Identification of the Orthopoxvirus p4c Gene, Which Encodes a Structural Protein That Directs Intracellular Mature Virus Particles into A-Type Inclusions

The orthopoxvirus gene p4c has been identified in the genomeof the vaccinia virus strain Western Reserve. This gene encodesthe 58-kDa structural protein P4c present on the surfaces ofthe intracellular mature virus (IMV) particles. The gene isdisrupted in the genome of cowpox virus Brighton Red (BR), demonstratingthat although the P4c protein may be advantageous for virusreplication in vivo, it is not essential for virus replicationin vitro. Complementation and recombination analyses with thep4c gene have shown that the P4c protein is required to directthe IMV into the A-type inclusions (ATIs) produced by cowpoxvirus BR. The p4c gene is highly conserved among most membersof the orthopoxvirus genus, including viruses that produce ATIs,such as cowpox, ectromelia, and raccoonpox viruses, as wellas those such as variola, monkeypox, vaccinia, and camelpoxviruses, which do not. The conservation of the p4c gene amongthe orthopoxviruses, irrespective of their capacities to produceATIs, suggests that the P4c protein provides functions in additionto that of directing IMV into ATIs. These findings, and thepresence of the P4c protein in IMV but not extracellular envelopedvirus (D. Ulaeto, D. Grosenbach, and D. E. Hruby, J. Virol.70:3372-3377, 1996), suggest a model in which the P4c proteinmay play a role in the retrograde movement of IMV particles,thereby contributing to the retention of IMV particles withinthe cytoplasm and within ATIs when they are present. In thisway, the P4c protein may affect both viral morphogenesis andprocesses of virus dissemination.

INTRODUCTION

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Introduction

One of the unusual features of the poxviruses is their abilityto produce virus particles of more than one type. The majorityof the orthopoxviruses can produce infectious virus particlesof four major types differing in either surface structure orsite of accumulation. Most abundant, often representing >90%of virus progeny, are the intracellular mature virus (IMV) particles,which are generally thought to possess a single membrane (6,18, 22), though an alternative view is that this membrane consistsof two membranes in tight apposition (16, 17, 70). A small proportionof the IMVs are converted to intracellular enveloped viruses(IEVs), which are IMVs wrapped in double membranes derived fromthe trans-Golgi network or tubular endosomes (20, 30, 63, 75).The IEVs are transported to the cell surface, where their outermembranes fuse with the plasma membrane to produce either thereleased extracellular enveloped viruses (EEVs) or the cell-associatedenveloped viruses (CEVs), which remain attached to the cellsurface (4). Those IMVs that are not converted into IEVs, CEVs,or EEVs remain in the cytoplasm of the cell, either as freeparticles or as particles embedded within A-type inclusions(ATIs). The ATIs are large, well-defined proteinaceous bodiesproduced in cells infected with certain strains of cowpox, ectromelia,raccoonpox, fowlpox, or canarypox viruses (9, 28, 37, 45, 53).The four different kinds of virus particles have distinct physical,immunological, and biological properties (reviewed in reference48), suggesting that particles of each type provide some advantagesfor virus replication; however, the exact in vivo roles of particlesof each of these types are not fully understood.

The least complex forms of infectious particles are the IMVs,which also constitutes the kernels of the other, more elaborateparticles. To provide these two different functions, the proteinson the surfaces of the IMVs are involved in a variety of processes,including the formation of the IMV, directing the incorporationof the IMV within IEV or the ATI, and facilitating viral infectionof cells. In keeping with the multiplicity of roles, >12proteins that participate in these processes have been identifiedeither at or near the surface of the IMV (reviewed in references48 and 68).

One of the first IMV surface proteins to be identified was theP4c protein, one of a triplet of major structural proteins (designated4a, 4b, and P4c) with apparent molecular masses of about 60kDa. These proteins were first resolved in analyses of the proteinsof the IMV particles of the Western Reserve strain of vacciniavirus (VV-WR) by Katz and Moss (40, 41). Subsequently, the P4cprotein was shown to be among the last proteins incorporatedinto the outer surface membranes of these particles (39, 61,62). The 4a and 4b proteins, which are generated by proteolyticcleavage of the precursor proteins P4a and P4b, function asthe two major proteins of the core of the virus particle (49,61).

