgi vesicles (Husain & Moss, 2001 ) and that F13L causes redistribution of B5R from the TGN to endosomal membranes unless the conserved PLD motif is mutated (Husain & Moss, 2001 ). Consistent with a requirement for PLD activity in VV morphogenesis, the PLD inhibitor butanol-1 inhibited VV morphogenesis but expression of cellular PLD could not substitute for loss of the F13L protein (Husain & Moss, 2002 ).
The other protein required for wrapping of IMV is B5R. This has four short consensus repeats (SCR) that are characteristic of regulators of complement activation (Takahashi-Nishimaki et al., 1991 ; Engelstad et al., 1992 ) (Fig. 2). The N-terminal signal peptide is proteolytically removed (Isaacs et al., 1992 ) and some of the protein is also cleaved near the transmembrane domain to produce a secreted 35 kDa protein of unknown function (Martinez-Pomares et al., 1993 ). The B5R protein is acylated (Payne, 1992 ) by addition of palmitic acid at Cys-301, and possibly a second unidentified site (Grosenbach et al., 2000 ), and forms higher molecular mass complexes in the absence of reducing agent (Engelstad et al., 1992 ; Payne, 1992 ). The B5R protein affects virus host-range in some cell types (Takahashi-Nishimaki et al., 1991 ; Martinez-Pomares et al., 1993 ).
Virus mutants lacking B5R are very inefficient at wrapping IMV to IEV, have 5- to 10-fold lower levels of EEV, form a small plaque and are attenuated in vivo (Takahashi-Nishimaki et al., 1991 ; Engelstad & Smith, 1993 ; Martinez-Pomares et al., 1993 ; Wolffe et al., 1993 ) (Table 1). The signals necessary for correct targeting of B5R to the Golgi membranes reside in the transmembrane/cytoplasmic tail since fusion of these domains to other proteins such as human immunodeficiency virus gp120 (Katz et al., 1997 ), green fluorescent protein (GFP) (Ward & Moss, 2000 ; Hollinshead et al., 2001 ; Rodger & Smith, 2002 ) or vesicular stomatitis virus G protein (Ward & Moss, 2000 ) directed these chimaeras to IEV and EEV. Deletion of one or more SCR domains impaired wrapping of IMV and caused a small plaque phenotype, but EEV production was enhanced 10- to 50-fold (Herrera et al., 1998 ; Mathew et al., 1998 ; Rodger & Smith, 2002 ). Loss of the cytoplasmic tail did not affect wrapping or reduce plaque size (Lorenzo et al., 1998 ; Mathew et al., 2001 ), but the protein was less rapidly transported through the exocytic pathway (Mathew et al., 2001 ). Another study reported reduced accumulation of the C-terminally truncated protein in the Golgi membranes and concluded that the cytoplasmic tail had a role in retrieving B5R from the plasma membrane (Ward & Moss, 2000 ). Addition of an ER retrieval sequence to the C terminus of B5R caused the relocation of B5R to the ER and a reduced plaque size, but did not prevent IEV and EEV formation (Mathew et al., 1999 ).
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Transport of IEV to the cell surface
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The IEV particle is an intermediate between IMV and CEV and functions to (i) transfer virus to the cell periphery and (ii) cloak the virus particle released from the cell with a membrane that shields IMV from antibody and complement, to which it is very sensitive.
The movement of a large virion (250x350 nm) through the cytoplasm by diffusion is a slow and inefficient process (Sodeik, 2000 ) and so to expedite egress, VV uses cellular transport pathways. Two mechanisms have been proposed. One was that IEV particles induce polymerization of actin to drive these virions to the cell surface (Cudmore et al., 1995 , 1996 ; Frischknecht et al., 1999b ). This proposal was consistent with the prior observations that VV-infected cells produce numerous actin bundles (diameter 0·3 µm) resembling filopodia or specialized microvilli with enveloped virions at their tip (Stokes, 1976 ; Hiller et al., 1979 , 1981b ; Krempien et al., 1981 ; Blasco et al., 1991 ). These structures were seen only late during infection and required VV particle formation (Hiller et al., 1979 ). However, the proposal by Cudmore et al. (1995) was problematic. First, cytochalasin D prevented actin tail formation but CEV particles were still found on the cell surface (Payne & Kristensson, 1982 ). Second, there are several virus mutants that are unable to produce actin tails (Table 1) but which still form CEV and EEV and in some cases with enhanced EEV levels. Third, the drug PP1, which inhibits tyrosine phosphorylation [which is necessary for actin tail formation (Frischknecht et al., 1999a , b )], did not prevent CEV formation (Hollinshead et al., 2001 ). Evidently, transport to the surface is not reliant on actin polymerization. Lastly, the actin tails are found on one side only of the virus particle. This is reminiscent of intracellular bacteria such as Listeria and Shigella that also induce actin polymerization, but in those cases a bacterial protein located at one end only of the bacterium directs actin polymerization to that site only (Goldberg & Theriot, 1995 ; Smith et al., 1995 ). Yet with VV, the A36R protein, which is required for actin tail polymerization (see below), is distributed evenly over the IEV surface (van Eijl et al., 2000 ); so how is the polymerization polar?
