Microtubule Reorganization during Herpes Simplex Virus Type 1 Infection Facilitates the Nuclear Localization of VP22, a Major Virion Tegument Protein

Abstract

Full-length VP22 is necessary for efficient spread of herpes simplex virus type 1 (HSV-1) from cell to cell during the course of productive infection. VP22 is a virion phosphoprotein, and its nuclear localization initiates between 5 and 7 h postinfection (hpi) during the course of synchronized infection. The goal of this study was to determine which features of HSV-1 infection function to regulate the translocation of VP22 into the nucleus. We report the following. The molecular mechanism of herpes simplex virus type 1 (HSV-1) tegument and envelope assembly is poorly understood. Most of the major tegument proteins exhibit nuclear or perinuclear distributions late in infection (1, 8, 10-12, 26, 33, 37, 43), and there is general agreement that primary envelopment occurs as the capsid exits the nucleus (7,19,21,42,46) (for a detailed review, see reference 17).Work in our laboratory has focused on the major tegument protein VP22 and its function in virion assembly and virus replication. Full-length VP22 is necessary for efficient spread of the virus from cell to cell, inasmuch as a recombinant virus producing a truncated form of the protein exhibits a decreased plaque size in Vero cells compared to that of wild-type virus (36). In an earlier study, we reported that the nuclear localization of VP22 initiates between 5 and 7 h postinfection (hpi) during the course of synchronized infection (37). This period corresponds to the peak of viral ␤ protein production and DNA synthesis (39). Recently, computer analyses predicted two nuclear localization signals (NLS) in the primary structure of VP22 (22). Although transiently expressed VP22 can be detected in the nuclei of transfected cells (15,22), these proposed NLS have not yet been characterized in nuclear import assays. VP22 is a 301-amino-acid protein with a predicted size of 32,000 Da (28). Thus, VP22 is below the size exclusion limit (40,000 to 45,000 Da) for passive diffusion through the nuclear pore (13). That VP22 accumulates in the cytoplasm of infected cells prior to 5 hpi suggests its translocation to the nucleus is regulated during infection. One possible means of regulation could be active retention through binding of VP22 to a cytoplasmic structure.In this study, we sought to determine which features of HSV-1 replication are involved in the redistribution of VP22 during productive infection. Our investigations show the following. Regulated VP22 nuclear localization initiates after 5 hpi with HSV-1 and is independent of viral DNA and true late protein synthesis. We propose that HSV-1 induced microtubule restructuring releases VP22 from the cytoskeleton, allowing its entry into the nucleus. Stabilization of microtubules during infection or in VP22-expressing cells increases VP22 retention in the cytoplasm. Based on these results, we conclude that microtubule reorganization during herpes HSV-1 infection acts to facilitate the nuclear localization of VP22. MATERIALS AND METHODS Cells and virus.African green monkey kidney (Vero) cells were obtained from the Ameri...

The major tegument structural protein VP22 targets areas of dispersed nucleolin and marginalized chromatin during productive herpes simplex virus 1 infection

“…We consistently observed in this (Fig. 2) and previous studies (Blouin and Blaho, 2001;Pomeranz and Blaho, 2000;Yedowitz et al, 2005) that the staining pattern of VP22 in V49 cells is homogeneous and remains stable at different times post seeding. Increased levels of VP22 staining at 4 and 8 hpi results from de novo VP22 expression from virus.…”

“…Close inspection of the infected cells at 4 hpi seemed to indicate that a subset of nuclear VP22 "surrounded" the regions of bright nucleolin staining. Although VP22 appears to be mostly present throughout the nucleus, as we have reported previously (Kotsakis et al, 2001;Pomeranz and Blaho, 1999;Yedowitz et al, 2005), regions exist in which there is slightly more intense VP22 staining over this background. In contrast, VP22 was undetectable at 2 hpi and the anti-nucleolin staining was uniform and circular, as in mock-infected cells.…”

“…We previously reported that this VP22 region possesses a pat4 NLS (Kotsakis et al, 2001), Thus, we set out to characterize the behavior of a GFP fusion possessing the VP22 pat4 motif (VP22pat4GFP) inside live cells in absence of other viral proteins. Vero cells were transfected with either plasmid pJB177, expressing VP22pat4GFP, or control plasmid pJB49 which expresses GFP alone.…”

“…pJB49 expresses a nonfused GFP only and was used as a control (Blouin and Blaho, 2001). DNAs of both plasmids were used in a modified CaCl 2 transfection protocol to transfect Vero cells (Pomeranz and Blaho, 2000).…”

“…Full-length VP22 is required for efficient viral cell-to-cell spread during infection in animals and cultured cells (Duffy et al, 2006;Pomeranz and Blaho, 2000). VP22 of HSV-1 possesses two nuclear localization signals (NLS) (Kotsakis et al, 2001) and the protein accumulates to high levels in the nuclei of either infected cells (Pomeranz and Blaho, 1999) or VP22-expressing stable cell lines (Pomeranz and Blaho, 2000). While characterizing a fusion of GFP with an NLS of VP22, we observed that this chimera was not excluded from nucleoli in transfected cells.…”