؊ protein synthesis defect, and the rescue by secondary mutations in vhs, occurred at the mRNA and/or translational levels, quantitative reverse transcriptase PCR (qRT-PCR) and polysome analyses were performed. We found that the absence of VP22 caused a small decrease in mRNA levels as well as a defect in polysome assembly that was independent of mRNA abundance. Both defects were complemented by the secondary mutations in vhs, indicating functional interplay between VP22 and vhs in both accumulation and translation of viral mRNAs.
The herpes simplex virus 1 (HSV-1) U L 21 gene encodes a 62-kDa tegument protein with homologs in the alpha-, beta-, and gammaherpesvirus subfamilies. In the present study, we characterized a novel U L 21-null virus and its genetic repair to determine whether this protein plays a role in early stages of the HSV-1 replication cycle. Single-step growth analyses, protein synthesis time courses, and mRNA quantifications indicated that the absence of U L 21 results in a delay early in the HSV-1 replication cycle.
VP22, encoded by the U L 49 gene, is one of the most abundant proteins of the herpes simplex virus 1 (HSV-1) tegument. In the present study we show VP22 is required for optimal protein synthesis at late times in infection. Specifically, in the absence of VP22, viral proteins accumulated to wild-type levels until ϳ6 h postinfection. At that time, ongoing synthesis of most viral proteins dramatically decreased in the absence of VP22, whereas protein stability was not affected. Of the individual proteins we assayed, VP22 was required for optimal synthesis of the late viral proteins gE and gD and the immediate-early protein ICP0 but did not have discernible effects on accumulation of the immediate-early proteins ICP4 or ICP27. In addition, we found VP22 is required for the accumulation of a subset of mRNAs to wild-type levels at early, but not late, times in infection. Specifically, the presence of VP22 enhanced the accumulation of gE and gD mRNAs until ϳ9 h postinfection, but it had no discernible effect at later times in infection. Also, VP22 did not significantly affect ICP0 mRNA at any time in infection. Thus, the protein synthesis and mRNA phenotypes observed with the U L 49-null virus are separable with regard to both timing during infection and the genes affected and suggest separate roles for VP22 in enhancing the accumulation of viral proteins and mRNAs. Finally, we show that VP22's effects on protein synthesis and mRNA accumulation occur independently of mutations in genes encoding the VP22-interacting partners VP16 and vhs.Herpes simplex virus 1 (HSV-1) virions are composed of a nucleocapsid enclosing the double-stranded linear DNA genome, a proteinaceous layer termed the tegument that surrounds the nucleocapsid, and a host-derived lipid membrane envelope that contains viral glycoproteins. The tegument is unique to herpesviruses and is composed of at least 20 different viral proteins of varied stoichiometries. Tegument proteins have been shown to play a variety of roles in infection, including the regulation of viral and host gene expression and the promotion of virus assembly and egress (1,16,34,38). Tegument proteins enter the cell upon fusion of the viral envelope with the host cell membrane during infection initiation. Thus, these proteins provide a potential means to modulate the host cell and advance viral infection at both very early times in infection upon tegument delivery and at late times in infection, when the tegument proteins are produced in high amounts.VP22, encoded by the U L 49 gene, is one of the most abundant HSV-1 tegument proteins, with an average stoichiometry of 2,000 copies per virion. A number of functions have been attributed to VP22, including association with and reorganization of microtubules in infected and uninfected cells (10,22) and incorporation of RNA into the virion (42). In previous studies, U L 49 truncation or deletion mutants propagated on VP22-expressing cell lines were shown to be significantly debilitated in production of infectious virus on noncomplementing cel...
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