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.     

Varicella-zoster virus (VZV) open reading frame 10 protein, the homolog of the essential herpes simplex virus protein VP16, is dispensable for VZV replication in vitro.

Varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein in the homolog of the herpes simplex virus type 1 (HSV-1) protein VP16. VZV ORF10 transactivates the VZV IE62 gene and is a tegument protein present in the virion. HSV-1 VP16, a potent transactivator of HSV-1 immediate-early genes and tegument protein, is essential for HSV-1 replication in vitro. To determine whether VZV ORF10 is required for viral replication in vitro, we constructed two VZV mutants which were unable to express ORF10. One mutant had a stop codon after the 61st codon of the ORF10 gene, and the other mutant was deleted for all but the last five codons of the gene. Both VZV mutants grew in cell culture to titers similar to that of the parental virus. To determine whether HSV-1 VP16 alters the growth of VZV, we constructed a VZV mutant in which VP16 was inserted in place of ORF10. Using immune electron microscopy, we found that HSV-1 VP16 was present in the tegument of the recombinant VZV virions. The VZV VP16 substitution mutant produced smaller plaques and grew to a lower titer than parental virus. Thus, VZV ORF10 is not required for growth of the virus in vitro, and substitution of HSV-1 VP16 for VZV ORF10 impairs the growth of VZV.     

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.     

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.     

Varicella-zoster virus open reading frame 4 protein is functionally distinct from and does not complement its herpes simplex virus type 1 homolog, ICP27.

Varicella-zoster virus (VZV) open reading frame 4 (ORF4) encodes a putative immediate-early protein which is homologous to herpes simplex virus type 1 (HSV-1) ICP27 on the basis of gene location and similarity in amino acid sequence. In transient expression assays, however, ORF4 and ICP27 exhibit different properties. ICP27 alone has little activity on target plasmids, but it acts as a transactivator or a transrepressor in the presence of other HSV-1 transactivators. In contrast, ORF4 directly transactivates plasmids containing homologous or heterologous promoters and has no apparent transrepressing activity. To further illuminate the functional similarities and differences between ORF4 and ICP27, Vero cell lines which express ORF4 under the inducible metallothionein promoter were constructed. Cell lines expressing functionally active ORF4 protein upregulated the expression of transfected VZV target plasmids but were unable to efficiently complement HSV-1 ICP27 mutants. These results indicate that, despite structural similarities, VZV ORF4 and HSV-1 ICP27 behave differently in transient expression assays and may play different roles in virus replication.     

Immunization with the immediate-early tegument protein (open reading frame 62) of varicella-zoster virus protects guinea pigs against virus challenge.

The IE62 protein, the primary regulatory protein of varicella-zoster virus (VZV) and the major component of the virion tegument, was an effective immunogen in the guinea pig model of VZV infection, whereas the ORF 29 gene product, a nonstructural DNA replication protein, did not elicit protection. All animals immunized with the ORF 29 protein had cell-associated viremia compared with 2 of 11 guinea pigs given the IE62 protein (P = 0.005). VZV was detected in ganglia from 38% of the animals given the ORF 29 protein and 44% of the control animals compared with 9% of the animals immunized with the IE62 protein (P = 0.04). In contrast to the IE62 protein, immunization with the ORF 29 protein did not prime the animals for an enhanced T-cell response upon challenge with infectious virus. The VZV IE62 protein has potential value as a vaccine component.     

Varicella-zoster virus open reading frame 66 protein kinase is required for efficient viral growth in primary human corneal stromal fibroblast cells

Varicella-zoster virus (VZV) open reading frame 66 (ORF66) encodes a serine/threonine protein kinase that is not required for VZV growth in most cell types but is needed for efficient growth in T cells. The ORF66 kinase affects nuclear import and virion packaging of IE62, the major regulatory protein, and is known to regulate apoptosis in T cells. Here, we further examined the importance of ORF66 using VZV recombinants expressing green fluorescent protein (GFP)-tagged functional and kinase-negative ORF66 proteins. VZV virions with truncated or kinase-inactivated ORF66 protein were marginally reduced for growth and progeny yields in MRC-5 fibroblasts but were severely growth and replication impaired in low-passage primary human corneal stromal fibroblasts (PCF). To determine if the growth impairment was due to ORF66 kinase regulation of IE62 nuclear import, recombinant VZVs that expressed IE62 with alanine residues at S686, the suspected target by which ORF66 kinase blocks IE62 nuclear import, were made. IE62 S686A expressed by the VZV recombinant remained nuclear throughout infection and was not packaged into virions. However, the mutant virus still replicated efficiently in PCF cells. We also show that inactivation of the ORF66 kinase resulted in only marginally increased levels of apoptosis in PCF cells, which could not fully account for the cell-specific growth requirement of ORF66 kinase. Thus, the unique short region VZV kinase has important cell-type-specific functions that are separate from those affecting IE62 and apoptosis.     

Mutational analysis of the human immunodeficiency virus vpr open reading frame.

Mutations were introduced by recombinant DNA techniques into the vpr open reading frame of an infectious molecular clone of human immunodeficiency virus type 1. The effect of these changes on the replicative and cytopathologic properties of the virus recovered from transfected cells was studied in several human CD4+ lymphocyte cell lines. In all cases, mutant viruses were infectious and cytopathic. However, when a low-input dose was used, mutants grew significantly more slowly than the wild-type virus. The growth kinetics of vpr mutants were distinct from those of vif and vpu mutants.     

