Open reading frame S/L of varicella-zoster virus encodes a cytoplasmic protein expressed in infected cells

We report the discovery of a novel gene in the varicella-zoster virus (VZV) genome, designated open reading frame (ORF) S/L. This gene, located at the left end of the prototype VZV genome isomer, expresses a polyadenylated mRNA containing a splice within the 3' untranslated region in virus-infected cells. Sequence analysis reveals significant differences between the ORF S/Ls of wild-type and attenuated strains of VZV. Antisera raised to a bacterially expressed portion of ORF S/L reacted specifically with a 21-kDa protein synthesized in cells infected with a VZV clinical isolate and with the original vaccine strain of VZV (Oka-ATCC). Cells infected with other VZV strains, including a wild-type strain that has been extensively passaged in tissue culture and commercially produced vaccine strains of Oka, synthesize a family of proteins ranging in size from 21 to 30 kDa that react with the anti-ORF S/L antiserum. MeWO cells infected with recombinant VZV harboring mutations in the C-terminal region of the ORF S/L gene lost adherence to the stratum and adjacent cells, resulting in an altered plaque morphology. Immunohistochemical analysis of VZV-infected cells demonstrated that ORF S/L protein localizes to the cytoplasm. ORF S/L protein was present in skin lesions of individuals with primary or reactivated infection and in the neurons of a dorsal root ganglion during virus reactivation.     

Functional Analysis of Vaccinia Virus B5R Protein: Essential Role in Virus Envelopment Is Independent of a Large Portion of the Extracellular Domain

Vaccinia virus has two forms of infectious virions: the intracellular mature virus and the extracellular enveloped virus (EEV). EEV is critical for cell-to-cell and long-range spread of the virus. The B5R open reading frame (ORF) encodes a membrane protein that is essential for EEV formation. Deletion of the B5R ORF results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo. The extracellular portion of B5R is composed mainly of four domains that are similar to the short consensus repeats (SCRs) present in complement regulatory proteins. To determine the contribution of these putative SCR domains to EEV formation, we constructed recombinant vaccinia viruses that replaced the wild-type B5R gene with a mutated gene encoding a B5R protein lacking the SCRs. The resulting recombinant viruses produced large plaques, indicating efficient cell-to-cell spread in vitro, and gradient centrifugation of supernatants from infected cells confirmed that EEV was formed. In contrast, phalloidin staining of infected cells showed that the virus lacking the SCR domains was deficient in the induction of thick actin bundles. Thus, the highly conserved SCR domains present in the extracellular portion of the B5R protein are dispensable for EEV formation. This indicates that the B5R protein is a key viral protein with multiple functions in the process of virus envelopment and release. In addition, given the similarity of the extracellular domain to complement control proteins, the B5R protein may be involved in viral evasion from host immune responses.     

vrrB, a hypervariable open reading frame in Bacillus anthracis

Bacillus anthracis appears to be the most molecularly homogeneous bacterial species known. Extensive surveys of worldwide isolates have revealed vanishingly small amounts of genomic variation. The biological importance of the resting-stage spore may lead to very low evolutionary rates and, perhaps, to the lack of potentially adaptive genetic variation. In contrast to the overall homogeneity, some gene coding regions contain hypervariability that is translated into protein variation. During marker analysis of diverse strains, we have discovered a novel ca. 750-nucleotide open reading frame (ORF) that contains in-frame, variable-number tandem-repeat sequences. Four distinct variable regions exist within vrrB, giving rise to 11 distinct alleles in eight different length categories among B. anthracis strains. This ORF putatively codes for a 241- to 265-amino-acid protein, rich in glutamine (13.2%), glycine (23.4%), and histidine (23.0%). The variable-region amino acids of the vrrB ORF are strongly hydrophilic. Coupled with putative transmembrane domains flanking the variable regions, this suggests a membrane-anchored cytosolic or extracellular location for the putative protein. Sequence analysis of the complete ORFs from three Bacillus cereus strains shows maintenance of the ORF across species boundaries, including strong conservation of the amino acid sequence and the capacity to vary among strains. The presence of 11 different alleles of the vrrB locus is in stark contrast to the near homogeneity of B. anthracis. Evolution of hypervariable genes can negate the lack of genetic variability in species such as B. anthracis and provide select rapid evolution in other more variable species.     

