Functional interactions between herpes simplex virus immediate-early proteins during infection: gene expression as a consequence of ICP27 and different domains of ICP4

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 C 3 HC 4 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 C 3 HC 4 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 ␣0 mutant viruses results partially from reduced levels of ICP27.

Section: Fig 5 Southern Blot Analysis Of Wild-type and Recombinant mentioning

confidence: 99%

“…ICP27, another essential protein, is required for the expression of ␤ and ␥ genes during the HSV life cycle. This protein appears to act primarily at the posttranscriptional level (47,59,62,65,67,73); however, recent evidence supports a more direct role for ICP27 in the regulation of gene expression (55).…”

mentioning

confidence: 99%

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

Lium

Silverstein

1997

“…The titers of infectious virus in stocks of all the ts mutant viruses were determined simultaneously on Vero cell monolayers at 34ЊC, and stocks of nonsense, deletion, and other mutant viruses were titrated on monolayers of the appropriate cell lines at 37ЊC. The ICP4-ICP27 double-mutant virus, d92, was derived by recombination between d120 and 5dl1.2 (36). Thus, d92 contains the d120 mutant ICP4 allele at both ICP4 loci and the 5dl1.2 mutant ICP27 allele at the single ICP27 locus and was propagated in the E26 cell line, which provides comparable levels of ICP4 and ICP27.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…Since transactivators function in the nucleus, the cytoplasmic localization of ICP0 in ICP4 mutant virus-infected cells would prevent ICP0 from performing its transactivating function. On the other hand, even though ICP0 localizes exclusively within the nucleus, the expression of E and L genes is more restricted in d92-infected cells than in cells infected with either d120 or 5dl1.2 (36). This observation suggests that the inability of ICP0 to activate the expression of E and L genes in ICP4 mutant virus-infected cells is not due solely to the cytoplasmic localization of ICP0 and that other factors, perhaps including ICP4 and/or ICP27, may also be involved in the control of the transactivating activity of ICP0 or the activities of the promoters of E and L genes in the context of the viral genome.…”

Section: Vol 70 1996mentioning

confidence: 99%

Overexpression of the herpes simplex virus type 1 immediate-early regulatory protein, ICP27, is responsible for the aberrant localization of ICP0 and mutant forms of ICP4 in ICP4 mutant virus-infected cells

Zhu

DeLuca

Schaffer

1996

Self Cite

ICP0 and ICP4 are immediate-early regulatory proteins of herpes simplex virus type 1. Previous studies by Knipe and Smith demonstrated that these two proteins are characteristically observed in the nuclei of wild-type virus-infected cells but predominantly in the cytoplasms of cells infected with several ICP4 temperaturesensitive (ts) mutant viruses at the nonpermissive temperature (NPT) (D. M. Knipe and J. L. Smith, Mol. Cell. Biol. 6:2371-2381, 1986). Consistent with this observation, it has been shown previously that ICP0 is present predominantly in the cytoplasms of cells infected with an ICP4 null mutant virus (n12) at high multiplicities of infection and that the level of ICP27, a third viral regulatory protein, plays an important role in determining the intracellular localization of ICP0 (Z. Zhu, W. Cai, and P. A. Schaffer, J. Virol. 68:3027-3040, 1994). To address whether the cytoplasmic localization of ICP0 is a common feature of cells infected with all ICP4 mutant viruses or whether mutant ICP4 polypeptides, together with ICP27, determine the intracellular localization of ICP0, we used double-staining immunofluorescence tests to examine the intracellular staining patterns of ICP0 and ICP4 in cells infected with an extensive series of ICP4 mutant viruses. In these tests, compared with the localization pattern of ICP0 in wild-type virus-infected cells, more ICP0 was detected in the cytoplasms of cells infected with all ICP4 mutants tested at high multiplicities of infection. Each of the mutant forms of ICP4 exhibiting predominantly cytoplasmic staining contains both the nuclear localization signal and the previously mapped ICP27-responsive region (Z. Zhu and P. A. Schaffer, J. Virol. 69: [49][50][51][52][53][54][55][56][57][58][59] 1995). No correlation between the intracellular staining patterns of ICP0 and mutant forms of ICP4 was demonstrated, suggesting that mutant ICP4 polypeptides per se are not responsible for retention of ICP0 in the cytoplasm. This observation was confirmed in studies of cells cotransfected with plasmids expressing ICP0 and mutant forms of ICP4, in which the staining pattern of ICP0 was not changed in the presence of mutant ICP4 proteins. Studies of cells infected at low multiplicities with a variety of ICP4 ts mutant viruses at the NPT showed that both ICP0 and ts forms of ICP4 were localized predominantly within the nucleus. These observations are a further indication that the aberrant localization of the ts forms of ICP4 at the NPT is not a direct result of specific mutations in the ICP4 gene. In the final series of tests, the localization of ICP0 in cells infected with a double-mutant virus unable to express either ICP4 or ICP27 was examined. In these tests, ICP0 was detected exclusively in the nuclei of Vero cells but in both the nuclei and the cytoplasms of ICP27-expressing cells infected with the double mutant. These results demonstrate that ICP27, rather than the absence of functional ICP4, is responsible for the cytoplasmic localization of ICP0 in ICP4 mutant virus-infec...