Various functions have been attributed to the P4c protein. First,it was proposed to be a component of the surface tubules visualizedon the surfaces of virus particles by conventional electronmicroscopy (72, 73, 82), though surface tubules have been visualizedon virions lacking intact P4c (31, 32) and the existence ofthe tubules has not been confirmed by cryoelectron microscopystudies (10). Second, the P4c protein has been implicated asa factor directing the inclusion of virus particles into theATIs. ATIs have two major phenotypes, those that contain virusparticles, the V⁺ ATIs, and those that lack virus particles,the V^- ATIs (28, 29). The V phenotype is a strain-specific property(28, 29). Usually only mature virions are found in ATIs, butimmature virions whose maturation has been arrested by treatmentwith rifampin can associate with ATIs (30). At least two genesare required for the formation of V⁺ ATIs: one, the ati gene(52, 53), encoding the ATI matrix protein and one or more encodingthe protein(s) required for the inclusion of virus particles,previously designated either the "In" or the "VO" factor. Cellscoinfected with V^- and V⁺ virus strains produce almost entirelyV⁺ ATIs, in which both V⁺ and V^- viruses are present (23, 24,29). Fusions between cells infected with V⁺ or V^- viruses werealso used to study viral association with ATIs (67). These studiesshowed that the VO factor is synthesized late in infection andhas rapid attachment to V⁺ viruses and that the residual portionin the cytoplasm attaches to V^- viruses after fusion. In comparingthe 16 most abundant proteins of V⁺ and V^- particles of cowpoxvirus and vaccinia virus, Shida et al. (67) noted a correlationbetween the amount of the P4c protein present in either theparticle or virus-free cell extracts and the VO activity, suggestingthat the p4c protein might constitute the VO factor. Third,Ulaeto et al. (76) have provided evidence that the P4c proteinis an IMV-specific protein, suggesting that IMVs that acquirethe P4c protein may not differentiate into IEVs, CEVs, or EEVs.

In this study, we have identified the orthopoxvirus gene encodingthe P4c protein. This gene is highly conserved among membersof the orthopoxvirus genus. The identification of this genehas allowed us to confirm that one of the roles of this proteinis to direct the insertion of virus particles into the ATI bodiesproduced in cells infected with orthopoxviruses encoding theAti protein.

MATERIALS AND METHODS

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Materials and Methods

Viruses and cells. VV-WR (provided by W. K. Joklik), the Copenhagen strain of vacciniavirus (provided by R. Drillien), and the Brighton Red strainof cowpox virus (CPV-BR) were used in this study. Strain 143human osteosarcoma cells or STO cells (mouse embryonic fibroblasts;ATCC strain CRL 1503) were used for virus culture and the selectionof recombinant viruses. For protein analyses, vaccinia viruseswere grown in spinner cultures of HeLa S3 cells cultured inJoklik's modification of minimal essential medium (Gibco-Invitrogen)containing 7.5% calf serum and purified as previously described(36).

Plasmid and recombinant virus constructions. Escherichia coli strains TG1 and JM103, originally providedby G. Winter (MRC Laboratory of Molecular Biology, Cambridge,United Kingdom), were cultured in Luria broth or tryptone-yeastextract broth. DNA fragments were inserted into plasmids derivedfrom pGEM7zf+ (Promega Biotec, Madison, Wis.), pUC4K, or pUC19(79) using standard techniques (57).

To subclone the VV-WR p4c gene, the 6.6-kbp XhoI-SalI fragment(containing the p4c gene) from the XhoI E fragment of VV-WRwas inserted into the XhoI site in pGEM7zf+, generating plasmidp1540. The 1.8-kbp EcoRV fragment from p1540 containing theentire p4c gene under the transcriptional control of its nativepromoter was inserted (using HindIII linkers) into the HindIIIsite of pGEM7zf+, generating plasmid p1542.

To generate a VV-WR containing an inactivated p4c gene, thefollowing procedure was used. The 1.8-kbp HindIII fragment containingthe p4c gene from the DNA of plasmid p1542, was inserted intothe HindIII site of a pUC19 derivative that lacks the rest ofthe pUC19 polylinker. The 2.0-kbp EcoRI fragment from plasmidpTK61-gpt (11), containing the E. coli xanthine-guanine phosphoribosyltransferase(gpt) gene under the control of the promoter of the vacciniavirus 7.5K gene, was inserted into the EcoRI site in the p4cgene to create plasmid p1546. This plasmid was used to constructa recombinant vaccinia virus containing a p4c gene disruptedby a functional copy of the gpt gene. The resulting vacciniavirus recombinant, A504, was isolated by positive gpt selectionaccording to the method of Falkner and Moss (11). The presenceof the insert of the gpt gene was verified by DNA hybridizationanalyses (71) of DNA extracted from cells infected with therecombinant virus. A wild-type p4c revertant of vaccinia virusA504 was generated by recombination of plasmid p1542 DNA withthe DNA of virus A504. The resulting recombinant vaccinia virus,A507, was isolated using negative gpt selection as describedpreviously (34).

To create a CPV-BR recombinant expressing the p4c gene, the1.8-kbp HindIII fragment containing the p4c gene from p1542was inserted (using ClaI linkers) into the ClaI site of plasmidp1249 (33), which contains the vaccinia virus thymidine kinasegene in pUC4K. This plasmid, p1544, was used to insert a functionalcopy of the p4c gene into the thymidine kinase gene of CPV-BR.The resulting recombinant cowpox virus, A505, was isolated byselection for viruses lacking a functional thymidine kinaseas described previously (44). The presence of the insert inthe tk gene was verified by DNA hybridization analyses (71)of DNA extracted from cells infected with the tk mutant virusisolates.