Varicella-zoster virus open reading frame 10 protein, the herpes simplex virus VP16 homolog, transactivates herpesvirus immediate-early gene promoters.

The varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein is the homolog of the herpes simplex virus type 1 (HSV-1) protein VP16. These are two virion tegument proteins that have extensive amino acid sequence identity in their amino-terminal and middle domains. ORF10, however, lacks the acidic carboxy terminus which is critical for transactivation by VP16. Earlier studies showed that VZV ORF10 does not form a tertiary complex with the TAATGARAT regulatory element (where R is a purine) with which HSV-1 VP16 interacts, suggesting that ORF10 may not have transactivating ability. Using transient-expression assays, we show that VZV ORF10 is able to transactivate VZV immediate-early (IE) gene (ORF62) and HSV-1 IE gene (ICP4 and ICP0) promoters. Furthermore, cell lines stably expressing ORF10 complement the HSV-1 mutant in1814, which lacks the transactivating function of VP16, and enhance the de novo synthesis of infectious virus following transfection of HSV-1 virion DNA. These results indicate that ORF10, like its HSV-1 homolog VP16, is a transactivating protein despite the absence of sequences similar to the VP16 carboxy-terminal domain. The transactivating function of the VZV ORF10 tegument protein may be critical for efficient initiation of viral infection.     

Contribution of protein p40 to hypovirus-mediated modulation of fungal host phenotype and viral RNA accumulation

The papain-like protease p29, derived from the N-terminal portion of the hypovirus CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, was previously shown to contribute to reduced pigmentation and sporulation by the infected host, the chestnut blight fungus Cryphonectria parasitica, while being dispensable for virus replication and attenuation of fungal virulence (hypovirulence). We now report that deletion of the C-terminal portion of p69, which encodes the highly basic protein p40, resulted in replication-competent mutant viruses that were, however, significantly reduced in RNA accumulation. While the Delta p40 mutants retained the ability to confer hypovirulence, Delta p40-infected fungal strains produced more asexual spores than strains infected with either wild-type CHV1-EP713 or a Delta p29 mutant virus. As observed for Delta p29-infected colonies, pigment production was significantly increased in Delta p40-infected fungal strains relative to that in CHV1-EP713-infected strains. Virus-mediated suppression of laccase production was not affected by p40 deletion. A gain-of-function analysis was employed to map the p40 symptom determinant to the N-terminal domain, encompassing p69 amino acid residues Thr(288) to Arg(312). Evidence that the gain of function was due to the encoded protein rather than the corresponding RNA sequence element was provided by introducing frameshift mutations on either side of the activity determinant domain. Moreover, restoration of symptoms correlated with increased accumulation of viral RNA. These results suggest that p40 indirectly contributes to virus-mediated suppression of fungal pigmentation and conidiation by providing an accessory function in hypovirus RNA amplification. A possible role for p40 in facilitating ORF B expression and the relationship between hypovirus RNA accumulation and symptom expression are discussed.     

Characterization of a vaccinia virus-encoded 42-kilodalton class I membrane glycoprotein component of the extracellular virus envelope.

Using a reverse genetic approach, we have demonstrated that the product of the B5R open reading frame (ORF), which has homology with members of the family of complement control proteins, is a membrane glycoprotein present in the extracellular enveloped (EEV) form of vaccinia virus but absent from the intracellular naked (INV) form. An antibody (C'-B5R) raised to a 15-amino-acid peptide from the translated B5R ORF reacted with a 42-kDa protein (gp42) found in vaccinia virus-infected cells and cesium chloride-banded EEV but not INV. Under nonreducing conditions, an 85-kDa component, possibly representing a hetero- or homodimeric form of gp42, was detected by both immunoprecipitation and Western immunoblot analysis. Metabolic labeling with [3H]glucosamine and [3H]palmitate revealed that the B5R product is glycosylated and acylated. The C-terminal transmembrane domain of the protein was identified by constructing a recombinant vaccinia virus that overexpressed a truncated, secreted form of the B5R ORF product. By N-terminal sequence analysis of this secreted protein, the site of signal peptide cleavage of gp42 was determined. A previously described monoclonal antibody (MAb 20) raised to EEV, which immunoprecipitated a protein with biochemical characteristics similar to those of wild-type gp42, reacted with the recombinant, secreted product of the B5R ORF. Immunofluorescence of wild-type vaccinia virus-infected cells by using either MAb 20 or C'-B5R revealed that the protein is expressed on the cell surface and within the cytoplasm. Immunogold labeling of EEV and INV with MAb 20 demonstrated that the protein was found exclusively on the EEV membrane.     

