Vaccinia Virus Cores Research Articles

Identification and Characterization of a Cleavage Site in the Proteolysis of Orf Virus 086 Protein.

The orf virus (ORFV) is among the parapoxvirus genus of the poxviridae family, but little is known about the proteolytic pathways of ORFV encoding proteins. By contrast, the proteolysis mechanism of the vaccinia virus (VV) has been extensively explored. Vaccinia virus core protein P4a undergoes a proteolytic process that takes place at a conserved cleavage site Ala-Gly-X (where X is any amino acid) and participates in virus assembly. Bioinformatics analysis revealed that an ORFV encoding protein, ORFV086, has a similar structure to the vaccinia virus P4a core protein. In this study, we focus on the kinetic analysis and proteolysis mechanism of ORFV086. We found, via kinetic analysis, that ORFV086 is a late gene that starts to express at 8 h post infection at mRNA level and 12–24 h post infection at the protein level. The ORFV086 precursor and a 21 kDa fragment can be observed in mature ORFV virions. The same bands were detected at only 3 h post infection, suggesting that both the ORFV086 precursor and the 21 kDa fragment are viral structural proteins. ORFV086 was cleaved from 12 to 24 h post infection. The cleavage took place at different sites, resulting in seven bands with differing molecular weights. Sequence alignment revealed that five putative cleavage sites were predicted at C-terminal and internal regions of ORFV086. To investigate whether those cleavage sites are involved in proteolytic processing, full length and several deletion mutant ORFV086 recombinant proteins were expressed and probed. The GGS site that produced a 21 kDa cleavage fragment was confirmed by identification of N/C-terminal FLAG epitope recombinant proteins, site-directed mutagenesis and pulse-chase analysis. Interestingly, chase results demonstrated that, at late times, ORFV086 is partially cleaved. Taken together, we concluded that GGS is a cleavage site in ORFV086 and produces a 21 kDa fragment post infection. Both ORFV086 precursor and the 21 kDa fragment are structural proteins of mature ORFV virions. ORFV086 and its cleaved products are indispensable for correct assembly of mature viral particles and this proteolytic processing of ORFV086 may play an essential role in viral morphogenic transition.

Isolation and Characterization of Monoclonal Antibodies Against a Virion Core Protein of Orf Virus Strain NA1/11 As Potential Diagnostic Tool for Orf Viruses.

Orf is caused by the orf virus (ORFV) and is a non-systemic, widespread disease afflicting sheep, goats, wild ruminants, and humans. Recent outbreaks in sheep and goats in Jilin and other northern Chinese provinces raise concerns about orf control in China. Thirty-five hybridoma clones were constructed from splenocytes of BALB/c mice immunized with natural orf virus protein. These hybridomas were used to produce antibodies targeting ORFV proteins. Immunological characterization of these monoclonal antibodies (MAb) showed that the 5F2D8 hybridoma line produced MAb that can recognize the 100, 70, and 20 kDa bands from total viral lysate. This hybridoma was further characterized by immunoprecipitation and peptide sequencing. The results indicate that 5F2D8 specifically recognizes orf virus encoded protein ORFV086, a late expression virion core protein that plays important roles in progeny virus particle assembly, morphogenesis, and maturity. Further experiments demonstrate that this MAb did not react with other viral proteins of ORFV orthopoxviruses, but reacted strongly to different field isolates of orf viruses from China. Additionally, this anti-ORFV086 MAb possesses ORFV neutralizing capability. Sequence alignments and phylogenetic analysis determined that ORFV086 of NA1/11, clustered together with NZ2 and IA82, is highly conserved and has structural similarities with the Vaccinia virus core protein P4a. As such, this MAb has great potential as a diagnostic tool for orf viruses, in the further exploration of orf pathogenesis, and in disease control and prevention.