Additional plasmids containing partial deletions of the p4cgene (from plasmid p1542) were generated for use as templatesin nucleotide sequence determinations. Plasmid p1548 was createdby the deletion of p1542 from the EcoRI site in the p4c genethrough the end of the gene to the EcoRI site in the pGEM7zf+vector. A deletion from the BamHI site in the pGEM7zf+ vectorthrough the start of the p4c gene to the AccI site in the p4cgene generated p1550. Plasmid p1552 was created by deletingthe sequences in p1542 from the AccI site in the p4c gene throughto the end of the gene to the ClaI site in the pGEM7zf+ vector.

Standard methods (3, 60, 74, 79) were used to determine thenucleotide sequences of both strands of the EcoRV fragmentscontaining the p4c genes of CPV-BR and VV-WR.

Metabolic labeling of proteins in infected cells. Human 143 cells (10⁶) in monolayer cultures were infected witha virus at a multiplicity of 5 PFU/cell. Eighteen hours afterinfection, the medium was removed and nascent proteins werelabeled by the addition of 20 µCi of [³⁵S]methionine in200 µl of Eagle's minimal essential medium at 37°Cfor 30 min. The medium was removed, the cells were washed twicewith cold phosphate-buffered saline, and the cells were lysedwith 0.3 ml of buffer containing 50 mM Tris (pH 7.0), 30 mMdithiothreitol, 5 mM EDTA, 1% sodium dodecyl sulfate (SDS),and 10% glycerol. Each lysate was passed through a 25-gaugeneedle five times to shear the DNA in the sample.

Electron microscopy of infected cells. Electron microscopy of cells infected with poxvirus was performedas described previously (47). For each infection, human 143cells at 80% confluence in two 30-cm² dishes were infected eitherwith a multiplicity of 10 PFU/cell for single-virus infectionsor with a multiplicity of 5 PFU of each virus/cell for coinfectionsof two different viruses. The cells were fixed 18 h after infectionby washing them twice with 3 ml of cold phosphate-buffered salineand adding 3 ml of CS buffer, a buffer of 0.1 M sodium cacodylatecontaining 3.4% sucrose (pH 7.4; 300 mosM), containing 2% glutaraldehyde.After glutaraldehyde fixation for 5 min, the cells were scrapedfrom the dishes with a rubber scraper and centrifuged at 10,000x g for 5 min in an Eppendorf microcentrifuge. The cells wereallowed to sit undisturbed as a pellet in the tube for an additionalhour. The supernatant was removed with a pipette, and the cellswere dried with a pointed piece of filter paper. The tip ofthe tube was cut off with a razor blade, and the pellet wasplaced onto Parafilm, where it was further dried to a thickpaste. It was then divided into clumps (0.5 mm³), and each clumpwas encased in molten agar cooled to 40°C. After the agarsolidified (10 min), the cell pellets were washed three times,each time for 3 min, with CS buffer. The cell pellets were postfixedwith 1% osmium tetroxide-0.1 M sodium cacodylate buffer for1 h. They were washed twice, each time for 10 min, in CS buffer.Two additional 10-min washes were performed with 0.11 M sodiumveronal acetate buffer, pH 7.4. Following en bloc staining with1% uranyl acetate-0.11 M sodium veronal acetate buffer for 1h, the pellets were washed with 0.11 M sodium veronal acetatebuffer, pH 7.4, for 5 min and then briefly with distilled water.The pellets were dehydrated in a graded series of ethanol (10min per wash), 30, 60, 80, and 95%, concluding with three finalwashes in 100% ethanol for 10 min. The pellets were infiltratedwith a mixture of 50% Spurr's resin in absolute ethanol for1 h followed by five changes of 100% resin for 10 min each.The resin was polymerized by baking it at 70°C for 8 h.Ultrathin sections were cut on a Reichert-Jung ultracut E microtomeusing a diamond knife. They were poststained in aqueous saturateduranyl acetate for 5 min, washed in water, and further stainedin lead citrate for 2 min. After a final wash in water, thesections were viewed and photographed in a Philips EM300 electronmicroscope.

Nucleotide sequence accession numbers. The nucleotide sequences of the EcoRV fragments of the PstIN fragment of VV-WR DNA and the corresponding region of CPV-BRDNA reported in this article have been assigned GenBank accessionnumbers AF464893 and AF464894.

