The null mutant of the U(L)31 gene of herpes simplex virus 1: construction and phenotype in infected cells

Abstract

Earlier studies have shown that the U L 31 protein is homogeneously distributed throughout the nucleus and cofractionates with nuclear matrix. We report the construction from an appropriate cosmid library a deletion mutant which replicates in rabbit skin cells carrying the U L 31 gene under a late (␥ 1 ) viral promoter. The mutant virus exhibits cytopathic effects and yields 0.01 to 0.1% of the yield of wild-type parent virus in noncomplementing cells but amounts of virus 10-to 1,000-fold higher than those recovered from the same cells 3 h after infection. Electron microscopic studies indicate the presence of small numbers of full capsids but a lack of enveloped virions. Viral DNA extracted from the cytoplasm of infected cells exhibits free termini indicating cleavage/packaging of viral DNA from concatemers for packaging into virions, but analyses of viral DNAs by pulsed-field electrophoresis indicate that at 16 h after infection, both the yields of viral DNA and cleavage of viral DNA for packaging are decreased. The repaired virus cannot be differentiated from the wild-type parent. These results suggest the possibility that U L 31 protein forms a network to enable the anchorage of viral products for the synthesis and/or packaging of viral DNA into virions. MATERIALS AND METHODS Cells and viruses. Rabbit skin cells and human 143TKϪ cells were obtained from John McLaren and Carlo Croce, respectively; Vero cells were obtained from American Type Culture Collection. Human lung fibroblast (HLF) cells were obtained from George Kemble (Aviron, Mountain View, Calif.). Cells were maintained in Dulbecco's modified Eagle medium supplemented with either 5% newborn calf serum (rabbit skin cells and Vero cells), 5% fetal bovine serum (143TK Ϫ cells), or 10% fetal bovine serum (HLF cells). Infected cells were maintained in medium consisting of mixture 199 supplemented with 1% calf serum (199V). Virus titer was measured on infected cells maintained in 199V plus 0.2% human immunoglobulin. RD14 and RD17 U L 31-expressing cell lines were two independent clones derived from rabbit skin cells and maintained in 10% fetal bovine serum. Herpes simplex virus 1 strain F [HSV-1(F)], a limitedpassage virus, serves as the prototype HSV-1 strain in this laboratory (16).Generation of U L 31 polyclonal antibody. A DNA fragment encoding U L 31 codons 1 to 163 was cloned into an Escherichia coli anthranilate synthetase (TrpE) expression vector (22). The fusion protein was induced after 2-h logphase growth of transformed E. coli with 20 g of indoleacrylic acid per ml. After 4 h of additional incubation, the bacteria were resuspended in lysis buffer (50 mM Tris-HCl [pH 7.5], 5 mM EDTA, 3 mg of lysozyme per ml) and stored on ice for 2 h. The lysate was then adjusted to 0.3 M NaCl and 0.65% Nonidet P-40 and centrifuged in a Sorvall SS34 rotor at 10,000 rpm for 10 min. The pellet was solubilized in disruption buffer (50 mM Tris-HCl [pH 7.0], 2% sodium dodecyl sulfate [SDS], 0.7 M ␤-mercaptoethanol, 2.75% sucrose) and subjected to electrophoresis in a ...

The Pivotal Roles of US3 Protein in Cell-to-Cell Spread and Virion Nuclear Egress of Duck Plague Virus

“…This complex recruits viral and cellular kinases, such as US3 or UL13, and protein kinase C (PKC) to phosphorylate and soften the nuclear lamina, subsequently allowing nucleocapsids to access the INM 31,41 . UL31 or UL34 deletion blocked virion nuclear egress, and all nucleocapsids accumulated in the nucleus 42,43 . Although UL47 deletion also blocks virion nuclear egress, the researchers suggested that UL47 protein played a regulatory role only in nucleocapsid nuclear egress 29 .…”

A Single Herpesvirus Protein Can Mediate Vesicle Formation in the Nuclear Envelope

“…This mode of nuclear escape has, however, only been observed after forced reversion analysis in cell culture. Differing roles for pUL31 and pUL34 in nuclear egress have been demonstrated (7,8), including recruitment of viral and cellular kinases that locally disrupt the lamina (15)(16)(17)(18)(19) as well as functions in herpesvirus replication beyond nuclear egress, including viral DNA binding and packaging (44,45) or cell-to-cell spread (46).…”

Functional Anatomy of Herpes Simplex Virus 1 Overlapping Genes Encoding Infected-Cell Protein 22 and U S 1.5 Protein

“…To conduct a comprehensive analysis of ICP22/U S 1.5, it was necessary to construct a virus from which the entire open reading frame of ␣22/U S 1.5 had been deleted. To delete the entire ␣22/U S 1.5 domain, we used a modified cosmid system as described previously (7,12) and in Materials and Methods. This cosmid set is illustrated in Fig.…”

“…Cosmids. The set of cosmids used in this study was derived from HSV-1(F) DNA as described elsewhere (7,12) and is illustrated in Fig. 1A, lines 2 through 6.…”