Replication of Herpes Simplex Virus Type 1 DNA is Inhibited in a Temperature-sensitive Mutant of BHK-21 Cells Lacking RCC1 (Regulator of Chromosome Condensation) and Virus DNA Remains Linear

Herpes simplex virus (HSV) immediate-early (IE) gene expression is initiated via the recruitment of the structural protein VP16 onto specific sites upstream of each IE gene promoter in a multicomponent complex (TRF.C) that also includes the cellular proteins Oct-1 and HCF. In vitro results have shown that HCF binds directly to VP16 and stabilizes TRF.C. Results from transfection assays have also indicated that HCF is involved in the nuclear import of VP16. However, there have been no reports on the role or the fate of HCF during HSV type 1 (HSV-1) infection. Here we show that the intracellular distribution of HCF is dramatically altered during HSV-1 infection and that the protein interacts with and colocalizes with VP16. Moreover, viral protein synthesis and replication were significantly reduced after infection of a BHK-21-derived temperaturesensitive cell line (tsBN67) which contains a mutant HCF unable to associate with VP16 at the nonpermissive temperature. Intracellular distribution of HCF and of newly synthesized VP16 in tsBN67-infected cells was similar to that observed in Vero cells, suggesting that late in infection the trafficking of both proteins was not dependent on their association. We constructed a stable cell line (tsBN67r) in which the temperature-sensitive phenotype was rescued by using an epitope-tagged wild-type HCF. In HSV-1-infected tsBN67r cells at the nonpermissive temperature, direct binding of HCF to VP16 was observed, but virus protein synthesis and replication were not restored to levels observed at the permissive temperature or in wild-type BHK cells. Together these results indicate that the factors involved in compartmentalization of VP16 alter during infection and that late in infection, VP16 and HCF may have additional roles reflected in their colocalization in replication compartments.One of the earliest regulatory events after a productive infection by herpes simplex virus (HSV) is the induction of virus immediate-early (IE) gene expression by VP16, a structural component of the viral particle (33). Transcriptional activation is initiated by the recruitment of VP16 (3), together with two cellular proteins, Oct-1 (31, 34, 41) and HCF (15, 50, 52), into a multicomponent complex formed on the regulatory sites, TAATGARAT motifs, present within each of the IE promoters (for reviews, see references 30 and 48). VP16 also plays an essential role later in infection, in virion morphogenesis (45). Although its precise function(s) at this stage remains to be elucidated, VP16 has been shown to interact with at least two virus proteins, VP22 (7) and the virion host shutoff protein (20, 39), both of which are also assembled into the tegument.With regard to its role in IE gene expression, previous results from several laboratories indicate that the first step in assembly of the multicomponent complex (called TRF.C in this work) is the formation of a binary complex between VP16 and HCF, which subsequently associates with Oct-1 already bound to the TAATGARAT motifs (8,15,46). While a precise mech...

Section: Discussionmentioning

confidence: 79%

Analysis of HCF, the Cellular Cofactor of VP16, in Herpes Simplex Virus-Infected Cells

Laboissiere

O’Hare

2000

“…Following entry into a cell, the virion linear double-stranded herpes simplex virus type 1 (HSV-1) genome undergoes a change in physical state in which it assumes a nonlinear, presumably circular form that has been referred to as "endless" (12,20,31). Consistent with the nonlinear, circular form of the input genomes, Strang and Stow (28) demonstrated that the circularization of the HSV genome occurs early in lytic infection.…”

mentioning

confidence: 73%

Evidence that the Immediate-Early Gene Product ICP4 Is Necessary for the Genome of the Herpes Simplex Virus Type 1 ICP4 Deletion Mutant Strain d 120 To Circularize in Infected Cells