RESULTS

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Results

Identification of vaccinia and cowpox virus p4c genes. Previous analyses of the nucleotide sequence of the gene immediatelyupstream of the ati gene in CPV-BR identified part of an openreading frame (Fig. 1, orf b) with the potential to encode aprotein containing 28 consecutive aspartic acids (52). Furthersequence analysis revealed that this open reading frame lacksan obvious transcriptional promoter region upstream of the predictedinitiation codon. In addition, it narrowly overlaps an upstreamopen reading frame (Fig. 1, orf a) which begins with the sequenceTAAATG, the consensus nucleotide sequence for the transcriptionalstart site of late promoters of poxviruses (7, 56, 81). An insertionof a single base in the overlapping region can put these twocoding regions in frame. These features suggested that the twoopen reading frames might be remnants of a single late genepredicted to encode a 58-kDa protein, possibly a virion protein,because many late proteins are structural components of thevirus particles. These properties suggested that the late genemight encode the P4c protein, because this is a structural proteinof about 60 kDa known to be present in some, but not all, strainsof cowpox virus (31, 67). Furthermore, although this proteinhad been identified in the virions of orthopoxviruses of severaltypes, its gene had not been identified. In the genome of CPV-BR,as recently described (GenBank accession number AF482758), openreading frame a corresponds to open reading frame CPXV161, andthe part of open reading frame b downstream of the first potentialinitiation codon corresponds to open reading frame CPXV159.

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FIG. 1. The p4c gene is intact in the genome of VV-WR but disrupted in the genome of CPV-BR. The rectangles correspond to the open reading frames of the ati, p4c, and 14K genes, where mutant alleles are designated ati1 and p4c1. The arrows indicate the position of the first potential initiation codon within each open reading frame. Each of these initiation codons, except for that in orf b of p4c1, is contained within a predicted late promoter element. The solid rectangle corresponds to the position of the 28 GAT repeat sequence predicted to encode a 28-aspartic-acid repeat (52). The scale is in base pairs.

The P4c protein was first described in VV-WR (40, 61). To determineif the VV-WR gene encoding the P4c protein is located in thecorresponding position (upstream of the ati locus) in the vacciniavirus genome, we first analyzed the DNA sequence of this region.This analysis identified an open reading frame predicted toencode a 58-kDa protein corresponding to the hypothetical productof an in-frame linkage of the two overlapping open reading framesin the cowpox virus DNA (Fig. 1). A specific genomic numberhas not been assigned to this open reading frame, because itsnumerical position among the open reading frames in the VV-WRgenome is not known.

Analyses of the protein components of purified virions of VV-WRand CPV-BR confirmed both the presence of a protein with theexpected properties (relative abundance and electrophoreticmobilities) of the P4c protein in the vaccinia virions and itsabsence from the cowpox virions. A continuous phosphate-bufferedelectrophoresis system similar to those used originally to identifythe P4c protein resolves the P4c protein (apparent molecularmass, 61 kDa) from the 4a and 4b proteins in the vaccinia virussample (Fig. 2A, lane 2). The P4c protein is not present amongproteins of cowpox virus (Fig. 2A, lane 1). This differenceis also apparent if the proteins are resolved by electrophoresisusing the discontinuous buffer system described by Laemmli (43).However, in that system, the relative electrophoretic mobilitiesof the 4a, 4b, and P4c proteins are altered such that, for example,the 4a protein migrates with an apparent molecular mass of 61kDa versus 59 kDa for the P4c protein (Fig. 2B, lane 2). Thesealterations in the relative electrophoretic mobilities of the4a, 4b, and P4c proteins in different electrophoresis buffersystems are distinguishing characteristics of these proteins(51).

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FIG. 2. The P4c protein is present in VV-WR particles but absent from particles of CPV-BR. Proteins present in virus particles (16 µg per sample), purified as described previously(36), were characterized as follows. (A) Virus particles were solubilized by boiling them for 3 min in a buffer containing 0.1 M sodium phosphate (pH 7.2), 6 M urea, 1 mM EDTA, 2% SDS, 5% ß-mercaptoethanol, and 10% glycerol. The solubilized proteins were resolved by electrophoresis in a 10% polyacrylamide gel containing 0.1 M sodium phosphate, 6 M urea, 1 mM EDTA, and 0.1% SDS (61) and visualized with Coomassie brilliant blue R-250 stain. (B) Virus particles were solubilized by boiling them for 3 min in a buffer containing 50 mM Tris (pH 7.0), 1% SDS, 30 mM dithiothreitol, 5 mM EDTA, and 10% glycerol. The solubilized proteins were resolved by electrophoresis in a 10% polyacrylamide gel using a discontinuous buffer system as described previously (43) and visualized with Coomassie brilliant blue R-250 stain. Lanes 1, proteins of CPV-BR; lanes 2, proteins of VV-WR; lanes 3, protein markers (in kilodaltons).

