Herpes simplex virus ICP27 activation of stress kinases JNK and p38

We previously reported that herpes simplex virus type 1 (HSV-1) can activate the stress-activated protein kinases (SAPKs) p38 and JNK. In the present study, we undertook a comprehensive and comparative analysis of the requirements for viral protein synthesis in the activation of JNK and p38. Infection with the UL36 mutant tsB7 or with UV-irradiated virus indicated that both JNK and p38 activation required viral gene expression. Cycloheximide reversal or phosphonoacetic acid treatment of wild-type virus-infected cells as well as infection with the ICP4 mutant vi13 indicated that only the immediate-early class of viral proteins were required for SAPK activation. Infection with ICP4, ICP27, or ICP0 mutant viruses indicated that only ICP27 was necessary. Additionally, we determined that in the context of virus infection ICP27 was sufficient for SAPK activation and activation of the p38 targets Mnk1 and MK2 by infecting with mutants deleted for various combinations of immediate-early proteins. Specifically, the d100 (0-/4-) and d103 (4-/22-/47-) mutants activated p38 and JNK, while the d106 (4-/22-/27-/47-) and d107 (4-/27-) mutants did not. Finally, infections with a series of ICP27 mutants demonstrated that the functional domain of ICP27 required for activation was located in the region encompassing amino acids 20 to 65 near the N terminus of the protein and that the C-terminal transactivation activity of ICP27 was not necessary.     

Mutations in DNA Binding and Transactivation Domains Affect the Dynamics of Parvovirus NS1 Protein

The multifunctional replication protein of autonomous parvoviruses, NS1, is vital for viral genome replication and for the control of viral protein production. Two DNA-interacting domains of NS1, the N-terminal and helicase domains, are necessary for these functions. In addition, the N and C termini of NS1 are required for activation of viral promoter P38. By comparison with the structural and biochemical data from other parvoviruses, we identified potential DNA-interacting amino acid residues from canine parvovirus NS1. The role of the identified amino acids in NS1 binding dynamics was studied by mutagenesis, fluorescence recovery after photobleaching, and computer simulations. Mutations in the predicted DNA-interacting amino acids of the N-terminal and helicase domains increased the intranuclear binding dynamics of NS1 dramatically. A substantial increase in binding dynamics was also observed for NS1 mutants that targeted the metal ion coordination site in the N terminus. Interestingly, contrary to other mutants, deletion of the C terminus resulted in slower binding dynamics of NS1. P38 transactivation was severely reduced in both N-terminal DNA recognition and in C-terminal deletion mutants. These data suggest that the intranuclear dynamics of NS1 are largely characterized by its sequence-specific and -nonspecific binding to double-stranded DNA. Moreover, binding of NS1 is equally dependent on the N-terminal domain and conserved β-loop of the helicase domain.     

The tetratricopeptide repeat domain and a C-terminal region control the activity of Ser/Thr protein phosphatase 5.

Protein Ser/Thr phosphatase 5 is a 58-kDa protein containing a catalytic domain structurally related to the catalytic subunits of protein phosphatases 1, 2A, and 2B and an extended N-terminal domain with three tetratricopeptide repeats. The activity of this enzyme is stimulated 4-14-fold in vitro by polyunsaturated fatty acids and anionic phospholipids. The structural basis for lipid activation of protein phosphatase 5 was examined by limited proteolysis and site-directed mutagenesis. Trypsinolysis removed the tetratricopeptide repeat domain and increased activity to approximately half that of lipid-stimulated, full-length enzyme. Subtilisin removed the tetratricopeptide repeat domain and 10 residues from the C terminus, creating a catalytic fragment with activity that was equal to or greater than that of lipid-stimulated, full-length enzyme. Catalytic fragments generated by proteolysis were no longer stimulated by lipid, and degradation of the tetratricopeptide repeat domain was decreased by association with lipid. A truncated mutant missing 13 C-terminal residues was also insensitive to lipid and was as active as full-length, lipid-stimulated enzyme. These results suggest that the C-terminal and N-terminal domain act in a coordinated manner to suppress the activity of protein phosphatase 5 and mediate its activation by lipid. These regions may be targets for the regulation of protein phosphatase 5 activity in vivo.     

The extreme carboxyl terminus of the equine herpesvirus 1 homolog of herpes simplex virus VP16 is essential for immediate-early gene activation.

Gene 12 of equine herpesvirus 1 (EHV-1), the homolog of herpes simplex virus (HSV) VP16 (alpha TIF, Vmw65), was cloned into a eukaryotic expression vector by PCR and used in transactivation studies of both the EHV-1 and HSV-1 IE1 promoters. Results demonstrated that the product of gene 12 is a potent transactivator of immediate-early gene expression of both viruses, which requires sequences in the upstream HSV-1 promoter for activity. Mutational analysis of the gene 12 open reading frame indicated that removal of the C-terminal 7 amino acids, which contain a short region of homology with the extreme C terminus of VP16, inactivated the protein. Within this region, only a single methionine residue appeared to be essential for activity, implying that gene 12 may have a modular array of organization similar to that of VP16. However, fusion of the gene 12 C terminus to a truncated form of VP16, which contained the complex formation domain, did not restore activity to the HSV-1 protein. These data demonstrate that the EHV-1 immediate-early transactivator may not be functionally colinear with VP16, with transactivation requiring both the C terminus and another region(s) present within the N-terminal portion.     

