Vaccinia virus E10R protein is associated with the membranes of intracellular mature virions and has a role in morphogenesis.

This study provides the initial biochemical, microscopic, and genetic characterization of the product of the vaccinia virus E10R gene, which belongs to the ERV1/ALR family of eukaryotic proteins, is conserved in all poxviruses and has homologs in other cytoplasmic DNA viruses. DNA encoding a short epitope tag was appended to the C-terminus of the 95-amino-acid open-reading frame without affecting virus reproduction. The E10R protein was synthesized after DNA replication and was associated with purified intracellular mature virions (IMV), from which it could be extracted with a nonionic detergent. Antibody to the tag decorated the surface of IMV, consistent with the anchorage of the E10R protein to the membrane via its hydrophobic N-terminus. Immunoelectron microscopy revealed that the E10R protein was associated with crescent membranes, immature virions, IMV, and extracellular particles. To investigate the role of E10R in the virus life cycle, we constructed an inducer-dependent null mutant. In the absence of inducer, the formation of infectious virus was severely inhibited and electron microscopy revealed an assembly block with accumulation of crescent membranes and immature virions. Cysteines 43 and 46, comprising a putative redox motif common to all poxvirus E10R homologs, were essential for complementation of the mutant virus by transfected E10R DNA.

Immune-defense molecules of molluscum contagiosum virus, a human poxvirus.

Molluscum contagiosum virus encodes more than 150 proteins including some involved in host interactions that might contribute to prolonged viral replication in the skin. These include homologs of a selenocysteine-containing glutathione peroxidase, a death effector domain protein, a chemokine, a major histocompatibility complex class I molecule and an interleukin-18-binding protein.

A viral member of the ERV1/ALR protein family participates in a cytoplasmic pathway of disulfide bond formation.

Proteins of the ERV1/ALR family are encoded by all eukaryotes and cytoplasmic DNA viruses for which substantial sequence information is available. Nevertheless, the roles of these proteins are imprecisely known. Multiple alignments of ERV1/ALR proteins indicated an invariant C-X-X-C motif, but no similarity to the thioredoxin fold was revealed by secondary structure predictions. We chose a virus model to investigate the role of these proteins as thiol oxidoreductases. When cells were infected with a mutant vaccinia virus in which the E10R gene encoding an ERV1/ALR family protein was repressed, the disulfide bonds of three other viral proteins-namely, the L1R and F9L proteins and the G4L glutaredoxin-were completely reduced. The same outcome occurred when Cys-43 or Cys-46, the putative redox cysteines of the E10R protein, was mutated to serine. These two cysteines were disulfide bonded during a normal virus infection but not if the synthesis of other viral late proteins was inhibited or the E10R protein was expressed by itself in uninfected cells, suggesting a requirement for an upstream viral thiol oxidoreductase. Remarkably, the cysteine-containing domains of the E10R and L1R viral membrane proteins and the glutaredoxin are in the cytoplasm, in which assembly of vaccinia virions occurs, rather than in the oxidizing environment of the endoplasmic reticulum. These data indicated a viral pathway of disulfide bond formation in which the E10R protein has a central role. By extension, the ERV1/ALR family may represent a ubiquitous class of cellular thiol oxidoreductases that interact with glutaredoxins or thioredoxins.

Domain structure, intracellular trafficking, and beta2-microglobulin binding of a major histocompatibility complex class I homolog encoded by molluscum contagiosum virus.

The MC80R gene of molluscum contagiosum virus (MCV) type 1 encodes a major histocompatibility complex (MHC) class I homolog that lacks several amino-acid residues critical for peptide binding by MHC molecules, contains an unusually long N-terminal hydrophobic domain possibly derived by triplication of a signal peptide, and has a C-terminal transmembrane domain with two glutamate residues. All of these features were present in the orthologous gene of MCV type 2. The MC80R gene was expressed as two glycosylated polypeptides of Mr 47,000 and 42,000. Pulse-chase experiments indicated that the larger polypeptide was a precursor of the shorter one and that the entire N-terminal domain was slowly removed, consistent with its function as a long signal peptide. The protein was largely sequestered in the endoplasmic reticulum and Golgi membranes, remained endoglycosidase-H sensitive, and was not detected on the cell surface. In addition, a genetically modified form of the MC80R protein lacking the transmembrane and cytoplasmic domains was not secreted. The roles of the MC80R protein domains were investigated by constructing chimera between the viral protein and the MHC class I protein HLA-A2. Expression studies confirmed that the N- and C-terminal hydrophobic regions of the MC80R protein served as signal and transmembrane domains, respectively. The central portion of the MC80R protein, corresponding to the alpha1-alpha3 extracellular domains of HLA-A2, was largely responsible for sequestering the protein in the endoplasmic reticulum or Golgi compartments. The MC80R protein, as well as HLA-A2 chimera with the central region of MC80R, formed stable intracellular complexes with beta2-microglobulin. Complex formation, however, was detected only by overexpression of the MC80R protein or beta2-microglobulin.

