The interaction of herpes simplex virus 1 regulatory protein ICP22 with the cdc25C phosphatase is enabled in vitro by viral protein kinases US3 and UL13

Earlier studies have shown that ICP22 and the U(L)13 protein kinase but not the U(S)3 kinase are required for optimal expression of a subset of late (gamma(2)) genes exemplified by U(L)38, U(L)41, and U(S)11. In primate cells, ICP22 mediates the disappearance of inactive isoforms of cdc2 and degradation of cyclins A and B1. Active cdc2 acquires a new partner, the viral DNA synthesis processivity factor U(L)42. The cdc2-U(L)42 complex recruits and phosphorylates topoisomerase IIalpha for efficient expression of the gamma(2) genes listed above. In uninfected cells, the cdc25C phosphatase activates cdc2 by removing two inhibitory phosphates. The accompanying report shows that in the absence of cdc25C, the rate of degradation of cyclin B1 is similar to that occurring in infected wild-type mouse embryo fibroblast cells but the levels of cdc2 increase, and the accumulation of a subset of late proteins and virus yields are reduced. This report links ICP22 with cdc25C. We show that in infected cells, ICP22 and U(S)3 protein kinase mediate the phosphorylation of cdc25C at its C-terminal domain. In in vitro assays with purified components, both U(L)13 and U(S)3 viral kinases phosphorylate cdc25C and ICP22. cdc25C also interacts with cdc2. However, in infected cells, the ability of cdc25C to activate cdc2 by dephosphorylation of the inactive cdc2 protein is reduced. Coupled with the phosphorylation of cdc25C by the U(S)3 kinase, the results raise the possibility that herpes simplex virus 1 diverts cdc25C to perform functions other than those performed in uninfected cells.     

Posttranslational processing of infected cell protein 22 mediated by viral protein kinases is sensitive to amino acid substitutions at distant sites and can be cell-type specific

Infected cell protein 22 (ICP22) is posttranslationally phosphorylated by the viral kinases encoded by U(S)3 and U(L)13 and nucleotidylylated by casein kinase II. In rabbit and rodent cells and in primary human fibroblasts infected with mutants from which the alpha 22 gene encoding ICP22 had been deleted, a subset of late (gamma(2)) gene products exemplified by U(L)38 and U(S)11 proteins are expressed at a reduced level, as measured by the accumulation of both mRNA and protein. The same phenotype was observed in cells infected with mutants lacking the U(L)13 gene. The focus of this report is on three serine- and threonine-rich domains of ICP22. Two of these domains are homologs located between residues 38 to 66 and 300 to 328. The third domain is near the carboxyl terminus and contains the sequence T374SS. The results were as follows. (i) Alanine substitutions in the amino-terminal homolog precluded the posttranslational processing of ICP22 in rabbit skin cells and in Vero cells but had no effect on the accumulation of either U(S)11 or U(L)38 protein. (ii) Alanine substitutions in the carboxyl-terminal homolog had no effect on posttranslational processing of ICP22 accumulating in Vero cells but precluded full processing of ICP22 accumulating in rabbit skin cells. The effect on accumulation of U(L)38 and U(S)11 proteins was insignificant in Vero cells and minimal in rabbit skin cells. (iii) Substitutions of alanine for the threonine and serines in the third domain precluded full processing of ICP22 and caused a reduction of accumulation of U(S)11 and U(L)38 proteins. These results indicate the following. (i) The posttranslational processing of ICP22 is sensitive to mutations within the domains of ICP22 tested and is cell-type dependent. (ii) Posttranslational processing of ICP22 is not required for accumulation of U(L)38 and U(S)11 proteins to the same level as that seen in cells infected with the wild-type virus. (iii) The T374SS sequence shared by ICP22 and the U(S)1.5 proteins is essential for the accumulation of a subset of gamma(2) proteins exemplified by U(S)11 and U(L)38 and is the first step in mapping of the sequences necessary for optimal accumulation of U(S)11 and U(L)38 proteins.     