Encephalitis in the Pediatric Population

1. Stephen J. Falchek, MD* 1. *Division of Pediatric Neurology, Alfred I. duPont Hospital for Children/Thomas Jefferson University, Wilmington, DE, and Philadelphia, PA. * Abbreviations: ADEM: : acute disseminated encephalomyelitis CEP: : California Encephalitis Project CNS: : central nervous system CPP: : cerebral perfusion pressure CSF: : cerebrospinal fluid HSV: : herpes simplex virus IVIG: : intravenous immune globulin MS: : multiple sclerosis PCR: : polymerase chain reaction WNV: : West Nile virus Management of encephalitis, which can be fatal, requires understanding of a broad range of causative agents, pathophysiologic mechanisms, clinical syndromes, and outcomes. After completing this article, readers should be able to: 1. Know the many causes of encephalitis, including infectious, parainfectious, and noninfectious disorders. 2. Recognize the clinical features of encephalitis. 3. Be able to conduct a diagnostic investigation of a patient with encephalitis by using clinical and laboratory criteria. 4. Know the anticipated clinical course of different types of encephalitis. 5. Be familiar with the modalities used to treat encephalitis. 6. Understand the factors that influence the outcome in a patient who has encephalitis. The broad definition of the term “encephalitis,” that is, inflammation of the brain , necessitates acknowledgment of the enormous inclusivity of the topic. The most common interpretation of the term implies a direct invasion of the brain by an infectious pathogen, most commonly, viral, fungal, or parasitic. The topic also includes examples of meningitis mediated by bacteria or other agents, which can produce extrameningeal symptoms such as lethargy or seizures, in which case, the combined term “meningoencephalitis” is used. There are also many examples of encephalitis not due to direct central nervous system (CNS) infections. Inflammatory processes due to an acute or chronic illness can result in an acute immune-mediated encephalitis, such as acute disseminated encephalomyelitis (ADEM), lupus cerebritis, and paraneoplastic syndromes. Agents or conditions that produce slowly progressive CNS symptoms, such as tertiary syphilis or “slow viruses” (the prion protein encephalopathies), also are considered examples of encephalitis. Table 1 lists only a limited number of the many pathogens and pathologic conditions that can cause either acute or subacute encephalitis. In this discussion, …

Vaccinia virus early gene transcription termination factors VTF and Rap94 interact with the U9 termination motif in the nascent RNA in a transcription ternary complex

The vaccinia virus core contains a 195 kb double stranded DNA genome, a multi-subunit RNA polymerase, transcription initiation and termination factors and mRNA processing enzymes. Upon infection, vaccinia virus early gene transcription takes place in the virus core. Transcription initiates at early promoters and terminates in response to a termination motif, UUUUUNU, in the nascent mRNA. Early gene transcription termination requires the vaccinia virus termination factor, VTF, a single stranded DNA-dependent ATPase, and NPH I, the Rap94 subunit of the virion RNA polymerase, as well as the presence of the UUUUUNU motif in the nascent RNA. The position of UUUUUNU in the ternary complex suggests that it serves as a site of interaction with one or more components of the transcription termination complex. In order to identify the factor(s) that interact with UUUUUNU a series of direct UV photo crosslinking and ribonuclease A protection studies were undertaken. Through these analyses both VTF and Rap94 were shown to interact with UUUUUNU in the isolated ternary complex. Evidence indicates that the interaction is not mutually exclusive. VTF was shown to bind to UUUUUNU through the N-terminal domain of the large D1 subunit. Furthermore, VTF protects from RNAse A digestion both the 5′ region of the nascent transcript as well as a large central component containing UUUUUNU. The addition of an oligonucleotide containing the 5BrU9 sequence both directly inhibits transcription termination, in vitro and inhibits UV photo crosslinking of VTF to the nascent RNA in the ternary complex. These results support a model in which the availability of the UUUUUNU motif outside of the transcribing RNA polymerase permits binding of both transcription termination factors, VTF and Rap94, to UUUUUNU. The assembly of this termination complex initiates the transcription termination sequence.

