Assembly of Infectious Herpes Simplex Virus Type 1 Virions in the Absence of Full-Length VP22

The herpesvirus tegument has historically been described as an amorphous protein layer located between the nucleocapsid and the envelope (50). However, many recent publications propose a more tightly controlled organization of this structure (33, 37, 55). Four viral proteins which compose the bulk of the tegument's mass (27, 28, 53) are encoded by genes that lie in a consecutive stretch in the unique long segment of the herpes simplex virus type 1 (HSV-1) genome. UL46, UL47, UL48, and UL49 encode VP11/12 (55), VP13/14 (38, 55), VP16 (9, 44), and VP22 (21), respectively. VP22 is the most abundant tegument protein in virus particles (27). Additionally, the virion host shutoff (VHS) protein, encoded by the UL41 gene (43), is also incorporated into virus particles (29, 30, 49, 51). Of these tegument proteins, only VP16 has been shown to provide an essential function in structural assembly of the virus in tissue culture (3, 49, 54, 55). The five tegument proteins mentioned above are phosphoproteins (5, 24), which may undergo additional posttranslational modifications at various stages of the infection cycle. VP11/12, VP13/14, VP16, VP22, and VHS have all been shown to incorporate radiolabeled phosphate in infected-cell extracts (5, 20, 51), while VP11/12, VP13/14, VP16, and VP22 can be radiolabeled in isolated virus particles (32). Phosphorylated forms of VP22 can be differentiated based on their migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the fastest-migrating form of VP22 is observed in purified virion preparations (5, 20, 45). VP13/14 and, to a lesser extent, VP22 were reported to be glycosylated in virion particles (39). VP13/14 and VP22 are nucleotidylated (5, 6), and VP22 is mono(ADP-ribosyl)ated (5, 48). While the roles of each of these modifications during infection remain unknown, recent results suggest that virion protein phosphorylation may regulate the assembly and dissociation of the tegument (41). A number of studies have examined the role of individual tegument proteins in virion assembly. Following infection with a recombinant virus expressing two copies of VP22, VP13/14 levels were decreased in purified virions, presumably to compensate for the increased levels of packaged VP22 (33). Virions devoid of VP11/12 exhibit increased incorporation of VP13/14 (55). In recombinant viruses in which synthesis of VP13/14 or both VP13/14 and VP11/12 is abolished, increased amounts of VP16 are incorporated into virions (55). Taken together, these results suggest that the total amount of tegument protein incorporated into the virion is constant but the relative ratio of individual tegument proteins is flexible. The present study was designed to examine the function of VP22 during infection, focusing on its potential role in tegument assembly. RF177, a recombinant strain of HSV-1 generated for the purposes of this study, synthesized low levels of a truncated form of VP22 (Δ212). Tegument proteins VP13/14, VP16, and VHS were synthesized and incorporated into RF177 virions at levels comparable to wild-type virus, while full-length VP22 was absent from purified RF177 particles. No obvious structural differences between RF177 and wild-type virus particles were observed in electron microscopic analyses. Single-step growth kinetics of RF177 in Vero cells were nearly identical to those of wild-type virus, although RF177 exhibited a slight reduction in titer at very late times postinfection. Finally, RF177 demonstrated smaller plaques than wild-type virus in Vero monolayers. Based on these results, we conclude that (i) tegument formation, virion assembly and maturation, and productive HSV-1 replication can occur in the absence of full-length VP22 and (ii) full-length VP22 in the viral tegument is required for efficient virus spread in Vero cells.