“…IE63 plays a key role in the switch from early to late gene expression (37,39), and this multifunctional protein has both trans-repressor and trans-activator functions (11,30,43,44). Transfection studies have demonstrated that IE63 acts synergistically with IE pro-teins IE175 and IE110 to repress expression from IE and early promoters and activate expression from late promoters (11,30,43); IE63 also affects the cellular localization of these two IE proteins (41,44). At the posttranscriptional level of gene regulation, enhancement of gene expression correlates with the ability of IE63 to stimulate 3Ј processing (28,29,42) at certain poly(A) sites whereas the repressor function is associated with an inhibition of splicing.…”

mentioning

confidence: 99%

Regulation of herpes simplex virus poly (A) site usage and the action of immediate-early protein IE63 in the early-late switch

McGregor

Phelan

Dunlop

et al. 1996

101

The essential herpes simplex virus type 1 (HSV-1) immediate-early protein IE63 (ICP27) is pleiotropic in function, promoting the switch from the early to late phase of virus gene expression, and has effects on the posttranscriptional processes of mRNA splicing and 3 processing. We have investigated the role of IE63 in the regulation of viral mRNA 3 processing and of late gene expression. Our in vitro 3 processing studies demonstrated that HSV-1 infection induces an activity, which requires IE63 gene expression, responsible for an observed increase in 3 processing of selected HSV-1 poly(A) sites. Processing efficiencies at the poly(A) sites of two late genes, UL38 and UL44, shown to be inherently weak processing sites, were increased by the IE63-induced activity. In contrast, 3 processing at the poly(A) sites of selected immediate-early and early genes, stronger processing sites, was unaffected by IE63 expression. UV cross-linking experiments demonstrated that HSV infection caused enhanced binding of protein factors, including the 64-kDa component of cleavage stimulation factor (CstF), to poly(A) site RNAs from virus genes of all temporal classes and that this enhanced binding required expression of IE63. By immunofluorescence, the homogeneous pattern of the 64-kDa CstF protein distribution became slightly clumped with infection, whereas the splicing small nuclear ribonucleoprotein particles were reorganized into a highly punctate distribution away from the sites of virus transcription. This effect could create an increase in the relative concentration of 3 processing factors available to pre-mRNAs. Western blot (immunoblot) analysis showed that IE63 was required for expression of several true late genes and for the efficient and timely expression of the UL29 and UL42 early genes, integral components of the viral DNA synthesis machinery. Our data are consistent with two effects of IE63 on late gene regulation: firstly, a stimulation of pre-mRNA 3 processing and, secondly, as a requirement for expression of functions necessary for viral DNA synthesis.