Vaccinia virus A12L protein and its AG/A proteolysis play an important role in viral morphogenic transition

Like the major vaccinia virus (VV) core protein precursors, p4b and p25K, the 25 kDa VV A12L late gene product (p17K) is proteolytically maturated at the conserved Ala-Gly-Ala motif. However, the association of the precursor and its cleavage product with the core of mature virion suggests that both of the A12L proteins may be required for virus assembly. Here, in order to test the requirement of the A12L protein and its proteolysis in viral replication, a conditional lethal mutant virus (vvtetOA12L) was constructed to regulate A12L expression by the presence or absence of an inducer, tetracycline. In the absence of tetracycline, replication of vvtetOA12L was inhibited by 80% and this inhibition could be overcome by transient expression of the wild-type copy of the A12L gene. In contrast, mutation of the AG/A site abrogated the ability of the transfected A12L gene to rescue, indicating that A12L proteolysis plays an important role in viral replication. Electron microscopy analysis of the A12L deficient virus demonstrated the aberrant virus particles, which were displayed by the AG/A site mutation. Thus, we concluded that the not only A12L protein but also its cleavage processing plays an essential role in virus morphogenic transition.

Characterization of vaccinia virus A12L protein proteolysis and its participation in virus assembly

Vaccinia virus (VV) undergoes a proteolytic processing to evolve from immature virus particles into intracellular mature virus particles. Most of structural core protein precursors such as p4a, p4b, and p25K are assembled into previrions and then proteolytically processed to yield core proteins, 4a, 4b, and 25 K, which become components of a mature virus particle. These structural rearrangements take place at a conserved cleavage motif, Ala-Gly-X (where X is any amino acid) and catalyzed by a VV encoded proteinase, the I7L gene product. The VV A12L gene product, a 25 kDa protein synthesized at late times during infection is cleaved at an N-terminal AG/A site, resulting in a 17 kDa cleavage product. However, due to the distinct characteristics of A12L proteolysis such as the localization of both the A12L full-length protein and its cleavage product in mature virions and two putative cleavage sites (Ala-Gly-Lys) located at internal and C-terminal region of A12L ORF, it was of interest to examine the A12L proteolysis for better understanding of regulation and function of VV proteolysis. Here, we attempted to examine the in vivo A12L processing by: determining the kinetics of the A12L proteolysis, the responsible viral protease, and the function of the A12L protein and its cleavage events. Surprisingly, the A12L precursor was cleaved into multiple peptides not only at an N-terminal AG/A but also at both an N- and a C-terminus. Despite the involvement of I7L proteinase for A12L proteolysis, its incomplete processing with slow kinetics and additional cleavages not at the two AG/K sites demonstrate unique regulation of VV proteolysis. An immunoprecipitation experiment in concert with N-terminal sequencing analyses and mass spectrometry led to the identification of VV core and membrane proteins, which may be associated with the A12L protein and suggested possible involvement of A12L protein and its cleavage products in multiple stages in virus morphogenesis.

Vaccinia Virus Entry into Cells via a Low-pH-Dependent Endosomal Pathway

Previous studies established that vaccinia virus could enter cells by fusion with the plasma membrane at neutral pH. However, low pH triggers fusion of vaccinia virus-infected cells, a hallmark of viruses that enter by the endosomal route. Here, we demonstrate that entry of mature vaccinia virions is accelerated by brief low-pH treatment and severely reduced by inhibitors of endosomal acidification, providing evidence for a predominant low-pH-dependent endosomal pathway. Entry of vaccinia virus cores into the cytoplasm, measured by expression of firefly luciferase, was increased more than 10-fold by exposure to a pH of 4.0 to 5.5. Furthermore, the inhibitors of endosomal acidification bafilomycin A1, concanamycin A, and monensin each lowered virus entry by more than 70%. This reduction was largely overcome by low-pH-induced entry through the plasma membrane, confirming the specificities of the drugs. Entry of vaccinia virus cores with or without brief low-pH treatment was visualized by electron microscopy of thin sections of immunogold-stained cells. Although some virus particles fused with the plasma membrane at neutral pH, 30 times more fusions and a greater number of cytoplasmic cores were seen within minutes after low-pH treatment. Without low-pH exposure, the number of released cores lagged behind the number of virions in vesicles until 30 min posttreatment, when they became approximately equal, perhaps reflecting the time of endosome acidification and virus fusion. The choice of two distinct pathways may contribute to the ability of vaccinia virus to enter a wide range of cells.