Identification of five major and two minor genotypes of varicella-zoster virus strains: a practical two-amplicon approach used to genotype clinical isolates in Australia and New Zealand

Whole genome phylogenetic analysis in this study resolved a total of five major genotypes among the 22 varicella-zoster virus (VZV) strains or isolates for which complete genomic sequences are available. Consistent with earlier publications we have designated these genotypes European 1 (E1), European 2 (E2), Japanese (J), mosaic 1 (M1), and mosaic 2 (M2). Single nucleotide polymorphism (SNP) analysis performed in a whole-genome alignment revealed that VZV isolates of all five genotypes can be accurately genotyped using SNPs from two amplicons: open reading frame 22 (ORF22) and either ORF21 or ORF50. This modified approach identifies all of the genotypes observed using any of the published genotyping protocols. Of 165 clinical varicella and zoster isolates from Australia and New Zealand typed using this approach, 67 of 127 eastern Australian isolates were E1, 30 were E2, 16 were J, 10 were M1, and 4 were M2; 25 of 38 New Zealand isolates were E1, 8 were E2, and 5 were M1. VZV strain diversity in eastern Australia is thus broader than has been described for any other region, including Europe, Africa, and North America. J strains were far more prevalent than previously observed in countries other than Japan. Two-amplicon typing was in complete accord with genotypes derived using SNP in multiple ORFs (ORFs 1, 21, 22, 38, 50, 54, and 62). Two additional minor genotypes, M3 and M4, could also be resolved using two-amplicon typing.     

Functions of the ORF9-to-ORF12 gene cluster in varicella-zoster virus replication and in the pathogenesis of skin infection

The gene cluster composed of varicella-zoster virus (VZV) open reading frame 9 (ORF9) to ORF12 encodes four putative tegument proteins and is highly conserved in most alphaherpesviruses. In these experiments, the genes within this cluster were deleted from the VZV parent Oka (POKA) individually or in combination, and the consequences for VZV replication were evaluated with cultured cells in vitro and with human skin xenografts in SCID mice in vivo. As has been reported for ORF10, ORF11 and ORF12 were dispensable for VZV replication in melanoma and human embryonic fibroblast cells. In contrast, deletion of ORF9 was incompatible with the recovery of infectious virus. ORF9 localized to the virion tegument and formed complexes with glycoprotein E, which is an essential protein, in VZV-infected cells. Recombinants lacking ORF10 and ORF11 (POKADelta10/11), ORF11 and ORF12 (POKADelta11/12), or ORF10, ORF11 and ORF12 (POKADelta10/11/12) were viable in cultured cells. Their growth kinetics did not differ from those of POKA, and nucleocapsid formation and virion assembly were not disrupted. In addition, these deletion mutants showed no differences compared to POKA in infectivity levels for primary human tonsil T cells. Deletion of ORF12 had no effect on skin infection, whereas replication of POKADelta11, POKADelta10/11, and POKADelta11/12 was severely reduced, and no virus was recovered from skin xenografts inoculated with POKADelta10/11/12. These results indicate that with the exception of ORF9, the individual genes within the ORF9-to-ORF12 gene cluster are dispensable and can be deleted simultaneously without any apparent effect on VZV replication in vitro but that the ORF10-to-ORF12 cluster is essential for VZV virulence in skin in vivo.     

Physical mapping and nucleotide sequence of a herpes simplex virus type 1 gene required for capsid assembly.