State and role of SRC family kinases in replication of herpes simplex virus 1

An earlier report showed that infected cell protein no. 0 (ICP0) of herpes simplex virus 1 (HSV-1) interacts with the SH3 domains of a recently discovered adaptor protein, CIN85. Here, we report the following. (i) ICP0 also interacts with other SH3 domain-containing proteins and, in particular, with nonneuronal members of the Src kinase family. (ii) HSV-1 infection enhanced the activating phosphorylation of Tyr416 of the members of the Src kinase family, modestly enhanced the kinase activity of Src, and posttranslationally modified at least one additional member of the Src kinase family by phosphorylation in a manner dependent on the viral gene products ICP0, unique short 3 (U(S)3), and unique long 13 (U(L)13). (iii) To define the roles of Src kinase family members, we examined the accumulation of viral proteins, DNA, and mRNA and virus yields from wild-type mouse embryo fibroblasts and sibling cells lacking Src, Fyn, and Yes (SYF-); a mutant cell line, +Src, in which Src was restored to SYF- cells; and the mutant cell line (CSK-) lacking the negative regulator Csk gene of the Src kinase family. Representative alpha, beta, and gamma2 proteins accumulated in the largest amounts in SYF- cells and the smallest amounts in +Src compared to wild-type cells. The CSK- cells yielded smaller amounts of the gamma2 protein and at least 10-fold less virus than wild-type cells. We conclude that HSV-1 proteins regulate the activities of Src family kinases to achieve optimal viral yields in the course of viral replication.     

Herpes simplex virus 1 ICP22 regulates the accumulation of a shorter mRNA and of a truncated US3 protein kinase that exhibits altered functions

The U(S)3 open reading frame of herpes simplex virus 1 (HSV-1) was reported to encode two mRNAs each directing the synthesis of the same protein. We report that the U(S)3 gene encodes two proteins. The predominant U(S)3 protein is made in wild-type HSV-1-infected cells. The truncated mRNA and a truncated protein designated U(S)3.5 and initiating from methionine 77 were preeminent in cells infected with a mutant lacking the gene encoding ICP22. Both the wild-type and truncated proteins also accumulated in cells transduced with a baculovirus carrying the entire U(S)3 open reading frame. The U(S)3.5 protein accumulating in cells infected with the mutant lacking the gene encoding ICP22 mediated the phosphorylation of histone deacetylase 1, a function of U(S)3 protein, but failed to block apoptosis of the infected cells. The U(S)3.5 and U(S)3 proteins differ with respect to the range of functions they exhibit.     

The products of the herpes simplex virus type 1 immediate-early US1/US1.5 genes downregulate levels of S-phase-specific cyclins and facilitate virus replication in S-phase Vero cells

Herpes simplex virus type 1 ICP22-/U(S)1.5- mutants initiate viral gene expression in all cells; however, in most cell types, the replication process stalls due to an inability to express gamma2 late proteins. Although the function of ICP22/U(S)1.5 has not been established, it has been suggested that these proteins activate, induce, or repress the activity of cellular proteins during infection. In this study, we hypothesized that cell cycle-associated proteins are targets of ICP22/U(S)1.5. For this purpose, we first isolated and characterized an ICP22-/U(S)1.5- mutant virus, 22/n199. Like other ICP22-/U(S)1.5- mutants, 22/n199 replicates in a cell-type-specific manner and fails to induce efficient gamma2 late gene expression in restrictive cells. Although synchronization of restrictive human embryonic lung cells in each phase of the cell cycle did not overcome the growth restrictions of 22/n199, synchronization of permissive Vero cells in S phase rendered them less able to support 22/n199 plaque formation and replication. Consistent with this finding, expression of cellular S-phase cyclins was altered in an ICP22/U(S)1.5-dependent manner specifically when S-phase Vero cells were infected. Collectively, these observations support the notion that ICP22/U(S)1.5 deregulates the cell cycle upon infection of S-phase permissive cells by altering expression of key cell cycle regulatory proteins either directly or indirectly.     

Small dense nuclear bodies are the site of localization of herpes simplex virus 1 U(L)3 and U(L)4 proteins and of ICP22 only when the latter protein is present

The herpes simplex virus 1 U(L)3 and U(L)4 open reading frames are expressed late in infection and are not essential for viral replication in cultured cells in vitro. An earlier report showed that the U(L)4 protein colocalizes with the products of the alpha22/U(S)1.5 genes in small nuclear dense bodies. Here we report that the U(L)3 protein also colocalized in these small nuclear dense bodies and the localization of U(L)3 and U(L)4 proteins in these bodies required the presence of alpha22/U(S)1.5 genes. In cells infected with a mutant lacking intact alpha22/U(S)1.5 genes, U(L)3 was diffused throughout the nucleus even though the overall accumulation of the gamma2 U(L)3 protein was decreased. The results suggest that ICP22 acts both as a regulator of U(L)3 accumulation and as the structural component and anchor of these small dense nuclear bodies.     