Avipoxvirus phylogenetics: identification of a PCR length polymorphism that discriminates between the two major clades

Avipoxvirus infections have been observed in an extensive range of wild, captive and domesticated avian hosts, yet little is known about the genome diversity and host-range specificity of the causative agent(s). Genome-sequence data are largely restricted to Fowlpox virus (FWPV) and Canarypox virus (CNPV), which have been sequenced completely, showing considerable divergence between them. It is therefore proving difficult, by empirical approaches, to identify pan-genus, avipoxvirus-specific oligonucleotide probes for PCR and sequencing to support phylogenetic studies. A previous preliminary study used the fpv167 locus, which encodes orthologues of vaccinia virus core protein P4b (A3). PCR per se did not discriminate between viruses, but restriction-enzyme or sequence analysis indicated that the avipoxviruses clustered either with FWPV or with CNPV. Here, further study of the P4b locus demonstrated a third cluster, from psittacine birds. A newly identified locus, flanking fpv140 (orthologue of vaccinia virus H3L), confirms the taxonomic structure. This locus is particularly useful in that viruses from the fowlpox-like and canarypox-like clusters can be discriminated by PCR on the basis of fragment size, whilst sequence comparison allows discrimination for the first time between Pigeonpox virus and Turkeypox virus. Except within the psittacines, virus and avian host taxonomies do not show tight correlation, with viruses from the same species located in very different clades. Nor are all the existing recognized avipoxvirus species, defined primarily by avian host species (such as CNPV and Sparrowpox virus), resolved within the present structure.

A vaccinia virus lacking A10L: viral core proteins accumulate on structures derived from the endoplasmic reticulum

The assembly of the intracellular mature virus (IMV) of vaccinia virus (VV), the prototype member of the poxviridae, is poorly understood and controversial. We have previously proposed that the IMV is composed of a continuous double-membraned cisterna derived from the smooth ER, whereby the genome-containing core is enwrapped by a part of this cisterna. In the present study we characterize a mutant virus in which the synthesis of the major core protein A10L can be conditionally expressed. Without A10L, IMVs are not made; immature viruses (IVs) and regularly stacked membrane structures that contain viral DNA, accumulate instead. By immunolabelling of thawed cryo-sections these stacks contain most of the viral core proteins and low levels of viral membrane proteins. Importantly, the stacked membranes could be labelled with antibodies to an ER marker protein, implying that they are derived from this cellular compartment. By electron tomography (ET) on semi-thin cryo-sections we show that the membranes of the stacks are continuous with the membranes of the IVs. Direct continuities with ER cisternae, to which the stacks are tightly apposed, were, however, not unequivocally seen. Finally, ET revealed how the IV membranes separated to become two-membrane profiles. Taken together, this study shows that VV core proteins and the viral DNA can coassemble onto ER-derived membranes that are continuous with the membranes of the IVs.

Development of an in vitro cleavage assay system to examine vaccinia virus I7L cysteine proteinase activity

Through the use of transient expression assays and directed genetics, the vaccinia virus (VV) I7L gene product has been implicated as the major maturational proteinase required for viral core protein cleavage to occur during virion assembly. To confirm this hypothesis and to enable a biochemical examination of the I7L cysteine proteinase, an in vitro cleavage assay was developed. Using extracts of VV infected cells as the source of enzyme, reaction conditions were developed which allowed accurate and efficient cleavage of exogenously added core protein precursors (P4a, P4b and P25K). The cleavage reaction proceeded in a time-dependent manner and was optimal when incubated at 25°C. I7L-mediated cleavage was not affected by selected inhibitors of metalloproteinases, aspartic acid proteinases or serine proteinases (EDTA, pepstatin, and PMSF, respectively), but was sensitive to several general cysteine proteinase inhibitors (E-64, EST, Iodoacetic acid, and NEM) as well as the I7L active site inhibitor TTP-6171 [C. Byrd et al., J. Virol. 78:12147–12156 (2004)]. Finally, in antibody pull down experiments, it could be demonstrated that monospecific αI7L serum depleted the enzyme activity whereas control sera including αG1L, directed against the VV metalloproteinase, did not. Taken together, these data provide biochemical evidence that I7L is a cysteine proteinase which is directly involved in VV core protein cleavage. Furthermore, establishment of this I7L-mediated in vitro cleavage assay should enable future studies into the enzymology and co-factor requirements of the proteolysis reaction, and facilitate antiviral drug development against this essential target.