In this report, we describe some phenotypic properties of a temperature-sensitive mutant of herpes simplex type 1 (HSV-1) and present data concerning the physical location and nucleotide sequence of the genomic region harboring the mutation. The effect of shifts from the permissive to the nonpermissive temperature on infectious virus production by the mutant A44ts2 indicated that the mutated function is necessary throughout, or late in, the growth cycle. At the nonpermissive temperature, no major differences were detected in viral DNA or protein synthesis with respect to the parent A44ts+. On the other hand, electron microscopy of mutant-infected cells revealed that neither viral capsids nor capsid-related structures were assembled at the nonpermissive temperature. Additional analyses employing the Hirt extraction procedure showed that A44ts2 is also unable to mature replicated viral DNA into unit-length molecules under nonpermissive conditions. The results of marker rescue experiments with intact A44ts2 DNA and cloned restriction fragments of A44ts+ placed the lesion in the coordinate interval 0.553 to 0.565 (1,837 base pairs in region UL) of the HSV-1 physical map. No function has previously been assigned to this region, although it is known to be transcribed into two 5' coterminal mRNAs which code in vitro for a 54,000-molecular-weight polypeptide (K. P. Anderson, R. J. Frink, G. B. Devi, B. H. Gaylord, R. H. Costa, and E. K. Wagner, J. Virol. 37:1011-1027, 1981). We sequenced the interval 0.551 to 0.565 and found an open reading frame (ORF) for a 50,175-molecular-weight polypeptide. The predicted product of this ORF exhibits strong homology with the product of varicella-zoster virus ORF20 and lower, but significant, homology with the product of Epstein-Barr virus BORF1. For the three viruses, the corresponding ORFs lie just upstream of the gene coding for the large subunit of viral ribonucleotide reductase. The ORF described here corresponds to the ORF designated UL38 in the recently published nucleotide sequence of the HSV-1 UL region (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988).     

The varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase is dispensable for viral replication and is not required for phosphorylation of ORF63 protein, the VZV homolog of herpes simplex virus ICP22.

To investigate the role of varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase during infection, a VZV mutant was generated in which two contiguous stop codons were introduced into ORF47, thus eliminating expression of the ORF47 kinase. ORF47 kinase was not essential for the growth of VZV in cultured cells, and the growth rate of the VZV mutant lacking ORF47 protein was indistinguishable from that of parental VZV. Nuclear extracts from cells infected with parental VZV contained several phosphorylated proteins which were not detected in extracts from cells infected with the ORF47 mutant. The herpes simplex virus type 1 (HSV-1) UL13 protein (the homolog of VZV ORF47 protein) is responsible for the posttranslational processing associated with phosphorylation of HSV-1 ICP22 (the homolog of VZV ORF63 protein). Immunoprecipitation of 32P-labeled proteins from cells infected with parental virus and those infected with ORF47 mutant virus yielded similar amounts of the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 (VZV gE), and the electrophoretic migration of these proteins was not affected by the lack of ORF47 kinase. Therefore, while the VZV ORF47 protein is capable of phosphorylating several cellular or viral proteins, it is not required for phosphorylation of the ORF63 protein in virus-infected cells.     

A simian varicella virus (SVV) homolog to varicella-zoster virus gene 21 is expressed in monkey ganglia latently infected with SVV.

We have sequenced a simian varicella virus (SVV) open reading frame (ORF), 3,123 bp in length, whose product has 51% amino acid homology with the sequence encoded by the ORF of varicella-zoster virus gene 21. Several regions are highly conserved between the two ORFs, with homologies of approximately 80%. The SVV gene is transcribed in tissue culture cells productively infected with SVV and in monkey ganglia latently infected with SVV.     

Identification and characterization of a human cytomegalovirus gene coding for a membrane protein that is conserved among human herpesviruses.