Mutational analysis of the herpes simplex virus type 1 ICP0 C3HC4 zinc ring finger reveals a requirement for ICP0 in the expression of the essential alpha27 gene.

The herpes simplex virus type 1 (HSV-1) immediate-early (IE) protein ICP0 has been implicated in the regulation of viral gene expression and the reactivation of latent HSV-1. Evidence demonstrates that ICP0 is an activator of viral gene expression yet does not distinguish between a direct or indirect role in this process. To further our understanding of the function of ICP0 in the context of the virus life cycle, site-directed mutagenesis of the consensus C3HC4 zinc finger domain was performed, and the effects of these mutations on the growth and replication of HSV-1 were assessed. We demonstrate that alteration of any of the consensus C3HC4 cysteine or histidine residues within this domain abolishes ICP0-mediated transactivation, alters the intranuclear localization of ICP0, and significantly increases its stability. These mutations result in severe defects in the growth and DNA replication of recombinant herpesviruses and in their ability to initiate lytic infections at low multiplicities of infection. These viruses, at low multiplicities of infection, synthesize wild-type levels of the IE proteins ICP0 and ICP4 at early times postinfection yet exhibit significant decreases in the synthesis of the essential IE protein ICP27. These findings reveal a role for ICP0 in the expression of ICP27 and suggest that the multiplicity-dependent growth of alpha0 mutant viruses results partially from reduced levels of ICP27.     

Herpes simplex virus type 1 ICP0 phosphorylation mutants impair the E3 ubiquitin ligase activity of ICP0 in a cell type-dependent manner

Herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) is a 110-kDa nuclear phosphoprotein that is required for both the efficient initiation of lytic infection and the reactivation of quiescent viral genomes from latency. The ability of ICP0 to act as a potent viral transactivator is mediated by its N-terminal zinc-binding RING finger domain. This domain confers E3 ubiquitin ligase activity to ICP0 and is required for the proteasome-dependent degradation of a number of cellular proteins during infection, including the major nuclear domain 10 (ND10) constituent protein promyelocytic leukemia. In previous work we mapped three phosphorylation regions within ICP0, two of which directly affected its transactivation capabilities in transient transfection assays (Davido et al., J. Virol. 79:1232-1243, 2005). Because ICP0 is a phosphoprotein, we initially sought to test the hypothesis that phosphorylation regulates the E3 ubiquitin ligase activity of ICP0. Although none of the mutations affected ICP0 E3 ligase activity in vitro, transient transfection analysis indicated that mutations within one or more of the phosphorylated regions impaired the ability of ICP0 to form foci with colocalizing conjugated ubiquitin and to disrupt ND10. Mutations within one of the regions also affected ICP0 stability, and all of these phenomena occurred in a cell type-dependent manner. In the context of viral infection, only one ICP0 phosphorylation mutant (P1) showed a significant defect in viral replication and enhanced protein stability compared to all the other viruses tested. This study suggests that specific cellular environments and context of expression (transfection versus infection) differentially regulate several activities of ICP0 related to its E3 ubiquitin ligase activity via phosphorylation.     

Identification of sequences in herpes simplex virus type 1 ICP22 that influence RNA polymerase II modification and viral late gene expression

Previous studies have shown that the herpes simplex virus type 1 (HSV-1) immediate-early protein ICP22 alters the phosphorylation of the host cell RNA polymerase II (Pol II) during viral infection. In this study, we have engineered several ICP22 plasmid and virus mutants in order to map the ICP22 sequences that are involved in this function. We identify a region in the C-terminal half of ICP22 (residues 240 to 340) that is critical for Pol II modification and further show that the N-terminal half of the protein (residues 1 to 239) is not required. However, immunofluorescence analysis indicates that the N-terminal half of ICP22 is needed for its localization to nuclear body structures. These results demonstrate that ICP22's effects on Pol II do not require that it accumulate in nuclear bodies. As ICP22 is known to enhance viral late gene expression during infection of certain cultured cells, including human embryonic lung (HEL) cells, we used our engineered viral mutants to map this function of ICP22. It was found that mutations in both the N- and C-terminal halves of ICP22 result in similar defects in viral late gene expression and growth in HEL cells, despite having distinctly different effects on Pol II. Thus, our results genetically uncouple ICP22's effects on Pol II from its effects on viral late gene expression. This suggests that these two functions of ICP22 may be due to distinct activities of the protein.