Vaccinia virus WR gene A5L is required for morphogenesis of mature virions

The vaccinia virus WR A5L open reading frame (corresponding to open reading frame A4L in vaccinia virus Copenhagen) encodes an immunodominant late protein found in the core of the vaccinia virion. To investigate the role of this protein in vaccinia virus replication, we have constructed a recombinant virus, vA5Li, in which the endogenous gene has been deleted and an inducible copy of the A5 gene dependent on isopropyl-beta-D-thiogalactopyranoside (IPTG) for expression has been inserted into the genome. In the absence of inducer, the yield of infectious virus was dramatically reduced. However, DNA synthesis and processing, viral protein expression (except for A5), and early stages in virion formation were indistinguishable from the analogous steps in a normal infection. Electron microscopy revealed that the major vaccinia virus structural form present in cells infected with vA5Li in the absence of inducer was immature virions. Viral particles were purified from vA5Li-infected cells in the presence and absence of inducer. Both particles contained viral DNA and the full complement of viral proteins, except for A5, which was missing from particles prepared in the absence of inducer. The particles prepared in the presence of IPTG were more infectious than those prepared in its absence. The A5 protein appears to be required for the immature virion to form the brick-shaped intracellular mature virion.     

Vaccinia virus A6 is essential for virion membrane biogenesis and localization of virion membrane proteins to sites of virion assembly

Poxvirus acquires its primary envelope through a process that is distinct from those of other enveloped viruses. The molecular mechanism of this process is poorly understood, but several poxvirus proteins essential for the process have been identified in studies of vaccinia virus (VACV), the prototypical poxvirus. Previously, we identified VACV A6 as an essential factor for virion morphogenesis by studying a temperature-sensitive mutant with a lesion in A6. Here, we further studied A6 by constructing and characterizing an inducible virus (iA6) that could more stringently repress A6 expression. When A6 expression was induced by the inducer isopropyl-β-D-thiogalactoside (IPTG), iA6 replicated normally, and membrane proteins of mature virions (MVs) predominantly localized in viral factories where virions were assembled. However, when A6 expression was repressed, electron microscopy of infected cells showed the accumulation of large viroplasm inclusions containing virion core proteins but no viral membranes. Immunofluorescence and cell fractionation studies showed that the major MV membrane proteins A13, A14, D8, and H3 did not localize to viral factories but instead accumulated in the secretory compartments, including the endoplasmic reticulum. Overall, our results show that A6 is an additional VACV protein that participates in an early step of virion membrane biogenesis. Furthermore, A6 is required for MV membrane protein localization to sites of virion assembly, suggesting that MV membrane proteins or precursors of MV membranes are trafficked to sites of virion assembly through an active, virus-mediated process that requires A6.     

Repression of Vaccinia Virus Holliday Junction Resolvase Inhibits Processing of Viral DNA into Unit-Length Genomes

The vaccinia virus A22R gene encodes a protein that is homologous to the bacterial enzyme RuvC and specifically cleaves and resolves four-way DNA Holliday junctions into linear duplex products. To investigate the role of the vaccinia virus Holliday junction resolvase during an infection, we constructed two recombinant viruses: vA22-HA, which has a short C-terminal epitope tag appended to the A22R open reading frame, and vA22i, in which the original A22R gene is deleted and replaced by an inducible copy. Polyacrylamide gel electrophoresis and Western blot analysis of extracts and purified virions from cells infected with vA22-HA revealed that the resolvase was expressed after the onset of DNA replication and incorporated into virion cores. vA22i exhibited a conditional replication defect. In the absence of an inducer, (i) viral protein synthesis was unaffected, (ii) late-stage viral DNA replication was reduced, (iii) most of the newly synthesized viral DNA remained in a branched or concatemeric form that caused it to be trapped at the application site during pulsed-field gel electrophoresis, (iv) cleavage of concatemer junctions was inhibited, and (v) virion morphogenesis was arrested at an immature stage. These data indicated multiple roles for the vaccinia virus Holliday junction resolvase in the replication and processing of viral DNA into unit-length genomes.     