Ultraviolet-induced cell death blocked by a selenoprotein from a human dermatotropic poxvirus.

Selenium, an essential trace element, is a component of prokaryotic and eukaryotic antioxidant proteins. A candidate selenoprotein homologous to glutathione peroxidase was deduced from the sequence of molluscum contagiosum, a poxvirus that causes persistent skin neoplasms in children and acquired immunodeficiency syndrome (AIDS) patients. Selenium was incorporated into this protein during biosynthesis, and a characteristic stem-loop structure near the end of the messenger RNA was required for alternative selenocysteine decoding of a potential UGA stop codon within the open reading frame. The selenoprotein protected human keratinocytes against cytotoxic effects of ultraviolet irradiation and hydrogen peroxide, providing a mechanism for a virus to defend itself against environmental stress.

The genome of molluscum contagiosum virus: analysis and comparison with other poxviruses.

Analysis of the molluscum contagiosum virus (MCV) genome revealed that it encodes approximately 182 proteins, 105 of which have direct counterparts in orthopoxviruses (OPV). The corresponding OPV proteins comprise those known to be essential for replication as well as many that are still uncharacterized, including 2 of less than 60 amino acids that had not been previously noted. The OPV proteins most highly conserved in MCV are involved in transcription; the least conserved include membrane glycoproteins. Twenty of the MCV proteins with OPV counterparts also have cellular homologs and additional MCV proteins have conserved functional motifs. Of the 77 predicted MCV proteins without OPV counterparts, 10 have similarity to other MCV proteins and/or distant similarity to proteins of other poxviruses and 16 have cellular homologs including some predicted to antagonize host defenses. Clustering poxvirus proteins by sequence similarity revealed 3 unique MCV gene families and 8 families that are conserved in MCV and OPV. Two unique families contain putative membrane receptors; the third includes 2 proteins, each containing 2 DED apoptosis signal transduction domains. Additional families with conserved patterns of cysteines and putative redox active centers were identified. Promoters, transcription termination signals, and DNA concatemer resolution sequences are highly conserved in MCV and OPV. Phylogenetic analysis suggested that MCV, OPV, and leporipoxviruses radiated from a common poxvirus ancestor after the divergence of avipoxviruses. Despite the acquisition of unique genes for host interactions and changes in GC content, the physical order and regulation of essential ancestral poxvirus genes have been largely conserved in MCV and OPV.

Death effector domain-containing herpesvirus and poxvirus proteins inhibit both Fas- and TNFR1-induced apoptosis.

To identify novel antiapoptotic proteins encoded by DNA viruses, we searched viral genomes for proteins that might interfere with Fas and TNFR1 apoptotic signaling pathways. We report here that equine herpesvirus type 2 E8 protein and molluscum contagiosum virus MC159 protein both show sequence similarity to the death effector domains (DEDs) of the Fas/TNFR1 signaling components FADD and caspase-8. Yeast two-hybrid analysis revealed that E8 protein interacted with the caspase-8 prodomain whereas MC159 protein interacted with FADD. Furthermore, expression of either E8 protein or MC159 protein protected cells from Fas- and TNFR1-induced apoptosis indicating that certain herpesviruses and poxviruses use DED-mediated interactions to interfere with apoptotic signaling pathways. These findings identify a novel control point exploited by viruses to regulate Fas- and TNFR1-mediated apoptosis.

Genome sequence of a human tumorigenic poxvirus: prediction of specific host response-evasion genes.

Molluscum contagiosum virus (MCV) commonly causes asymptomatic cutaneous neoplasms in children and sexually active adults as well as persistent opportunistic acquired immunodeficiency syndrome (AIDS)-associated disease. Sequencing the 190-kilobase pair genome of MCV has now revealed that the virus potentially encodes 163 proteins, of which 103 have homologs in the smallpox virus. MCV lacks counterparts to 83 genes of the smallpox virus, including those important in suppression of host responses to infection, nucleotide biosynthesis, and cell proliferation. MCV possesses 59 genes that are predicted to encode previously uncharacterized proteins, including major histocompatibility complex class I, chemokine, and glutathione peroxidase homologs, which suggests that there are MCV-specific strategies for coexistence with the human host.