Role of the I7 protein in proteolytic processing of vaccinia virus membrane and core components.

Certain core and membrane proteins of vaccinia virus undergo proteolytic cleavage at consensus AG/X sites. The processing of core proteins is coupled to morphogenesis and is inhibited by the drug rifampin, whereas processing of the A17 membrane protein occurs at an earlier stage of assembly and is unaffected by the drug. A temperature-sensitive mutant with a lesion in the I7L gene exhibits blocks in morphogenesis and in cleavage of core proteins. We found that the mutant also failed to cleave the A17 membrane protein. To further investigate the role of the putative I7 protease, we constructed a conditional lethal mutant in which the I7L gene was regulated by the Escherichia coli lac repressor. In the absence of an inducer, the synthesis of I7 was repressed, proteolytic processing of the A17 membrane protein and the L4 core protein was inhibited, and virus morphogenesis was blocked. Under these conditions, expression of the wild-type I7 protein in trans restored protein processing. In contrast, rescue did not occur when the putative protease active site residue histidine 241 or cysteine 328 of I7 was converted to alanine. The mutation of an authentic AG/A and an alternative AG/S motif of L4 prevented substrate cleavage. Similarly, when AG/X sites of A17 were mutated, I7-induced cleavages at the N and C termini failed to occur. In conclusion, we provide evidence that I7 is a viral protease that is required for AG/X-specific cleavages of viral membrane and core proteins, which occur at early and late stages of virus assembly, respectively.

UUUUUNU oligonucleotide inhibition of RNA synthesis in vaccinia virus cores

Recent results from this laboratory demonstrated the ability of U5NU-containing oligonucleotides to stimulate premature termination of early gene transcription in vitro. Further studies on the oligonucleotide sequence and structural requirements for stimulating premature termination demonstrated that only oligonucleotides possessing ribouracil U9 with a phosphodiester linkage are active. Because an oligonucleotide as short as 9 bases serves as an effective stimulator of premature transcription termination, we reasoned that short U5NU-containing oligonucleotides might serve as efficacious anti-poxvirus agents because they would prevent the synthesis of full-sized early mRNA. To be useful in vivo, the oligonucleotides must not only be taken up by the infected cells, but also be able to enter the virus core, the site of early gene transcription, and retain their ability to stimulate premature termination. The ability of U9-containing oligonucleotides to inhibit virus core RNA synthesis was evaluated. The U5NU oligonucleotides exhibited a dramatic sequence-specific inhibition of core RNA synthesis, consistent with their ability to stimulate premature termination of early gene transcription. Moreover, the concentration of U5NU oligonucleotide required to exhibit half maximal inhibition of RNA synthesis was found to be less for a 9 mer RNA than it was for a 17 or 22 mer RNA. This suggests the possibility that the smaller oligonucleotides may have easier access to the core. This observation lends support to the notion that such oligonucleotides might serve as effective anti-poxvirus therapeutic agents.

Vaccinia virus cores are transported on microtubules.

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

The vaccinia virus I7L gene product is the core protein proteinase.

Maturation of vaccinia virus (VV) core proteins is required for the production of infectious virions. The VV G1L and I7L gene products are the leading candidates for the viral core protein proteinase (vCPP). Using transient-expression assays, data were obtained to demonstrate that the I7L gene product and its encoded cysteine proteinase activity are responsible for vCPP activity.

Microtubule-dependent Organization of Vaccinia Virus Core–derivd Early mRNAs into Distinct Cytoplasmic Structures

Vaccinia virus (vv) early transcription can be reconstituted in vitro from purified virions; in this assay mRNAs are made inside the viral core and subsequently extruded. Although the in vitro process has been extensively characterized, relatively little is known about vv early transcription in vivo. In the present study the fate of vv early mRNAs in infected HeLa cells was followed by BrUTP transfection and confocal and electron microscopy. The extruded vv early mRNAs were found to be organized into unique granular cytoplasmic structures that reached a size up to 1 microm. By EM these structures appeared as amorphous electron-dense cytoplasmic aggregates that were surrounded by ribosomes. Confocal images showed that the RNA structures were located some distance away from intracellular cores and that both structures appeared to be aligned on microtubules (MTs), implying that MT tracks connected mRNAs and cores. Accordingly, intact MTs were found to be required for the typical punctate organization of viral mRNAs. Biochemical evidence supported the notion that vv mRNAs were MT associated and that MT depletion severely affected viral (but not cellular) mRNA synthesis and stability. By confocal microscopy the viral mRNA structures appeared to be surrounded by molecules of the translation machinery, showing that they were active in protein synthesis. Finally, our data suggest a role for a MT and RNA-binding viral protein of 25 kDa (gene L4R), in mRNA targeting away from intracellular cores to their sites of cytoplasmic accumulation.