To confirm that the gene identified in the vaccinia virus genomeencodes the P4c protein, a recombinant vaccinia virus (A504)was constructed in which this gene was disrupted by the insertionof a DNA fragment containing the gpt gene under the transcriptionalcontrol of an early poxvirus promoter (11). Analysis of theproteins of purified virus particles showed that the insertionalinactivation of the predicted p4c gene in vaccinia virus A504led to the production of virus particles lacking the P4c protein(Fig. 3A, lane 3). The A504 virus particles also contained an $~$ 34-kDa protein (Fig. 3B, lane 3) that may be the product ofthe disrupted p4c gene, corresponding to the N-terminal portionof the P4c protein encoded by the region upstream of the insertionpoint of the gpt gene.

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FIG. 3. Insertional inactivation of the p4c gene leads to loss of intact P4c protein in the purified virus. Proteins present in virus particles (16 µg per sample), purified as described previously(36), were characterized as described in the legend to Fig. 2. (A) Solubilized proteins were resolved by electrophoresis in an 8% polyacrylamide gel containing a continuous phosphate buffer system (61). (B) Solubilized proteins were resolved by electrophoresis in a 12.5% polyacrylamide gel using a discontinuous buffer system as described previously (43). Lanes 1, protein markers (sizes in kilodaltons); lanes 2, proteins of VV-WR; lanes 3, proteins of vaccinia virus A504; lanes 4, proteins of CPV-BR. The arrow indicates the position of an $~$ 34-kDa protein present only in vaccinia virus A504 (panel B, lane 3).

The expression of the p4c gene can also be detected in virus-infectedcells pulse-labeled with [³⁵S]methionine during the late phaseof virus replication, when most newly synthesized proteins arevirus encoded. In the absence of a chase period, the nascentP4a and P4b proteins are not proteolytically processed to theforms 4a and 4b, which can obscure the P4c protein in one-dimensionalgel electrophoretic analyses. The expression of viral proteinsin cells infected with different orthopoxviruses that containor lack the predicted p4c gene is shown in Fig. 4. A late proteinwith electrophoretic mobilities identical to those of the virionP4c protein is synthesized in cells infected with VV-WR (lane1), absent in cells infected with vaccinia virus A504 (lane2), and present in cells infected with vaccinia virus A507,a revertant of virus A504 (lane 3). An $~$ 34-kDa protein that maybe the product of the disrupted p4c gene is also evident amongproteins synthesized in cells infected with vaccinia virus A504(lane 2) but not the revertant virus A507 (lane 3). As expected,CPV-BR does not direct the synthesis of a 59-kDa P4c protein(lane 5), but cowpox virus A505, containing a copy of the vacciniavirus p4c gene including its predicted promoter region, does(lane 4).

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FIG. 4. Expression of the P4c protein in cells infected with recombinant vaccinia and cowpox viruses. Human 143B cells were infected and metabolically labeled with [³⁵S]methionine for 30 min 18 h after infection, as described in Materials and Methods. The solubilized labeled proteins were resolved by electrophoresis in a 12.5% polyacrylamide gel using a discontinuous buffer system as described previously (43) and visualized by autoradiography of the dried gel. The lanes contain proteins from cells infected with VV-WR (lane 1), vaccinia virus A504 (lane 2), vaccinia virus A507 (lane 3), cowpox virus A505 (lane 4), and CPV-BR (lane 5). Lane 6 contains protein markers (in kilodaltons).

Collectively, these results show that the open reading framesimmediately upstream of the ati genes in vaccinia and cowpoxviruses are the coding regions of the p4c genes in these viruses.In CPV-BR, we designated this gene p4c1 to indicate that thisis a variant of the wild-type p4c gene.

The P4c protein facilitates the formation of V⁺ ATI bodies. The identification of the p4c gene allowed us to test the hypothesisthat the P4c protein is required for V⁺ ATI formation. The hypothesispredicts that orthopoxviruses lacking a functional p4c genewill have a V^- phenotype, whereas orthopoxviruses containinga functional p4c gene will have a V⁺ phenotype.

First, we used complementation assays, as described by Ichihashiand Matsumoto (29), to examine these predictions. Cells wereinfected with one or two strains of poxviruses. Eighteen hourslater, the cells were fixed, stained, and examined by electronmicroscopy to identify the phenotype of the ATIs present inthe cytoplasm. The ATIs of at least 30 cells were examined ineach sample. If all of the ATIs were V^-, the phenotype of thevirus was scored as V^-. The phenotype of the virus was scoredas V⁺ if any of the ATIs were V⁺, though in practice, infectionof a cell with a V⁺ virus resulted in the production of ATIsof which >90% contained virus particles. Ichihashi and Matsumoto(29) similarly noted the dominance of the inclusion of particlesover the exclusion of particles in complementation assays.