A rabbit antiserum was raised against envelope material from purified human cytomegalovirus strain AD169. The serum recognized polypeptides 200, 170, 160, 75, 58, and 45 kilodaltons in size. It was used to screen a cDNA library constructed from poly(A)+ RNA from human cytomegalovirus-infected cells in the expression vector lambda gt11. A recombinant bacteriophage expressing cytomegalovirus-specific sequences was identified, and the corresponding gene was mapped to the HindIII R fragment. The gene is transcribed into a late 1.5-kilobase RNA. The nucleotide sequence of the coding region was determined. Computer analysis of the gene product revealed a polypeptide containing multiple potential membrane-spanning domains, representing a type of protein not identified in the envelope of herpesviruses before. The protein shows homology on the amino acid level to hypothetical proteins from reading frames BBRF3 of Epstein-Barr virus, UL10 of herpes simplex virus type 1, and ORF50 of varicella-zoster virus. By using an antiserum raised against procaryote-expressed parts of the cytomegalovirus membrane protein, a 45-kilodalton structural component of the virus was identified as the gene product.     

Varicella-Zoster Virus Open Reading Frame 48 Encodes an Active Nuclease

Based on a DNA sequence and relative genomic position similar to those other herpesviruses, varicella-zoster virus (VZV) open reading frame 48 (ORF48) is predicted to encode an alkaline nuclease. Here we report the cloning, expression, purification, and characterization of recombinant VZV ORF48 protein and a VZV ORF48 point mutation (T172P). Protein encoded by wild-type ORF48, but not mutant protein, displayed both endo- and exonuclease activity, identifying ORF48 as a potential therapeutic target in VZV disease since efficient viral replication requires viral nuclease activity.     

Murine gammaherpesvirus 68 open reading frame 45 plays an essential role during the immediate-early phase of viral replication

Murine gammaherpesvirus 68 (MHV-68) has been developed as a model for the human gammaherpesviruses Epstein-Barr virus and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV), which are associated with several types of human diseases. Open reading frame 45 (ORF45) is conserved among the members of the Gammaherpesvirinae subfamily and has been suggested to be a virion tegument protein. The repression of ORF45 expression by small interfering RNAs inhibits MHV-68 viral replication. However, the gene product of MHV-68 ORF45 and its function have not yet been well characterized. In this report, we show that MHV-68 ORF45 is a phosphorylated nuclear protein. We constructed an ORF45-null MHV-68 mutant virus (45STOP) by the insertion of translation termination codons into the portion of the gene encoding the N terminus of ORF45. We demonstrated that the ORF45 protein is essential for viral gene expression immediately after the viral genome enters the nucleus. These defects in viral replication were rescued by providing ORF45 in trans or in an ORF45-null revertant (45STOP.R) virus. Using a transcomplementation assay, we showed that the function of ORF45 in viral replication is conserved with that of its KSHV homologue. Finally, we found that the C-terminal 23 amino acids that are highly conserved among the Gammaherpesvirinae subfamily are critical for the function of ORF45 in viral replication.     

Strain variation of R5 direct repeats in the right-hand portion of the long unique segment of varicella-zoster virus DNA

We located a region of interstrain size variability in a short segment in an area at the right-hand end of the long unique sequence of the varicella-zoster viral genome. Varicella-zoster virus strains isolated in a district of Japan were classified into three groups on the basis of the size of this segment. Sequence comparison of the variable segment among strains from different groups revealed that the tandem direct repeat, R5, in the segment was variable among strains. R5, which was first discovered in a European strain (Dumas), contained a direct duplication of 88-base-pair (bp) elements separated by a 24-bp element (A.J. Davison and J.E. Scott, J. Gen. Virol. 67:1759-1816, 1986). We found that one 88-bp element and one 24-bp element constitute a repeating unit whose copy number varied from one to three among strains. The simplest R5 we detected was similar to that of Dumas, but there were a few base mismatches between these two R5 structures.     

The 160,000-Mr virion protein encoded at the right end of the herpesvirus saimiri genome is homologous to the 140,000-Mr membrane antigen encoded at the left end of the Epstein-Barr virus genome.