The vaccinia virus superoxide dismutase-like protein (A45R) is a virion component that is nonessential for virus replication.

A characterization of the A45R gene from vaccinia virus (VV) strain Western Reserve is presented. The open reading frame is predicted to encode a 125-amino-acid protein (M(r), of 13,600) with 39% amino acid identity to copper-zinc superoxide dismutase (Cu-Zn SOD). Sequencing of the A45R gene from other orthopoxviruses, here and by others, showed that the protein is highly conserved in all viruses sequenced, including 16 strains of VV, 2 strains of cowpox virus, camelpox virus, and 4 strains of variola virus. In all cases the protein lacks key residues involved in metal ion binding that are important for the catalytic activity. The A45R protein was expressed in Escherichia coli, purified, and tested for SOD activity, but neither enzymatic nor inhibitory SOD activity was detected. Additionally, no virus-encoded SOD activity was detected in infected cells or purified virions. A monoclonal antibody raised against the A45R protein expressed in E. coli identified the A45R gene product as a 13.5-kDa protein that is expressed late during VV infection. Confocal microscopy of VV-infected cells indicated that the A45R protein accumulated predominantly in cytoplasmic viral factories. Electron microscopy and biochemical analyses showed that the A45R protein is incorporated into the virion core. A deletion mutant lacking the majority of the A45R gene and a revertant virus in which the deleted gene was restored were constructed and characterized. The growth properties of the deletion mutant virus were indistinguishable from those of wild-type and revertant viruses in all cell lines tested, including macrophages. Additionally, the virulence and pathogenicity of the three viruses were also comparable in murine and rabbit models of infection. A45R is unusual in being the first VV core protein described that affects neither virus replication nor virulence.

Characterization of vaccinia virus intracellular cores: implications for viral uncoating and core structure.

The entry of vaccinia virus (VV) into the host cell results in the delivery of the double-stranded DNA genome-containing core into the cytoplasm. The core is disassembled, releasing the viral DNA in order to initiate VV cytoplasmic transcription and DNA replication. Core disassembly can be prevented using the VV early transcription inhibitor actinomycin D (actD), since early VV protein synthesis is required for core uncoating. In this study, VV intracellular cores were accumulated in the presence of actD and isolated from infected cells. The content of these cores was analyzed by negative staining EM and by Western blotting using a collection of antibodies to VV core and membrane proteins. By Western blot analyses, intracellular actD cores, as well as cores prepared by NP-40-dithiothreitol treatment of purified virions (NP-40/DTT cores), contained the core proteins p25 (encoded by L4R), 4a (A10L), 4b (A3L), and p39 (A4L) as well as small amounts of the VV membrane proteins p32 (D8L) and p35 (H3L). While NP-40/DTT cores contained the major putative DNA-binding protein p11 (F17R), actD cores entirely lacked this protein. Labeled cryosections of cells infected for different periods of time in the presence or absence of actD were subsequently used to follow the fate of VV core proteins by EM. These EM images confirmed that p11 was lost at the plasma membrane upon core penetration. The cores that accumulated in the presence of actD were labeled with antibodies to 4a, p39, p25, and DNA at all times examined. In the absence of the drug the cores gradually lost their electron-dense inner part, concomitant with the loss of p25 and DNA labeling. The remaining core shell still labeled with antibodies to p39 and 4a/4b, implying that these proteins are part of this structure. These combined data are discussed with respect to the structure of VV as well as core disassembly.

A vaccinia virus core protein, p39, is membrane associated.