Ectromelia virus RING finger protein is localized in virus factories and is required for virus replication in macrophages.

We have previously described a gene of ectromelia virus (EV) that codes for a 28-kDa RING zinc finger-containing protein (p28) that is nonessential for virus growth in cell culture but is critical for EV pathogenicity in mice (T. G. Senkevich, E. V. Koonin, and R. M. L. Buller, Virology 198:118-128; 1994). Here, we show that, unlike all tested cell cultures, the expression of p28 is required for in vitro replication of EV in murine resident peritoneal macrophages. In macrophages infected with the p28- mutant, viral DNA replication was not detected, whereas the synthesis of at least two early proteins was observed. Immunofluorescence and biochemical analyses showed that in EV-infected macrophages or BSC-1 cells, p28 is associated with virus factories. By use of a vaccinia virus expression system to examine different truncated versions of p28, it was shown that the disruption of the specific structure of the RING domain had no influence on the intracellular localization of this protein. When viral DNA replication was inhibited with cytosine arabinoside, p28 was found in distinct, focal structures that may be precursors to the factories. We hypothesize that in macrophages, which are highly specialized, nondividing cells, p28 substitutes for an unknown cellular factor(s) that may be required for viral DNA replication or a stage of virus reproduction between the expression of early genes and the onset of DNA synthesis. In the absence of p28, the attenuation of EV pathogenicity can be explained by a failure of the virus to replicate in macrophage lineage cells at all successive steps in the spread of virus from the skin to its target organ, the liver.

A poxvirus protein with a RING zinc finger motif is of crucial importance for virulence.

The DNA sequence of a 2060-bp fragment from the left-hand end of the ectromelia (mousepox) virus genome was determined. Two genes were identified coding for 28 kDa (p28) and 16 kDa (p16) proteins, both of which are disrupted in vaccinia virus but conserved in variola (smallpox) virus. p16 contains an N-terminal hydrophobic region and may be a membrane or secreted protein. p28 contains a C3HC4 (RING) zinc finger motif that has been found in a large family of proteins involved mostly in transcription regulation. p28 was expressed in bacteria and shown to bind Zn in vitro. Disruption of the p28 gene had no appreciable effect on the multiplication of the virus in cell culture but abolished its lethality for susceptible mice. The p28- mutant virus replicated to significantly lower titers than the wild-type virus in different organs of infected mice. It is proposed that the p28 gene is an important determinant of orthopoxvirus pathogenicity, and its product may positively or negatively regulate expression of host or viral gene(s) involved in virus-host interaction.

Nucleotide sequence of XhoI O fragment of ectromelia virus DNA reveals significant differences from vaccinia virus.

The nucleotide sequence of the 3913 base pair XhoI O fragment located in an evolutionary variable region adjacent to the right end of the genome of ectromelia virus (EMV) was determined. The sequence contains two long open reading frames coding for putative proteins of 559 amino acid residues (p65) and 344 amino acid residues (p39). Amino acid database searches showed that p39 is closely related to vaccinia virus (VV), strain WR, B22R gene product (C12L gene product of strain Copenhagen), which belongs to the family of serine protease inhibitors (serpins). Despite the overall high conservation, differences were observed in the sequences of p39, B22R, and C12L in the site known to interact with proteases in other serpins, suggesting that the serpins of EMV and two strains of VV may all inhibit proteases with different specificities. The gene coding for the ortholog of p65 is lacking in the Copenhagen strain of vaccinia virus; the WR strain contains a truncated variant of this gene (B21R) potentially coding for a small protein (p16) corresponding to the C-terminal region of p65. p65 is a new member of the family of poxvirus proteins including vaccinia virus proteins A55R, C2L and F3L, and a group of related proteins of leporipoxviruses, Shope fibroma and myxoma viruses (T6, T8, T9, M9). These proteins are homologous to the Drosophila protein Kelch involved in egg development. Both Kelch protein and the related poxvirus proteins contain two distinct domains. The N-terminal domain is related to the similarly located domains of transcription factors Ttk, Br-C (Drosophila), and KUP (human), and GCL protein involved in early development in Drosophila. The C-terminal domain consists of an array of four to five imperfect repeats and is related to human placental protein MIPP. Phylogenetic analysis of the family of poxvirus proteins showed that their genes have undergone a complex succession of duplications, and complete or partial deletions.

