Phosphorylation of herpes simplex virus type 1 Us11 protein is independent of viral genome expression

Abstract

The Us11 protein is a true late gene product of herpes simplex virus type 1 (HSV-1), whose exact function is unknown but which exhibits RNA-binding properties and which is phosphorylated on serine residues. In order to determine whether the Us11 protein is phosphorylated by cellular kinase(s) or by virally encoded kinase(s), the Us11 gene has been cloned and transiently expressed in HeLa cells. In addition, HeLa-derived cell lines have been selected for their ability to express Us11 protein constitutively. 32P-Labeling and analysis by two-dimensional electrophoresis of transiently and constitutively expressed Us11 protein demonstrated that, indeed, multiple phosphorylation of the protein occurs in absence of HSV-1 genome expression, indicating that the protein behaves as a natural substrate for cellular kinase(s). In addition, a sequence heterogeneity of the Us11 protein, due to a difference in the number of SPREPR repeats, has been characterized between different strains of HSV-1.

Unique Motif for Nucleolar Retention and Nuclear Export Regulated by Phosphorylation

“…6E) spots, respectively, corresponding to proteins differing in charge. In this electrophoresis system, the phosphorylated derivatives of US11 protein migrated to the left of the main spot, as we have already reported (14,51). All together, these results indicated that, at least in the absence of viral infection, Ser 129 was phosphorylated in US11 wild-type protein (both KOS and MP strains).…”

“…Four XPR repeats account for this additional sequence, which contains an extra serine residue (Fig. 6A) (51). Transfected cells were labeled with 32 P, and US11 proteins were immunoprecipitated, separated by SDS-PAGE, and transferred to a nitrocellulose membrane for Western blot analysis using anti-US11 antibodies (Fig.…”

Distinct domains in herpes simplex virus type 1 US11 protein mediate post-transcriptional transactivation of human T-lymphotropic virus type I envelope glycoprotein gene expression and specific binding to the Rex responsive element.

“…Construction of the recombinant fusion proteins was achieved by cloning different parts of the US11 coding sequence (HSV-1 strain KOS) in-frame to the glutathione S-transferase (GST) coding sequence of pGEX-2T (Pharmacia). DNA fragments containing portions of the US11 coding sequence were obtained by PCR amplification using pG-3 (Simonin et al, 1995), a subclone of pSG-25 (Goldin et al, 1981), as a template plasmid. The deduced amino acid sequence of US11 protein from the KOS strain is almost identical to that of strain 17 (McGeoch et al, 1985), the main difference being the two SPREPR repeats located at the C terminus, as previously described (Simonin et al, 1995).…”

“…DNA fragments containing portions of the US11 coding sequence were obtained by PCR amplification using pG-3 (Simonin et al, 1995), a subclone of pSG-25 (Goldin et al, 1981), as a template plasmid. The deduced amino acid sequence of US11 protein from the KOS strain is almost identical to that of strain 17 (McGeoch et al, 1985), the main difference being the two SPREPR repeats located at the C terminus, as previously described (Simonin et al, 1995). US11 protein from the KOS strain is 149 amino acids long, while US11 proteins from strains 17 and…”

Uncoupling ribosome biogenesis regulation from RNA polymerase I activity during herpes simplex virus type 1 infection

“…Some of them are specifically phosphorylated: RPS2a, RPS2, RPS3a, RPS6, and especially a unique case of phosphorylation for RPL30 (Fenwick and Walker 1979;Kennedy et al 1981;Garcin et al 1990;Masse et al 1990a,b;Simonin et al 1995a). (3) Nonribosomal cellular (PABP1) (Greco et al 2001) and viral (US11, VP16, VP19C) proteins are specifically associated to ribosomes (Simonin et al 1995b;Diaz et al 1996). Therefore, taken together, these pieces of evidence allow us to postulate that the complex post-transcriptional regulation of ribosome biogenesis that we have unraveled in this study and that takes place after infection could permit the synthesis and assembly of several kinds of ribosomal machineries (indicated by ribosomes marked by a star in Fig.…”