The sequence of 4.4 kilobase pairs (kbp) from the conventional right terminus of the A + T-rich light-DNA (L-DNA) sequences of the herpesvirus saimiri (HVS) genome contains a leftward-directed open reading frame (ORF) for a 1,299-residue protein. The molecular weight predicted for the protein (143,000) is in good agreement with the estimates of 150,000 to 160,000 for the major nonglycosylated polypeptide of the virion tegument (the 160K polypeptide), previously shown to be encoded by this region of the genome. The first initiation codon of the ORF is only 250 nucleotides from the junction of the L-DNA component with the G + C-rich terminal reiterations (i.e., heavy or H-DNA) of the genome. An unusually A + T-rich sequence (43 of 45 nucleotides are A or T, relative to a mean composition of 40% G + C for the ORF) occurs some 75 bp 5' to this initiation codon, and the first adenylation signal (AATAAA) on this DNA strand occurs 18 bp 3' to the termination codon. The amino acid sequence predicted for the 160K protein of HVS is homologous over most of its length to the 1,318-residue protein encoded by the leftmost major ORF of the G + C-rich genome of Epstein-Barr virus (BNRF1, the 140K nonglycosylated membrane antigen). No homology to either of these proteins is evident among the products predicted from the complete sequence of the alpha herpesvirus varicella-zoster virus. Thus gamma herpesviruses with coding sequences which differ in mean nucleotide composition by some 20% G + C have homologous proteins encoded at similar positions with respect to genome termini, with the right end of HVS being homologous to the left end of Epstein-Barr virus.     

Varicella-zoster virus open reading frame 61 protein is functionally homologous to herpes simplex virus type 1 ICP0.

The varicella-zoster virus (VZV) open reading frame 61 (ORF61) protein is thought to be the homolog of herpes simplex virus type 1 (HSV-1) ICP0, based on gene location and limited amino acid homology. However, HSV-1 ICP0 trans activates HSV-1 genes, while VZV ORF61 protein trans represses the function of VZV trans activators on VZV promoters in transient expression assays. To investigate the functional relatedness of HSV-1 ICP0 and VZV ORF61 protein, we established Vero and MeWo cell lines which stably express VZV ORF61 under the control of a metallothionein promoter and performed complementation studies with an HSV-1 ICP0 deletion mutant (7134). Mutant 7134 is impaired for plaque formation and replication at a low multiplicity of infection in cell culture, but these defects were complemented by up to 200-fold in Vero cell lines expressing VZV ORF61. Likewise, the efficiency of plaque formation was improved by up to 100-fold in MeWo cell lines expressing VZV ORF61. A cell line expressing another VZV immediate-early gene product (ORF62) was unable to complement mutant 7134. HSV-1 mutants which are deleted for other HSV-1 immediate-early gene products (ICP4, ICP27) were unable to grow in VZV ORF61-expressing cell lines. These results indicate that, despite marked differences in their sequences and in effects on their cognate promoters in transient expression assays, VZV ORF61 protein is the functional homolog of HSV-1 ICP0.     

Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons.

The complete nucleotide sequence of plasmid pGI3 from Bacillus thuringiensis subsp. thuringiensis H1.1. was obtained. Although this 11,365-bp molecule contained at least 11 putative open reading frames (ORFs), extensive database searches did not reveal any homologous sequences with the exception of ORF6, which displayed similarity to the largest ORF of pSTK1, a 1,883-bp cryptic plasmid isolated from Bacillus stearothermophilus. Deletion analysis to determine the pGI3 minimal replicon revealed that ORF6 is the rep gene. Replication occurred via a single-stranded DNA (ssDNA) intermediate, as demonstrated by S1 treatment and Southern hybridization in nondenaturating conditions. Interestingly, however, no homology was found between the pGI3 (ORF6) and pSTK1 (ORF3) rep genes and those from other single-stranded DNA plasmids, nor was there any DNA similarity to the double-strand origins of replication characterized so far, indicating that pGI3 and pSTK1 form another, new family of ssDNA plasmids. PCR analysis revealed that the pGI3 rep gene is largely distributed among B. thuringiensis strains but can also be found in B. cereus and B. mycoides strains, albeit at a lower frequency. Finally, segregation experiments performed with B. subtilis and B. thuringiensis showed that the pGI3 derivatives, including the minimal replicon, were segregationally stable at temperatures suitable for B. thuringiensis growth (<43 degrees C).