Fowlpox virus encodes a protein related to human deoxycytidine kinase: further evidence for independent acquisition of genes for enzymes of nucleotide metabolism by different viruses.

It is demonstrated that fowlpox virus (FPV) protein FP26 located in the HindIII D fragment of the genome is related to the human deoxycytidine kinase (dCK) and probably possesses the same enzymatic activity. A homologous protein is not encoded by vaccinia virus. A multiple alignment of the amino acid sequences of the human and FPV dCKs, the thymidine kinases (TK) of herpesviruses, and cellular and vaccinia virus thymidylate kinases (ThyK) was generated and the conserved motifs, at least two of which are implicated in ATP binding, were characterized. An apparent duplication of ATP-binding motif B in the dCKs was revealed, leading to the reassignment of one of the catalytic residues. Phylogenetic analysis based on the multiple alignment suggested that the putative dCK of FPV probably has diverged from the common ancestor with the human dCK at a later stage of evolution than the herpesvirus TKs, with the ThyKs being peripheral members of the family. These results are compatible with hypothesis that genes for enzymes of nucleotide metabolism could be acquired independently by different DNA viruses (Koonin, E.V. and Senkevich, T.G., Virus Genes 6:187-196, 1992).

Gene A32 product of vaccinia virus may be an ATPase involved in viral DNA packaging as indicated by sequence comparisons with other putative viral ATPases.

Statistically significant sequence similarity was revealed between the gene A32 product of vaccinia virus (VV), gene I products (gpI) of filamentous single-stranded DNA bacteriophages, and IVa2 gene products of adenoviruses. Four conserved sequence motifs were delineated, the two N-proximal of which correspond to the A and B motifs of the purine NTP-binding pattern. Based on the role of gpI and IVa2 proteins in virion morphogenesis, and on the conservation of the NTP-binding pattern in these proteins, we hypothesize that the A32 gene product might be involved in an ATP-consuming function in VV virion formation, e.g., packaging of the DNA in the virus particle.

[Search for unique segments of the ectromelia virus genome by cross blot hybridization with DNA from other orthopoxviruses]. / Poisk unikal'nykh uchastkov genoma virusa éktromelii metodom perekrestnoi blot-gibridizatsii s DNK drugikh ortopoksvirusov]

In order to identify ectromelia virus (EMV) genome regions which may contain genes responsible for the specific pathogenicity of this virus, blot cross-hybridization of EMV DNA with those of other orthopoxviruses was performed. Two hybridization schemes were employed: one of them included hybridization of labelled cloned fragments of EMV with digests of other viral DNAs, the other, reciprocal, consisted in hybridization of labelled total DNAs of various orthopoxviruses with digests of the region of EMV DNA adjacent to the right-terminal inverted repeat. It was demonstrated that the counterpart to an approximately 8-kilobase pair portion of EMV genome flanking the inverted repeat could be detected only in the cowpox virus genome but not in the genomes of vaccinia and rabbitpox viruses. XhoI-O and XhoI-K fragments of EMV DNA contained, along with genes found in other poxviruses, certain genes which appeared to be unique for EMV. It is postulated that some of these genes may determine the specific biological properties of EMV, including its pathogenicity for mice.

Evolution of thymidine and thymidylate kinases: the possibility of independent capture of TK genes by different groups of viruses.

Phylogenetic analysis of viral and cellular thymidine and thymidylate kinases was performed using computer-assisted methods. Multiple alignments and tentative phylogenetic trees were generated for the two families of these enzymes, which include a) thymidine kinases (TK) of mammals, poxviruses, African swine fever virus, E. coli, and bacteriophage T4; and b) thymidylate kinases (ThyK) of yeast and poxviruses and distantly related herpesvirus proteins with both enzymatic activities. Analysis of the alignment of the TKs of the first family highlighted three strongly conserved segments. Two of these corresponded to the A and B motifs of the purine NTP-binding pattern. The third, C-terminal segment, showing the highest conservation, encompassed a modified Zn finger motif. It is speculated that this motif might be involved in TK oligomerization. Phylogenetic trees constructed by three different methods suggested that cellular TK genes could be captured independently by T4 bacteriophage, African swine fever virus, fowlpox virus, and the other poxviruses. The observed tree topologies appear to contradict the popular virus-host coevolution schemes and to imply that different subdivisions of poxviruses diverged at earlier stages of evolution than their hosts did. It was shown that deoxynucleoside monophosphate kinase of bacteriophage T4 is related to the ThyK family. Phylogenetic analysis suggested that ThyK genes probably have been acquired independently by phage T4, poxviruses, and herpes-viruses.