Complementation assays were initially used to determine theV phenotypes of two strains of vaccinia virus whose genotypeswith respect to both the p4c gene and the ati gene are known.VV-WR contains the ati1 gene and the intact p4c gene. The vacciniavirus ati1 gene produces a variant of the cowpox virus ati genecontaining a stop codon that results in the synthesis of a truncated94-kDa version of the ATI protein that does not form ATIs (1,8, 53). Vaccinia virus strain Copenhagen contains partiallydeleted variants of the ati and p4c genes. Major portions ofboth genes are missing so that the 5' end of the residual portionof the p4c gene is fused to the 3' end of the ati gene, generatinga fusion gene, ${Phi}$ (p4c'- ${Delta}$ ati), containing the A26L open readingframe (14). CPV-BR contains the ati and p4c1 genes, where theati gene is functional, producing a 160-kDa protein that formsATIs (52, 53), and the p4c1 gene is the disrupted form of thep4c gene shown in Fig. 1. Human 143B cells were infected withVV-WR or vaccinia virus Copenhagen, either alone or togetherwith CPV-BR. As expected, neither VV-WR nor vaccinia virus Copenhagenproduced ATIs (Fig. 5A and C). Coinfection of VV-WR (ati1 p4c)with CPV-BR (ati p4c1) resulted in the formation of V⁺ ATIs(Fig. 5B), whereas the coinfection of vaccinia virus Copenhagen[ ${Phi}$ (p4c'- ${Delta}$ ati)] with CPV-BR (ati p4c1) resulted in the formationof V^- ATIs (Fig. 5D).

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FIG. 5. VV-WR expressing the p4c gene can provide a factor needed to direct the inclusion of IMV within ATIs. Human 143B cells were infected with 10 PFU of one strain of poxvirus/cell (A and C) or with 5 PFU each of two strains of poxvirus/cell (B and D). Eighteen hours after the cells were infected, they were fixed, stained, sectioned, and examined by electron microscopy to identify the phenotype of the ATIs present in the infected cells. Shown are sections of cells infected with VV-WR (p4c ati1) (A); VV-WR and CPV-BR (p4c1 ati), where the large mass is a V⁺ ATI (B); vaccinia virus Copenhagen [ ${Phi}$ (p4c'- ${Delta}$ ati)] (C); and vaccinia virus Copenhagen and CPV-BR, where the large mass is a V^- ATI (D). Bars, 1,000 nm.

Insertional inactivation of the p4c gene in VV-WR, producingvaccinia virus A504, abrogated the ability of this virus toinduce the formation of V⁺ ATIs in coinfections with CPV-BR(Fig. 6B). A revertant of this virus, vaccinia virus A507, whichwas generated by repairing the disrupted p4c gene of vacciniavirus A504 by recombination with the wild-type p4c gene in plasmidp1542, regained the ability both to express the P4c protein(Fig. 4, lane 3) and to induce the formation of V⁺ ATIs in cellscoinfected with CPV-BR (Fig. 6D).

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FIG. 6. The p4c gene is required for the formation of V⁺ ATIs in cells coinfected with VV-WR and CPV-BR. Human 143B cells were infected with either 10 PFU of one strain of poxvirus/cell (A and C) or 5 PFU each of two strains of poxvirus/cell (B and D). Eighteen hours after the cells were infected, they were fixed, stained, sectioned, and examined by electron microscopy. Shown are sections of cells infected with vaccinia virus A504 (p4c::gpt ati1) containing a p4c gene disrupted by the insertion of a selectable marker gene, the gpt gene (A); vaccinia virus A504 and CPV-BR (p4c1 ati), where the large masses are V^- ATIs (B); vaccinia virus A507 (p4c ati1), which is a revertant of vaccinia virus A504 containing an intact p4c gene (C); and vaccinia virus A507 and CPV-BR, where the large masses are V⁺ ATIs (D). Bars, 1,000 nm.

These results predicted that a recombinant CPV-BR expressingthe VV-WR p4c gene would have a V⁺ phenotype. To test this hypothesis,we examined the phenotype of recombinant cowpox virus A505,which contains a functional copy of the VV-WR p4c gene underthe transcriptional control of its endogenous promoter. Thisp4c gene was recombined into the thymidine kinase gene of thecowpox virus as described in Materials and Methods, generatingcowpox virus A505 (ati p4c1 p4c), which expresses the vacciniavirus P4c protein (Fig. 4, lane 4). As shown in Fig. 7, theinsertion of the functional p4c gene into the genome of CPV-BRresulted in the embedding of virus particles within the ATIs.

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FIG. 7. Insertion of the vaccinia virus p4c gene into the genome of CPV-BR is sufficient to convert the phenotype of the ATIs from V^- to V⁺. Human 143B cells were infected with 10 PFU of cowpox virus/cell. Eighteen hours after infection, the cells were fixed, stained, sectioned, and examined by electron microscopy. Shown are sections of cells infected with CPV-BR (p4c1 ati), which produces V^- ATIs (A), and cowpox virus A505 (p4c1 p4c ati), which is a recombinant cowpox virus containing the VV-WR p4c gene under the control of its own promoter (B). ATIs in cells infected with cowpox virus A505 have a V⁺ phenotype. Bars, 1,000 nm.