The second model proposes that IEV move to the cell surface on microtubules, and actin tails form at the cell surface beneath CEV particles. The proposal that actin tails form only at the cell surface was based on the observation that the A36R protein was on IEV and beneath CEV on the cytosolic face of the plasma membrane, but was absent from CEV and EEV (van Eijl et al., 2000 ). This location is ideal to induce actin tail formation to drive the particle away from the cell. Subsequently, several groups utilizing GFP-labelled virions reported that IEV movement to the cell surface requires microtubules (Geada et al., 2001 ; Hollinshead et al., 2001 ; Rietdorf et al., 2001 ; Ward & Moss, 2001 ). IEV were found to move along defined pathways rather than randomly in the cytosol, their movement was inhibited reversibly by nocodazole, and they moved in a stop–start manner with an average speed of 60 µm/min characteristic of microtubular transport but 20-fold greater than VV movement on actin tails (2·8 µm/min) (Cudmore et al., 1995 ).
These observations demonstrate that microtubules are used at two stages during VV egress: first, for transport of IMV from the virus factories toward the MTOC, and second, for transport of IEV from the MTOC to the cell surface (Fig. 1). Evidence that disruption of dynein-dynactin (Ploubidou et al., 2000 ) inhibited IMV movement, while disruption of kinesin inhibited IEV movement (Rietdorf et al., 2001 ), and the presence of different proteins on the surface of IMV and IEV is consistent with this. For IMV, the A27L protein is implicated directly or indirectly in microtubular movement, but which IEV proteins are involved? IEV proteins F12L, F13L and A36R are candidates because they are predominantly cytosolic (Fig. 3). Of these, F13L is required for IEV formation (Blasco & Moss, 1991 ) whereas F12L and A36R are not. In the absence of A36R, IEV are transported to the cell surface and CEV are visible by confocal and electron microscopy (Sanderson et al., 1998a ; Wolffe et al., 1998 ; van Eijl et al., 2000 ; Hollinshead et al., 2001 ), although another study reported that A36R is needed for IEV movement on microtubules (Rietdorf et al., 2001 ). The third protein, F12L, seems a better candidate for microtubular movement.
The F12L protein is a 65–70 kDa protein that is conserved in chordopoxviruses (Zhang et al., 2000 ). Immunoelectron microscopy using an epitope-tagged F12L revealed the protein is located on the IEV surface and is absent from CEV and EEV. In this respect it resembles A36R, but one difference is the absence of F12L beneath CEV at the cell surface (van Eijl et al., 2002 ). A mutant lacking the F12L protein made IEV, but IEV were not transported to the cell surface, EEV levels were reduced, the plaque size was small and the virus was highly attenuated (Zhang et al., 2000 ; van Eijl et al., 2002 ). Disruption of the corresponding gene in fowlpox virus also caused a small plaque phenotype and decreased EEV production (Ogawa et al., 1993 ). The VV F12L deletion mutant is the only mutant reported to make IEV particles that are not transported and thus is a prime candidate for interactions with microtubules. The mode of interaction of F12L with the IEV is not understood.
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Actin tail formation
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Once IEV reach the cell surface the outer envelope fuses with the plasma membrane exposing CEV on the cell surface. Electron micrographs showed an array of electron-dense material on the cytosolic face of the plasma membrane beneath CEV (Ichihashi et al., 1971 ) and immunoelectron microscopy showed that the A36R protein becomes concentrated here and is absent from other parts of the plasma membrane and CEV (Fig. 3) (van Eijl et al., 2000 ). The mechanism(s) for this and how the A36R protein is located only on the outer IEV membrane, so that after fusion at the cell surface the protein is absent from CEV, are unknown. But A36R is a key protein for induction of actin tails and it is possible that the increased concentration of A36R beneath CEV is the trigger for its phosphorylation by Src-like kinases that initiates the cascade of interactions resulting in actin polymerization (Frischknecht et al., 1999b ). The failure to polymerize actin on IEV particles might be due to insufficient A36R concentration or absence of other components.