We describe herein the characterization of p39, the product of the A4L gene of vaccinia virus. By immunolabelling of thawed cryosections from infected HeLa cells, we show that this protein is initially located in the central region, or viroplasm, of the viral factories, as well as in the immature virions, with very small amounts of labelling observed on the surrounding membranes. The localization of p39 changes dramatically during the transition of the immature virion to the intracellular mature virus (IMV), coincident with the appearance of the core structure in the center of the IMV, with p39 located between this core and the surrounding membranes. Complementary biochemical data, such as partitioning into the Triton X-114 detergent phase and stripping of the viral membranes with Nonidet P-40 and dithiothreitol, suggest that p39 is associated with the innermost of the two membranes surrounding the core. Sodium carbonate treatment also indicates that p39 is associated with membranes, even at the early stages of viral assembly. However, following in vitro translation of p39 in the presence of microsomal membranes, we failed to detect any association of the independently expressed protein with membranes. We also failed to detect any posttranslational acylation of p39 with myristate or palmitate, suggesting that p39 does not achieve its membrane association through lipid anchors. Therefore, p39 is most likely membrane associated through an interaction with an integral membrane protein(s) present in the innermost of the two membranes surrounding the IMV. These data, together with our recent data showing that p39 colocalizes with the spike-like protrusions on the IMV core (N. Roos, M. Cyrklaff, S. Cudmore, R. Blasco, J. Krijnse-Locker, and G. Griffiths, EMBO J. 15:2343-2355, 1996), suggest that p39 may form part of this spike and that it possibly functions as a matrix-like linker protein between the core and the innermost of the two membranes surrounding the IMV.

Vaccinia virions lacking core protein VP8 are deficient in early transcription.

When synthesis of the 25-kDa vaccinia virus core protein VP8 is repressed, mature virus particles of normal appearance are produced to approximately 80% of wild-type levels but these particles are over 100-fold less infectious than wild-type particles (D. Wilcock and G. L. Smith, Virology 202:294-304, 1994). Here we show that virions which lack VP8 can bind to and enter cells but the levels of steady-state RNA are greatly reduced in comparison with those for wild-type infections. In vitro assays using permeabilized virions demonstrated that VP8-deficient virions had drastically reduced rates of transcription (RNA synthesis was decreased by 80 to 96%) and that the extrusion of RNA transcripts from these virions was also decreased. Low concentrations of sodium deoxycholate extracted proteins more efficiently from VP8-deficient virions than from wild-type virions. The increased fragility of VP8-deficient virions and their slower RNA extrusion rates suggest that VP8 may be required for the correct formation of the core. Virions which lack VP8 were shown to contain a full complement of transcription enzymes, and soluble extracts from these virions were active in transcription assays using either single-stranded M13 DNA or exogenous plasmid template containing a vaccinia virus early promoter. Thus, the defect in transcription is due not to a lack of specific transcriptional enzymes within virions but rather to the inability of these enzymes to efficiently transcribe the DNA genome packaged within VP8-deficient virions. These results suggest that VP8 is required for the correct packaging of the viral DNA genome and/or for the efficient transcription of packaged virion DNA, which has a higher degree of structural complexity than plasmid templates. Possible roles for VP8 in these processes are discussed.

Vaccinia protein kinase 2: a second essential serine/threonine protein kinase encoded by vaccinia virus.

The major protein kinase activity from vaccinia virus core particles was purified to near homogeneity. The protein kinase is a 50-kDa polypeptide that is shown here to phosphorylate primarily seryl residues in alpha-casein, a casein kinase I-specific peptide substrate, and itself through autophosphorylation. The sequence of four peptides derived from the protein kinase demonstrated that it is encoded by the vaccinia virus F10L gene. Expression of the F10L gene product in bacteria as a fusion with glutathione S-transferase confirmed that the vaccinia F10L gene encodes the protein kinase. We have termed this enzyme vaccinia protein kinase 2 (VPK2) to distinguish it from the protein kinase encoded by the vaccinia B1R gene. Targeted disruption of the VPK2 gene with a positive selectable marker demonstrated that all viruses with a disrupted gene also possessed a wild-type gene, suggesting that VPK2 is essential for viability. The discovery of a second essential protein kinase encoded by vaccinia virus, in addition to a protein phosphatase, underscores the importance of protein phosphorylation in poxvirus biogenesis.