The vaccinia virus gene I2L encodes a membrane protein with an essential role in virion entry

The previously unstudied vaccinia virus gene I2L is conserved in all orthopoxviruses. We show here that the 8-kDa I2 protein is expressed at late times of infection, is tightly associated with membranes, and is encapsidated in mature virions. We have generated a recombinant virus in which I2 expression is dependent upon the inclusion of tetracycline in the culture medium. In the absence of I2, the biochemical events of the viral life cycle progress normally, and virion morphogenesis culminates in the production of mature virions. However, these virions show an ~400-fold reduction in specific infectivity due to an inability to enter target cells. Several proteins that have been previously identified as components of an essential entry/fusion complex are present at reduced levels in I2-deficient virions, although other membrane proteins, core proteins, and DNA are encapsidated at normal levels. A preliminary structure/function analysis of I2 has been performed using a transient complementation assay: the C-terminal hydrophobic domain is essential for protein stability, and several regions within the N-terminal hydrophilic domain are essential for biological competency. I2 is thus yet another component of the poxvirus virion that is essential for the complex process of entry into target cells.     

A glutaredoxin, encoded by the G4L gene of vaccinia virus, is essential for virion morphogenesis

Vaccinia virus encodes two glutaredoxins, O2L and G4L, both of which exhibit thioltransferase and dehydroascorbate reductase activities in vitro. Although O2L was previously found to be dispensable for virus replication, we now show that G4L is necessary for virion morphogenesis. RNase protection and Western blotting assays indicated that G4L was expressed at late times after infection and was incorporated into mature virus particles. Attempts to isolate a mutant virus with a deleted G4L gene were unsuccessful, suggesting that the protein was required for virus replication. This interpretation was confirmed by the construction and characterization of a conditional lethal recombinant virus with an inducible copy of the G4L gene replacing the original one. Expression of G4L was proportional to the concentration of inducer, and the amount of glutaredoxin could be varied from barely detectable to greater than normal amounts of protein. Immunogold labeling revealed that the induced G4L protein was associated with immature and mature virions and adjacent cytoplasmic depots. In the absence of inducer, the production of infectious virus was severely inhibited, though viral late protein synthesis appeared unaffected except for decreased maturation-dependent proteolytic processing of certain core components. Electron microscopy of cells infected under nonpermissive conditions revealed an accumulation of crescent membranes on the periphery of electron-dense globular masses but few mature particles. We concluded that the two glutaredoxin homologs encoded by vaccinia virus have different functions and that G4L has a role in virion morphogenesis, perhaps by acting as a redox protein.     

Temperature-sensitive mutants with lesions in the vaccinia virus F10 kinase undergo arrest at the earliest stage of virion morphogenesis.

Vaccinia virus encodes two protein kinases; the B1 kinase is expressed early and appears to play a role during DNA replication, whereas the F10 kinase is expressed late and is encapsidated in virions. Here we report that the F10 kinase gene is the locus affected in a complementation group of temperature-sensitive mutants composed of ts15, ts28, ts54, and ts61. Although these mutants have a biochemically normal phenotype at the nonpermissive temperature, directing the full program of viral gene expression, they fail to form mature virions. Electron microscopic analysis indicates that morphogenesis undergoes arrest at a very early stage, prior to the formation of membrane crescents or immature virions. An essential role for the F10 protein kinase in orchestrating the onset of virion assembly is implied.     

Vaccinia virus J1R protein: a viral membrane protein that is essential for virion morphogenesis

Vaccinia virus, a member of the poxvirus family, contains a conserved J1R open reading frame that encodes a late protein of 17.8 kDa. The 18-kDa J1R protein is associated mainly with the membrane fraction of intracellular mature virus particles. This study examines the biological function of J1R protein in the vaccinia virus life cycle. A recombinant vaccinia virus was constructed to conditionally express J1R protein in an isopropyl-beta-D-galactopyranoside (IPTG)-inducible manner. When J1R is not expressed during vaccinia virus infection, the virus titer is reduced approximately 100-fold. In contrast, J1R protein is not required for viral gene expression, as indicated by protein pulse-labeling. J1R protein is also not required for DNA processing, as the resolution of the concatemer junctions of replicated viral DNA was detected without IPTG. A deficiency of J1R protein caused a severe delay in the processing of p4a and p4b into mature core proteins 4a and 4b, indicating that J1R protein participates in virion morphogenesis. Infected cells grown in the absence of IPTG contained very few intracellular mature virions in the cytoplasm, and enlarged viroplasm structures accumulated with viral crescents attached at the periphery. Abundant intermediate membrane structures of abnormal shapes were observed, and many immature virions were either empty or partially filled, indicating that J1R protein is important for DNA packaging into immature virions. J1R protein also coimmunoprecipited with A45R protein in infected cells. In summary, these results indicate that vaccinia virus J1R is a membrane protein that is required for virus growth and plaque formation. J1R protein interacts with A45R protein and performs an important role during immature virion formation in cultured cells.     