Phosphorylation of varicella-zoster virus open reading frame (ORF) 62 regulatory product by viral ORF 47-associated protein kinase.

Varicella-zoster virus (VZV) encodes within its unique long region a gene product with protein kinase motifs. In a previous study, we demonstrated that immunoprecipitated VZV open reading frame (ORF) 47 protein was associated with a functional protein kinase activity, on the basis of its ability to both autophosphorylate and phosphorylate artificial substrates. To further define potential substrates of ORF 47-associated protein kinase, we analyzed individual viral phosphoproteins to determine whether any were modified by the viral protein kinase. These candidates included gene products of VZV ORFs 4, 61, 62, and 63, which are homologs of herpes simplex virus type 1 (HSV-1) immediate-early proteins. Each of the above VZV proteins was coimmunoprecipitated with ORF 47 kinase, and the immune complex was incubated in a protein kinase assay. Under these conditions, only the VZV immediate-early ORF 62 protein was phosphorylated by ORF 47-associated protein kinase. The specificity of this phosphorylation event was analyzed by a competition assay in which a recombinant ORF 47 protein lacking enzymatic activity was able to reduce the amount of phosphorylation of ORF 62 protein by VZV ORF 47-associated kinase. To provide an additional evaluation of specificity, the experiment was repeated with [32P]GTP instead of [32P]ATP, because the VZV ORF 47 kinase has the distinctive property of using GTP as a phosphate donor. Again the ORF 62 substrate was phosphorylated. In summary, the VZV ORF 47-associated protein kinase (the HSV-1 UL13 homolog) catalyzed the in vitro phosphorylation of the VZV ORF 62 protein, the homolog of the HSV-1 ICP4 regulatory protein.     

Human T-lymphotropic virus type 1 open reading frame I p12(I) is required for efficient viral infectivity in primary lymphocytes

Human T-lymphotropic virus type 1 (HTLV-1) is a complex retrovirus encoding regulatory and accessory genes in four open reading frames (ORF I to IV) of the pX region. Emerging evidence indicates an important role for the pX ORF I-encoded accessory protein p12(I) in viral replication, but its contribution to viral pathogenesis remains to be defined. p12(I) is a conserved, membrane-associated protein containing four SH3-binding motifs (PXXP). Its interaction with the interleukin-2 (IL-2) receptor beta- and gamma-chains implies an involvement of p12(I) in intracellular signaling pathways. In addition, we have demonstrated that expression of pX ORF I p12(I) is essential for persistent infection in rabbits. In contrast, standard in vitro systems have thus far failed to demonstrate a contribution of p12(I) to viral infectivity and ultimately cellular transformation. In this study we developed multiple in vitro coculture assays to evaluate the role of p12(I) in viral infectivity in quiescent peripheral blood mononuclear cells to more accurately reflect the virus-cell interactions as they occur in vivo. Using these assays, we demonstrate a dramatic reduction in viral infectivity in quiescent T lymphocytes for a p12 mutant viral clone (ACH.p12) in comparison to the wild-type clone ACH. Moreover, addition of IL-2 and phytohemagglutinin during the infection completely rescued the ability of ACH.p12 to infect primary lymphocytes. When newly infected primary lymphocytes are used to passage virus, ACH.p12 also exhibited a reduced ability to productively infect activated lymphocytes. Our data are the first to demonstrate a functional role for pX ORF I in the infection of primary lymphocytes and suggest a role for p12(I) in activation of host cells during early stages of infection.     

Varicella-zoster virus (VZV) ORF17 protein induces RNA cleavage and is critical for replication of VZV at 37 degrees C but not 33 degrees C

Varicella-zoster virus (VZV) open reading frame 17 (ORF17) is homologous to herpes simplex virus (HSV) UL41, which encodes the viral host shutoff protein (vhs). HSV vhs induces degradation of mRNA and rapid shutoff of host protein synthesis. An antibody to ORF17 protein detected a 46-kDa protein in VZV-infected cells. While HSV vhs is located in virions, VZV ORF17 protein was not detectable in virions. ORF17 protein induced RNA cleavage, but to a substantially lesser extent than HSV-1 vhs. Expression of ORF17 protein did not inhibit expression from a beta-galactosidase reporter plasmid, while HSV type 1 vhs abolished reporter expression. Two VZV ORF17 deletion mutants were constructed to examine the role of ORF17 in virus replication. While the ORF17 VZV mutants grew to peak titers that were similar to those of the parental virus at 33 degrees C, the ORF17 mutants grew to 20- to 35-fold-lower titers than parental virus at 37 degrees C. ORF62 protein was distributed in a different pattern in the nuclei and cytoplasm of cells infected with an ORF17 deletion mutant at 37 degrees C compared to 33 degrees C. Inoculation of cotton rats with the ORF17 deletion mutant resulted in a level of latent infection similar to that produced by inoculation with the parental virus. The importance of ORF17 protein for viral replication at 37 degrees C but not at 33 degrees C suggests that this protein may facilitate the growth of virus in certain tissues in vivo.