Functional anatomy of herpes simplex virus 1 overlapping genes encoding infected-cell protein 22 and US1.5 protein

Earlier studies have shown that (i) the coding domain of the alpha22 gene encodes two proteins, the 420-amino-acid infected-cell protein 22 (ICP22) and a protein, US1.5, which is initiated from methionine 147 of ICP22 and which is colinear with the remaining portion of that protein; (ii) posttranslational processing of ICP22 mediated largely by the viral protein kinase UL13 yields several isoforms differing in electrophoretic mobility; and (iii) mutants lacking the carboxyl-terminal half of the ICP22 and therefore DeltaUS1.5 are avirulent and fail to express normal levels of subsets of both alpha (e.g., ICP0) or gamma2 (e.g., US11 and UL38) proteins. We have generated and analyzed two sets of recombinant viruses. The first lacked portions of or all of the sequences expressed solely by ICP22. The second set lacked 10 to 40 3'-terminal codons of ICP22 and US1. 5. The results were as follows. (i) In cells infected with mutants lacking amino-terminal sequences, translation initiation begins at methionine 147. The resulting protein cannot be differentiated in mobility from authentic US1.5, and its posttranslational processing is mediated by the UL13 protein kinase. (ii) Expression of US11 and UL38 genes by mutants carrying only the US1.5 gene is similar to that of wild-type parent virus. (iii) Mutants which express only US1. 5 protein are avirulent in mice. (iv) The coding sequences Met147 to Met171 are essential for posttranslational processing of the US1.5 protein. (v) ICP22 made by mutants lacking 15 or fewer of the 3'-terminal codons are posttranslationally processed whereas those lacking 18 or more codons are not processed. (vi) Wild-type and mutant ICP22 proteins localized in both nucleus and cytoplasm irrespective of posttranslational processing. We conclude that ICP22 encodes two sets of functions, one in the amino terminus unique to ICP22 and one shared by ICP22 and US1.5. These functions are required for viral replication in experimental animals. US1.5 protein must be posttranslationally modified by the UL13 protein kinase to enable expression of a subset of late genes exemplified by UL38 and US11. Posttranslational processing is determined by two sets of sequences, at the amino terminus and at the carboxyl terminus of US1.5, respectively, a finding consistent with the hypothesis that both domains interact with protein partners for specific functions.     

Herpes Simplex Virus 1 ICP22 but Not US1.5 Is Required for Efficient Acute Replication in Mice and VICE Domain Formation

The herpes simplex virus 1 (HSV-1) immediate-early protein, infected cell protein 22 (ICP22), is required for efficient replication in restrictive cells, for virus-induced chaperone-enriched (VICE) domain formation, and for normal expression of a subset of viral late proteins. Additionally, ICP22 is important for optimal acute viral replication in vivo. Previous studies have shown that the U_S1 gene that encodes ICP22, produces an in-frame, N-terminally truncated form of ICP22, known as U_S1.5. To date, studies conducted to characterize the functions of ICP22 have not separated its functions from those of U_S1.5. To determine the individual roles of ICP22 and U_S1.5, we made viral mutants that express either ICP22 with an M90A mutation in the U_S1.5 initiation codon (M90A) or U_S1.5 with three stop codons introduced upstream of the U_S1.5 start codon (3×stop). Our studies showed that, in contrast to M90A, 3×stop was unable to replicate efficiently in the eyes and trigeminal ganglia of mice during acute infection, to efficiently establish a latent infection, or to induce VICE domain formation and was only mildly reduced in its replication in restrictive HEL-299 cells and murine embryonic fibroblasts (MEFs). Both mutants enhanced the expression of the late viral proteins virion host shutoff (vhs) and glycoprotein C (gC) and inhibited viral gene expression mediated by HSV-1 infected cell protein 0 (ICP0). When we tested our mutants'' sensitivity to type I interferon (beta interferon [IFN-β]) in restrictive cells, we noticed that the plating of the ICP22 null (d22) and 3×stop mutants was reduced by the addition of IFN-β. Overall, our data suggest that U_S1.5 partially complements the functions of ICP22.