The vaccinia virus I1 protein is essential for the assembly of mature virions.

The product of the vaccinia virus I1 gene was characterized biochemically and genetically. This 35-kDa protein is conserved in diverse members of the poxvirus family but shows no homology to nonviral proteins. We show that recombinant I1 binds to both single-stranded and double-stranded DNA in a sequence-nonspecific manner in an electrophoretic mobility shift assay. The protein is expressed at late times during infection, and approximately 700 copies are encapsidated within the virion core. To determine the role of the I1 protein during the viral life cycle, a inducible viral recombinant in which the I1 gene was placed under the regulation of the Escherichia coli lac operator/repressor was constructed. In the absence of isopropyl-beta-D-thiogalactopyranoside, plaque formation was abolished and yields of infectious, intracellular virus were dramatically reduced. Although all phases of gene expression and DNA replication proceeded normally during nonpermissive infections, no mature virions were produced. Electron microscopic analysis confirmed the absence of mature virion assembly but revealed that apparently normal immature virions accumulated. Thus, I1 is an encapsidated DNA-binding protein required for the latest stages of vaccinia virion morphogenesis.     

Vaccinia virus envelope H3L protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo

An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L(-)) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L(-) mutant virus. IMV from the H3L(-) mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L(-) mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.     

IPTG-dependent vaccinia virus: identification of a virus protein enabling virion envelopment by Golgi membrane and egress.

A novel method has been developed to study the functional roles of individual vaccinia virus gene products that is neither limited by the possible essentiality of the target gene nor by the availability of conditional lethal mutants. The system utilises the E. coli lac repressor protein, the operator sequence to which it binds and the specific inducer IPTG. It allows the generation of recombinant viruses in which the expression of any chosen gene, and hence virus replication, can be externally controlled. In principle, this system is broadly applicable to the functional analysis of genes in any large DNA virus. This approach has demonstrated that the gene encoding the 14 kDa membrane protein of vaccinia virus is non-essential for the production of infectious intracellular virus particles, but essential for the envelopment of intracellular virions by Golgi membrane and for egress of mature extracellular viral particles. This is the first vaccinia virus protein shown to be specifically required for these processes. In vivo this system may prove useful as a means of attenuating recombinant vaccinia virus vaccines by preventing virus spread without reducing the amount of the foreign antigen expressed in each infected cell. Attenuation of other live virus vaccines may be developed in a similar way.     

Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts

Vaccinia virus infects a wide variety of mammalian cells from different hosts, but the mechanism of virus entry is not clearly defined. The mature intracellular vaccinia virus contains several envelope proteins mediating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance of plasma membrane lipid rafts in the mature intracellular vaccinia virus infection process by using biochemical and fluorescence imaging techniques. A raft-disrupting drug, methyl-beta-cyclodextrin, inhibited vaccinia virus uncoating without affecting virion attachment, indicating that cholesterol-containing lipid rafts are essential for virion penetration into mammalian cells. To provide direct evidence of a virus and lipid raft association, we isolated detergent-insoluble glycolipid-enriched membranes from cells immediately after virus infection and demonstrated that several viral envelope proteins, A14, A17L, and D8L, were present in the cell membrane lipid raft fractions, whereas the envelope H3L protein was not. Such an association did not occur after virions attached to cells at 4 degrees C and was only observed when virion penetration occurred at 37 degrees C. Immunofluorescence microscopy also revealed that cell surface staining of viral envelope proteins was colocalized with GM1, a lipid raft marker on the plasma membrane, consistent with biochemical analyses. Finally, mutant viruses lacking the H3L, D8L, or A27L protein remained associated with lipid rafts, indicating that the initial attachment of vaccinia virions through glycosaminoglycans is not required for lipid raft formation.     