DISCUSSION

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Discussion

This study has identified the orthopoxvirus p4c gene and confirmedthat one of the roles of this gene is to direct the inclusionof IMV particles within the ATIs.

The p4c gene is highly conserved among most if not all speciesof orthopoxvirus, including those that do not produce ATIs.For example, the p4c gene is present in the genomes of strainsof variola virus major and variola virus minor that have beencharacterized by nucleotide sequence analysis (46, 64, 66),where the protein (encoded by open reading frame A29L or A30L,depending on the virus strain) shows $~$ 94% (473 of 500 amino acids)identity with the vaccinia virus P4c protein. The p4c gene ispresent as open reading frame A28L in monkeypox virus strainZaire-96-I-16, where it is predicted to encode a protein with $~$ 91% (476 of 520 amino acids) identity to the vaccinia virusP4c protein (65). It is present in camelpox virus M-96 (19),where it is predicted to show 95% identity (476 of 500 aminoacids) to the vaccinia virus P4c protein. It is also presentin a variety of characterized VV-WR isolates, as well as theIHD-J strain of vaccinia virus (72). These data are consistentwith those of early phenotypic analyses showing that 18 differentstrains of cowpox, vaccinia, ectromelia, variola, and monkeypoxviruses each expressed an inclusion factor capable of directingthe formation of V⁺ ATIs during coinfection with a V^- cowpoxvirus (27). The high degree of sequence conservation of thep4c gene among the various types of orthopoxvirus, irrespectiveof whether the virus produces ATIs, suggests that the P4c proteinis advantageous for viral replication in vivo, even in the absenceof ATIs. The disruption of the p4c gene in many of the well-characterizedstrains and isolates of vaccinia virus, such as the Copenhagenstrain (14), the MVA strain (2), the Tian Tan strain (GenBankaccession number AF095689), and at least one isolate of theWR strain (1), may reflect the passage histories of these viruses.Whereas the expression of the p4c gene during replication invivo may be advantageous to the virus, this may not be the casefor virus replication in tissue culture.

The predicted sequences of the P4c proteins yield few insightsinto the structures and functions of these proteins. The PSORTII algorithm (50) predicts one potential transmembrane portionbetween residues 112 and 130, with the N-terminal portion ofthe protein predicted to be within the inner side of the IMVmembrane. Consistent with this model, a novel protein of about34 kDa, which may correspond to the predicted 37-kDa productof the insertionally inactivated p4c gene (equivalent to theN-terminal portion of the P4c protein), is present both in thepurified particles of A504 virus (Fig. 3B, lane 3) and amongproteins synthesized during the late phase of replication ofA504 (Fig. 4, lane 2). This model suggests that the COOH-terminalportion of the P4c protein is required to facilitate the embeddingof IMV particles within the ATIs. In this context, two featuresof the COOH-terminal regions of the P4c proteins are noteworthy.First, this portion of the cowpox virus P4c protein containsthe region predicted to contain 28 consecutive aspartic acidresidues. A similarly positioned tract of 23 aspartic acidsis present in the predicted sequence of the monkeypox virusP4c protein (65). However, this region is absent from the P4cproteins of variola virus and vaccinia virus, showing that thispolyaspartate tract is not required for the V⁺ phenotype. Thepresence or absence of the polyaspartate tract will affect themolecular masses of the P4c proteins, giving rise to P4c proteinsthat are slightly larger in cowpox and monkeypox viruses thanin some of the other orthopoxviruses. Second, the amino acidsequence of a region near the COOH terminus of the VV-WR P4cprotein (441-CCDTAAVDRLEHHIETLGQYAVILARKINMQT-472) shows 44%identity (underlined amino acids) with the segment of the 14Kfusion protein that binds to the 21-kDa protein (the A17L proteinin VV-WR) in the membrane of the IMV (78). Whether this region,or another region of the P4c protein, mediates an associationbetween the P4c protein and other proteins in the IMV membraneremains to be determined. Ichihashi (25) previously reportedthat the P4c protein associates via a disulfide linkage witha 16-kDa viral membrane protein.

The presence of the P4c protein on the surface of the IMV isconsistent with its role in the process that places virus particlesinto the ATIs. However, the mechanism involved in this processhas yet to be determined. Ulaeto et al. (76) have shown thata 59-kDa protein is a specific marker for IMV of vaccinia virus(IHD-J strain). Antibodies against the 59-kDa IMV protein specificallyprecipitated the P4c protein encoded by cowpox virus A505, confirmingthe coidentity of the 59-kDa and P4c proteins in these two studies.The P4c protein was not detected in EEV, leading Ulaeto andcolleagues to suggest that virion acquisition of the P4c proteinmarks this particle for maturation as an IMV, presumably eitherby the prevention of processes leading to the formation of IEVparticles or by the facilitation of an alternative maturationpathway.