The A36R protein was originally described as a component of the EEV surface based upon its co-purification with EEV and its sensitivity to digestion by exogenous trypsin (Parkinson & Smith, 1994 ). However, subsequent analysis showed that the protein was absent from CEV and EEV (van Eijl et al., 2000 ). The A36R protein has a type 1b membrane topology with the majority of the amino acids in the cytosol (Fig. 2) (Röttger et al., 1999 ; Grosenbach et al., 2000 ; van Eijl et al., 2000 ), explaining why the six potential sites for attachment of N-linked carbohydrate are unused (Parkinson & Smith, 1994 ). Although the A36R protein is expressed by all orthopoxviruses examined, the protein varies in length and sequence near the C terminus (Pulford et al., 2002 ) and in ectromelia virus strain MP1 the protein is significantly shorter (160 amino acids versus 220 in VV). Mutagenesis demonstrated that truncated versions of A36R can still induce actin tail formation, but phosphorylation of Tyr-112 [an amino acid conserved in all sequenced A36R proteins (Pulford et al., 2002 )] is essential and can be inhibited by PP1 (Frischknecht et al., 1999b ). After phosphorylation A36R interacts with Nck leading to recruitment of N-WASP to the site of actin assembly (Frischknecht et al., 1999b ). The recruitment of A36R to IEV requires the A33R protein, which functions as a chaperone and with which A36R forms a non-covalent complex (Wolffe et al., 2001 ). Tyrosine phosphorylation of A36R is reduced in the absence of A34R or F13L and inhibited in the absence of A33R (Wolffe et al., 2001 ). The A36R protein is also phosphorylated on serine and threonine residues (Wolffe et al., 2001 ) and is acylated via Cys-25 (Grosenbach et al., 2000 ). Deletion of A36R causes a dramatic attenuation (Parkinson & Smith, 1994 ) comparable to that resulting from loss of F12L (Zhang et al., 2000 ).
A direct comparison of the plaque-size phenotype of all mutants listed in Table 1 (Law et al., 2002 ) highlighted the role for actin tails in cell-to-cell spread. These mutants include those lacking F12L (Zhang et al., 2000 ), F13L (Blasco & Moss, 1991 ; Cudmore et al., 1995 ), A33R (Roper et al., 1998 ), A34R (Duncan & Smith, 1992 ; McIntosh & Smith, 1996 ; Wolffe et al., 1997 ; Sanderson et al., 1998a ), A36R (Parkinson & Smith, 1994 ; Sanderson et al., 1998a ; Wolffe et al., 1998 ; Frischknecht et al., 1999b ; Röttger et al., 1999 ) and B5R (Engelstad & Smith, 1993 ; Wolffe et al., 1993 ; Mathew et al., 1998 ; Sanderson et al., 1998a ; Röttger et al., 1999 ). One report that a mutant lacking the SCR domains of B5R produced a normal size plaque but failed to produce actin tails (Herrera et al., 1998 ) did not fit with this model, but upon re-examination this mutant was found to form a small plaque (Rodger & Smith, 2002 ). The A56R protein is the only IEV/CEV/EEV protein not needed for efficient actin tail formation (Sanderson et al., 1998a ).
Two other observations are noteworthy regarding VV-induced actin tail formation. First, VV gene A42R encodes a profilin-like protein (an actin-binding protein) but this is not required for formation of actin tails or for virus maturation and egress (Blasco et al., 1991 ). Second, VV infection induces cell migration and subsequent cellular projections up to 160 µm long that often are branched and require drastic rearrangement of actin cytoskeleton of the host cell (Sanderson et al., 1998b ). Cell migration required early virus gene expression only, whereas formation of projections required both early and late virus gene expression (Sanderson et al., 1998b ).
Actin tails can continue to grow from the cell surface for considerable distances (Hiller et al., 1979 ) and facilitate virus penetration of surrounding cells. These tails can also re-enter the same cell (Hollinshead et al., 2001 ). Eventually, as the tail grows longer, it may be detached from the cell still containing the CEV at its tip. Alternatively, the CEV may be released to form EEV.
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Release of EEV
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Several factors influence the release of EEV. Payne (1979 , 1980 ) showed that both the strain of virus and the host cell influence EEV release: thus the IHD-J strain produces more EEV than the WR or Lister strains and the greatest yields were from RK13 cells. The enhanced release of EEV by the IHD-J strain was due to a Lys-151 to Glu mutation in the A34R protein (Blasco et al., 1993 ) and the majority of VV strains and VAR have the WR genotype (McIntosh & Smith, 1996 ). Furthermore, deletion of A34R caused a 25-fold increase in EEV, although the specific infectivity of these EEV was reduced 5-fold (McIntosh & Smith, 1996 ) (Table 1). Other proteins affecting EEV formation or release are A33R, A36R and B5R. Loss of A33R caused a 3-fold incre
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Abstract
Introduction
