An SR-protein induced by HSVI binding to cells functioning as a splicing inhibitor of viral pre-mRNA

The interaction between a virus and its specific receptor on the membrane of the host cell mimics the physiological combination of signal ligand and its receptor, and initiates the specific signal transduction from this activated receptor to induce a relative gene response. During the investigation of the interaction between Herpes simplex virus I (HSVI) and human fibroblast via the virus binding to its receptor complex on the cellular membrane, a new gene of cellular response against the specific stimulation of HSVI binding to fibroblasts was cloned from a cDNA library established from mRNA of an early gene response. This gene encoded a protein of 14.9kDa with the structural characteristics of Arg-rich and RS repeats. The analysis of the role of this protein in the infection by HSVI indicated that this protein, expressed only in G(1)/S phase and phosphorylated, functioned as a splicing inhibitor of HSVI pre-mRNA. The details of the mechanism of this inhibition of HSVI pre-mRNA splicing is still unclear.     

Analysis of HCF, the cellular cofactor of VP16, in herpes simplex virus-infected cells

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 temperature-sensitive 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.     

A 3'' co-terminal family of mRNAs from the herpes simplex virus type 1 short region: two overlapping reading frames encode unrelated polypeptide one of which has highly reiterated amino acid sequence.

We have used DNA sequencing, mRNA mapping and in vitro translation to characterise three partially overlapping genes in the genome of herpes simplex virus (HSV) type 1. These genes specify three mRNAs with distinct 5' termini but a common 3' terminus, the longest of which is immediate-early (IE) mRNA-5. The 12,000 MW (12K) IE polypeptide encoded by IEmRNA-5 is translated from an 88 codon open reading frame, leaving a 1200 base 3' non-translated region. The second mRNA (mRNA-B) is initiated within the coding sequence of IEmRNA-5, and encodes a 21K polypeptide. The 12K and 21K polypeptide coding regions do not overlap. The third mRNA (mRNA-C) is initiated within the coding region of mRNA-B, and encodes a 33K polypeptide. The reading frame for 33K has a 110 codon out-of-frame overlap with the 21K reading frame. This is the first instance of overlapping genes described for HSV. The 21K polypeptide is thought to be a DNA binding protein and is remarkable for an array of 24 tandem repeats of the sequence X/Pro/Arg (where X represents predominantly Glu, Asp, Thr, Ser or Val) in its C-terminal portion. This array, which occupies most of the region of overlap with 33K, can vary in repeat number between virus strains.     

Function of dynein and dynactin in herpes simplex virus capsid transport

After fusion of the viral envelope with the plasma membrane, herpes simplex virus type 1 (HSV1) capsids are transported along microtubules (MTs) from the cell periphery to the nucleus. The motor ATPase cytoplasmic dynein and its multisubunit cofactor dynactin mediate most transport processes directed toward the minus-ends of MTs. Immunofluorescence microscopy experiments demonstrated that HSV1 capsids colocalized with cytoplasmic dynein and dynactin. We blocked the function of dynein by overexpressing the dynactin subunit dynamitin, which leads to the disruption of the dynactin complex. We then infected such cells with HSV1 and measured the efficiency of particle binding, virus entry, capsid transport to the nucleus, and the expression of immediate-early viral genes. High concentrations of dynamitin and dynamitin-GFP reduced the number of viral capsids transported to the nucleus. Moreover, viral protein synthesis was inhibited, whereas virus binding to the plasma membrane, its internalization, and the organization of the MT network were not affected. We concluded that incoming HSV1 capsids are propelled along MTs by dynein and that dynein and dynactin are required for efficient viral capsid transport to the nucleus.     

Echovirus 1 replication, not only virus binding to its receptor, VLA-2, is required for the induction of cellular immediate-early genes.

Induction of immediate-early genes c-jun, junB, and c-fos was demonstrated during echovirus 1 infection in a human osteogenic sarcoma (HOS) cell line. Tenfold induction was seen at 10 h postinfection, corresponding approximately to the end of the first replication cycle of the virus. Echovirus 1 uses VLA-2 integrin as its cellular receptor, and ligand binding by integrin is known to trigger signal transduction pathways ultimately activating immediate-early genes. In the present study, however, VLA-2 binding alone was not sufficient to induce their expression; viral replication was needed. This conclusion was based on the observations that no stimulation of the immediate-early genes occurred in the MG-63 cell line where the virus attached only to VLA-2 but was not able to replicate and that induction of these genes was observed when the HOS cells were infected with echovirus type 7, known to use a different cellular receptor. Induction was not seen in the presence of the antiviral compound WIN 54954, which evidently inhibits the uncoating but not receptor binding of echovirus 1, suggesting that viral replication triggers the activation of the immediate-early genes. The induction of these genes may have a role in viral replication and in the pathogenesis of infection.