Vaccinia virus E2L null mutants exhibit a major reduction in extracellular virion formation and virus spread

The vaccinia virus E2L (VACWR058) gene is conserved in all sequenced chordopoxviruses and is predicted to encode an 86-kDa protein with no recognizable functional motifs or nonpoxvirus homologs. Although the region immediately upstream of the open reading frame lacked optimal consensus promoter motifs, expression of the E2 protein occurred after viral DNA replication. Transfection studies, however, indicated that the promoter was weak compared to well-characterized intermediate and late promoters. The E2 protein was present in mature virions purified from infected cells but was more abundant in extracellular enveloped forms. Despite the conservation of the E2L gene in chordopoxviruses, deletion mutants could be isolated from both the WR and IHD-J strains of vaccinia virus. These null mutants produced very small plaques in all cell lines tested, reduced amounts of mature infectious virions, and very low numbers of extracellular virions. Nevertheless, viral protein synthesis appeared qualitatively and quantitatively normal. The defect in extracellular virus formation was corroborated by electron microscopy, which also showed some aberration in the wrapping of virions by cisternal membranes. Extracellular virions that did form, however, were able to induce actin tail formation.     

Cell biological and functional characterization of the vaccinia virus F10 kinase: implications for the mechanism of virion morphogenesis

The vaccinia virus F10 protein is one of two virally encoded protein kinases. A phenotypic analysis of infections involving a tetracycline-inducible recombinant (vDeltaiF10) indicated that F10 is involved in the early stages of virion morphogenesis, as previously reported for the mutants ts28 and ts15. The proteins encoded by ts28 and ts15 have primary defects in enzymatic activity and thermostability, respectively. Using a transient complementation assay, we demonstrated that the enzymatic activity of F10 is essential for its biological function and that both its enzymatic and biological functions depend upon N-terminal sequences that precede the catalytic domain. An execution point analysis indicated that in addition to its role at the onset of morphogenesis, F10 is also required at later stages, when membrane crescents surround virosomal contents and develop into immature virions. The F10 protein is phosphorylated in vivo, appears to be tightly associated with intracellular membranes, and can bind to specific phosphoinositides in vitro. When F10 is repressed or impaired, the phosphorylation of several cellular and viral proteins appears to increase in intensity, suggesting that F10 may normally intersect with cellular signaling cascades via the activation of a phosphatase or the inhibition of another kinase. These cascades may drive the F10-induced remodeling of membranes that accompanies virion biogenesis. Upon the release of ts28-infected cultures from a 40 degrees C-induced block, a synchronous resumption of morphogenesis that culminates in the production of infectious virus can be observed. The pharmacological agents H89 and cerulenin, which are inhibitors of endoplasmic reticulum exit site formation and de novo lipid synthesis, respectively, block this recovery.     

Vaccinia Virus A19 Protein Participates in the Transformation of Spherical Immature Particles to Barrel-Shaped Infectious Virions

The A19L open reading frame of vaccinia virus encodes a 9-kDa protein that is conserved in all sequenced chordopoxviruses, yet until now it has not been specifically characterized in any species. We appended an epitope tag after the start codon of the A19L open reading frame without compromising infectivity. The protein was synthesized after viral DNA replication and was phosphorylated independently of the vaccinia virus F10 kinase. The A19 protein was present in purified virions and was largely resistant to nonionic detergent extraction, suggesting a location within the core. A conditional lethal mutant virus was constructed by placing the A19 open reading frame under the control of the Escherichia coli lac repressor system. A19 synthesis and infectious virus formation were dependent on inducer. In the absence of inducer, virion morphogenesis was interrupted, and spherical dense particles that had greatly reduced amounts of the D13 scaffold accumulated in place of barrel-shaped mature virions. The infectivity of purified A19-deficient particles was more than 2 log units less than that of A19-containing virions. Nevertheless, the A19-deficient particles contained DNA, and except for the absence of A19 and decreased core protein processing, they appeared to have a similar protein composition as A19-containing virions. Thus, the A19 protein participates in the maturation of immature vaccinia virus virions to infectious particles.     