The IEV particles are produced after anterograde microtubule-mediatedtransport of IMVs to the sites where they become enveloped bymembranes of the trans-Golgi network or tubular endosomes. Thisis a process that is mediated by direct or indirect interactionsbetween the 14K protein and kinesin motors (59). Subsequently,the IEVs are propelled toward the plasma membrane by kinesin-dependentmovement on microtubules, finally being propelled to and throughthe plasma membrane by actin bundles (5, 13, 21, 54, 55, 58,77, 80). In contrast, the IMVs are either localized within thecytoplasm, often in the perinuclear region, or embedded withinATIs.

For the same reasons that active transport mechanisms are neededto move IMVs and IEVs to the plasma membrane, similar mechanismsmay be necessary for the retrograde transport of both IMVs andthe viral ATI proteins to the sites where the IMVs and ATIscolocalize. Several features of the cytoskeleton, the IMV, andATIs are consistent with such a model. First, the use of microfilamentsto move virus particles in one direction suggests that the virusmay use the microfilaments to move in the opposite direction,because movement of cargoes (virus particles, organelles, mRNAs,protein, and nucleoprotein complexes) by kinesin superfamilyproteins, myosins, and dyneins along a microfilament can occurin either the forward or reverse direction, depending on thenature of the motor protein (reviewed in references 15 and 38).Viruses of several types, including poxviruses, are now knownto employ microfilament-mediated transport mechanisms to movesubviral components in anterograde or retrograde directions(54), with the herpesviruses providing prime examples of virusesthat employ both modes of transport to effect either entry oregress from the infected cell (reviewed in reference 69). Second,when P4c is absent in cells containing ATIs, there is a clearsegregation of IMVs from the V^- ATIs, with IMVss typically observedclose to the ATIs but not within the ATIs themselves (Fig. 5to 7). However, in cells containing both P4c and ATIs, mostof the IMVs are observed within the ATIs in a cytoplasm containingimmature virus particles but largely devoid of IMVs. Similardistributions are evident in micrographs of previous studiesof V⁺ and V^- ATIs (23, 26, 29, 30, 67). Indeed, in some previousstudies, particularly those involving fusions between cellscontaining V^- ATIs and cells containing virus expressing "inclusionfactor," it was common to find a special type of ATI, designatedV⁺' ATIs, in which the ATI lacks internal IMVs but is completelyencrusted with IMVs on its surface within a field of cytoplasmdevoid of IMVs (67). Furthermore, in these assays yielding V⁺ATIs, all ATIs in the cell were V⁺ or V⁺' (29, 67). These featuresare consistent with a mechanism involving the active transportof P4c-containing IMVs to the ATI. Such a mechanism could efficientlysegregate IMVs containing P4c from IMVs lacking P4c and destinedto become IEV, consistent with the results of Ulaeto et al.(76). Third, the ATIs themselves resemble aggresomes, whichare microtubule-dependent cytoplasmic inclusion bodies thatare metabolically stable, insoluble aggregates of proteins commonlyfound in cells within lesions in a variety of degenerative diseases(reviewed in references 35 and 42).

According to this model, one function of the P4c protein inorthopoxviruses, including those such as variola virus thatencode a P4c protein but not an Ati protein, may be to directthe retention of most progeny viruses as IMVs rather than IEVs,CEVs, or EEVs. Retention of IMVs within ATIs takes this sequestrationone step further. A mechanism to promote the intracellular retentionof progeny virus as IMV particles may seem to be counterproductive,because during the early phase of an infection in vivo, EEVsmay be the most efficient particles for dissemination of thevirus in the host (reviewed in reference 68). However, IMV,as the more robust particle, may be more important than EEVfor transmission of the virus from host to host (77). In addition,even for transmission within a host, as adaptive immune responsescome into play, the dissemination of cells containing virus,rather than the transport of free virus, may become the moreimportant mode of virus dissemination (reviewed in reference12). This form of viral transport, which has important ramificationsfor viral pathogenesis, could be facilitated by the productionof the P4c protein.

The identification of the p4c gene will allow these models ofthe potential roles of the P4c protein in viral morphogenesisand pathogenesis to be tested.

ACKNOWLEDGMENTS

We are grateful to Susan Hester for her help in preparing theelectron micrographs. The shared core facilities of the DukeUniversity Comprehensive Cancer Center were used in this study.

This work was supported by grants AI32982, 2T32CA09111-16A10051,and 5T32GM07184-14 021 (T.A.M.) from the National Institutesof Health.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, Box 3020, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-2480. Fax: (919) 684-8735. E-mail: picku001{at}mc.duke.edu.

REFERENCES

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