Vaccinia virus encodes four putative DNA and/or RNA helicases distantly related to each other.

Computer-assisted analysis of the amino acid sequences of vaccinia virus proteins containing the purine NTP-binding pattern revealed the seven motifs typical of the DNA (RNA) helicase superfamily II in the proteins I8R and A18R. Together with the previously described putative helicases D6R and D11L, the number of putative helicases of this superfamily encoded by the genome of vaccinia virus now reaches four. Aside from the helicase motifs, the sequences of I8R and A18R showed no strong similarity to each other, nor to D6R and D11L. Statistically significant similarity was demonstrated between I8R and the putative RNA helicases involved in pre-mRNA splicing in yeast, PRP2, PRP16 and PRP22, whereas A18R appeared to be related to the putative helicases encoded by the human DNA repair gene ERCC3 and the D10 gene of bacteriophage T5. These findings suggest that I8R may be an RNA helicase. Based on the known properties of the virion NTPases of vaccinia virus, it is possible that the I8R protein may be identical to the previously characterized virion NTPase II. A18R is likely to possess DNA and/or RNA helicase activity. Circumstantial evidence suggests that this activity might be involved in melting duplex structures in late mRNAs. The possibility of independent acquisition of the putative helicases I8R, A18R and a common progenitor to D6R and D11L by an ancestral poxvirus is discussed.

[Vaccinia and ectromelia recombinant viruses, causing an infection, characteristic for ectromelia, in mice]. / Rekombinanty virusov ospovaktsiny i éktromelii, vyzyvaiushchie kharakternye dlia virusa éktromelii porazheniia u myshei.

Ten recombinants between the viruses of vaccinia and ectromelia were isolated that cause the ectromelia virus specific lesions in mice. The structure of recombinant viral genomes, the efficiency of viral propagation in mice, the nature of lesions induced by viruses have been studied. Eight of obtained recombinants have a DNA insertion originating from the right end of ectromelia viral genome, nine recombinants have an insertion originating from the left end, seven recombinants possess both insertions. The latter recombinants have more pronounced pathogenicity for mice. Both revealed regions are supposed to define the specific pathogenicity of ectromelia virus for mice.

Biologic properties and genome structure of the recombinants between ectromelia and rabbitpox viruses.

Administration of rabbitpox virus (RPV) DNA, cleaved into 2 fragments by SmaI restrictase, into ectromelia virus (EMV)-infected chick fibroblast cells yielded recombinants whose properties were characteristic of both parents. Some recombinants capable of producing RPV-type lesions upon intracutaneous (i.c.) infection of rabbits could also produce EMV-specific lesions upon footpad inoculation of mice. The analysis of some recombinants as well as vaccinia virus strains has shown that the ability of the virus to reproduce when injected into the mouse footpad is a necessary, but not a sufficient condition for production of EMV-type lesions. According to restrictase analysis of recombinant DNA, the genome of recombinants mainly consists of RPV DNA sequences with insertions of small EMV DNA fragments.

[Physical mapping of DNA-temperature-sensitive mutations of vaccinia virus]. / Fizicheskoe kartirovanie DNK-temperaturochuvstvitel'nykh mutatsii virusa ospovaktsiny.

Four DNA-temperature-sensitive (ts) mutations were mapped in the genome of vaccinia virus (VV). Physical mapping of these mutations was performed by restriction analysis of the genomes of recombinants between VV DNA- ts mutants and ectromelia virus as well as by the marker rescue with cloned restriction fragments of VV DNA. One of the mutations was mapped on the HindIII-E-fragment. Biochemical studies of this mutant indicate that the mutation is not in the DNA polymerase gene which is located on the same fragment. The other three mutations were mapped in a 10 kilobase region in the middle of the HindIII-D-fragment. As shown previously, these mutations inactivate different genes, and the products of these genes participate directly in the DNA synthesis. Thus, at least three proteins involved in the VV DNA synthesis are encoded by neighboring genes in the central part of the viral genome.