Evidence for an essential catalytic role of the F10 protein kinase in vaccinia virus morphogenesis

Temperature-sensitive mutants of vaccinia virus, with genetic changes that map to the open reading frame encoding the F10 protein kinase, exhibit a defect at an early stage of viral morphogenesis. To further study the role of the enzyme, we constructed recombinant vaccinia virus vF10V5i, which expresses inducible V5 epitope-tagged F10 and is dependent on a chemical inducer for plaque formation and replication. In the absence of inducer, viral membrane formation was delayed and crescents and occasional immature forms were detected only late in infection. When the temperature was raised from 37 to 39 degrees C, the block in membrane formation persisted throughout the infection. The increased stringency may be explained by a mild temperature sensitivity of the wild-type F10 kinase, which reduced the activity of the very small amount expressed in the absence of inducer, or by the thermolability of an unphosphorylated kinase substrate or uncomplexed F10-interacting protein. Further analyses demonstrated that tyrosine and threonine phosphorylation of the A17 membrane component was inhibited in the absence of inducer. The phosphorylation defect could be overcome by transfection of plasmids that express wild-type F10, but not by plasmids that express F10 with single amino acid substitutions that abolished catalytic activity. Although the mutated forms of F10 were stable and concentrated in viral factories, only the wild-type protein complemented the assembly and replication defects of vF10V5i in the absence of inducer. These studies provide evidence for an essential catalytic role of the F10 kinase in vaccinia virus morphogenesis.     

Vaccinia virus A28L gene encodes an essential protein component of the virion membrane with intramolecular disulfide bonds formed by the viral cytoplasmic redox pathway

We report the initial characterization of the product of the vaccinia virus A28L gene, which is highly conserved in all sequenced poxviruses. Our studies showed that the A28 protein is expressed at late times during the virus replication cycle and is a membrane component of the intracellular mature virion. An N-terminal hydrophobic sequence, present in all poxvirus A28 orthologs, anchors the protein in the virion surface membrane so that most of it is exposed to the cytoplasm. The cytoplasmic domain contains four conserved cysteines, which form two intramolecular disulfide bonds. Disulfide bond formation depended on the expression of three viral proteins, E10, A2.5, and G4, which together comprise a conserved cytoplasmic redox pathway. A28 is the third identified substrate of this pathway; the others are the L1 and F9 proteins. We constructed a conditional-lethal recombinant vaccinia virus with an inducible A28L gene. The recombinant virus was propagated in the presence of inducer but was unable to replicate and spread in its absence. During a single round of an abortive infection in the absence of inducer, the synthesis and processing of viral proteins, assembly of intra- and extracellular virions, and formation of actin tails occurred normally. In another paper (T. Senkevich, B. M. Ward, and B. Moss, J. Virol. 78:2357-2366, 2004), we have demonstrated that virions assembled without A28 cannot carry out a second round of infection because they are defective in cell penetration.     

The Red clover necrotic mosaic virus capsid protein N-terminal lysine-rich motif is a determinant of symptomatology and virion accumulation

The interaction between viral capsid protein (CP) and its cognate viral RNA modulates many steps in the virus infection cycle, such as replication, translation and assembly. The N-terminal 50 amino acids of the Red clover necrotic mosaic virus (RCNMV) CP are rich in basic residues (especially lysine) and are essential for the core functions of the CP, namely RNA binding and virion assembly. To further elucidate additional biological roles for these basic residues, a series of alanine substitution mutations was introduced into infectious clones of RCNMV RNA-1 and assayed for symptomatology, virion formation and systemic infection. Infectivity assays conducted in Nicotiana benthamiana revealed that all nine alanine substitution mutants (ASMs) were competent for systemic infection. Two ASMs (K4A and K7A/K8A) induced severe symptoms and delayed the systemic spread of viral genomes when compared with wild-type RCNMV. However, these ASMs were still competent for virion formation. Three other ASMs (K25A, K33A and K38A) displayed milder symptoms and significant reductions in virion accumulation when compared with wild-type RCNMV, but retained the ability to spread systemically. Evidence from these last three ASMs, as well as a CP null mutant, showed that RCNMV is able to move systemically in N. benthamiana as a nonvirion form. These observations reaffirm the necessity of the N-terminal lysine-rich residues of the RCNMV CP for efficient virion accumulation. They also reveal additional roles for the CP in the modulation of host symptomatology, independent of its role in virion assembly and the rate of systemic viral movement in N. benthamiana.