Zhang

Wang

et al. 2006

Following infection, the physical state of linear herpes simplex virus (HSV) genomes may change into an "endless" or circular form. In this study, using Southern blot analysis of the HSV genome, we provide evidence that immediate-early protein ICP4 is involved in the process of converting the linear HSV-1 ICP4-deleted mutant strain d120 genome into its endless form. Under conditions where de novo viral DNA synthesis was inhibited, the genome of the ICP4 deletion mutant d120 failed to assume an endless conformation following infection of Vero cells (compared with the ability of wild-type strain KOS). This defect was reversed in the Vero-derived cell line E5, which produces the ICP4 protein, suggesting that ICP4 is necessary and sufficient to complement the d120 defect. When ICP4 protein was provided by the replication-defective DNA polymerase mutant HP66, the genomes of mutant d120 could assume an endless conformation in Vero cells. Western blot analysis using antibody specific to the ICP4 protein showed that although the d120 virions contained ICP4 protein, the majority of that ICP4 protein was in a 40-kDa truncated form, with only a small fraction present as a full-length 175-kDa protein. When expression of ICP4 protein from E5 cells was inhibited by cycloheximide, the d120 virion-associated ICP4 protein was unable to mediate endless formation after infection of E5 cells. Collectively, these data suggest that ICP4 protein has an important role in mediating the endless formation of the HSV-1 genome upon infection and that this function can be provided in trans.

“…We cannot, however, exclude the possibility that circularization by homologous recombination can occur when direct terminal repeats are present. The only cellular gene so far implicated as having a possible role in the circularization of input HSV-1 genomes is RCC1 (regulator of chromosome condensation), but it remains unclear whether its involvement is direct or indirect (44).…”

Section: Discussionmentioning

confidence: 99%

Circularization of the Herpes Simplex Virus Type 1 Genome upon Lytic Infection

Strang

Stow

2005

For many years, the generally accepted model for the replication of the double-stranded DNA genome of herpes simplex virus type 1 (HSV-1) incorporated initial circularization of linear molecules in the cell nucleus. Ensuing DNA synthesis resulted in the generation of head-to-tail concatemers which were subsequently cleaved into monomeric units and packaged into the nascent viral capsid. Recently, however, it has been proposed that circularization of HSV-1 genomes does not occur at the onset of lytic infection and moreover that this event is specifically inhibited by the HSV-1 transcriptional transactivator, ICP0 (S.A. Jackson and N.A. DeLuca, Proc. Natl. Acad. Sci. USA 100:7871-7876, 2003). To further investigate genome circularization, we have generated HSV-1 derivatives in which the viral a sequences, which contain the cleavage-packaging signals, have been replaced by a minimal packaging element located in the thymidine kinase gene. In contrast to wild-type HSV-1, fusion of the genomic termini of these viruses produces a novel fragment in circular or concatemeric DNA which can be detected by Southern blot hybridization. Utilizing these viruses, we demonstrate that fusion of the genomic termini occurred rapidly upon infection and in the presence of inhibitors of viral DNA or protein synthesis. We provide evidence indicating that the end joining represented circularization rather than concatemerization of input molecules and that circularized molecules functioned as templates for replication. Since the termini of these viruses lack direct repeats, our findings indicate that circularization can be mediated by direct end-to-end ligation of linear input genomes.Herpes simplex virus type 1 (HSV-1), the prototype virus of the family Herpesviridae, possesses a linear double-stranded DNA genome of approximately 153 kbp and is widely used as a model for the study of herpesvirus DNA replication and recombination. Like those of other members of the herpesvirus family, the HSV-1 genome is characterized by the presence of unique and repeated sequences (Fig. 1a). Two covalently joined segments, L and S, each comprise a unique region (U L and U S ) flanked by a set of inverted repeats (TR L and IR L , TR S and IR S , respectively). A region of approximately 400 bp, the a sequence, is present as a direct repeat at the genomic termini and in an inverted orientation at the junction between the L and S segments. The L and S segments of the genome have the capacity to invert relative to each other at high frequency, resulting in the appearance of four equimolar isomeric forms of virion DNA (reviewed in reference 35).Following infection, the HSV-1 genome is released into the nucleus and may either be retained in a latent state or enter into the lytic cycle (reviewed in reference 32). During the establishment of latency in both in vivo and tissue culture systems, the ends of the viral genome become joined, and there is now strong evidence for persistence as a nonreplicating circular episomal form (5,17,18,27,32,34,42). The lytic cyc...