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The UL11 gene of herpes simplex virus 1 was reported to encode a myristylated protein (C. A. MacLean, B. Clark, and D. J. McGeoch, J. Gen. Virol. 70:3147-3157, 1989). To determine the function of the gene product, a recombinant virus (R7219) lacking 61% of the codons (176 bp of the 288-bp coding domain) was genetically engineered. The deletion mutant replicated in all cell lines tested, albeit to titers 30- to 250-fold lower than those obtained from cells infected with wild-type virus. Electron microscopic analyses indicated that both full and empty capsids accumulated in the nuclei, juxtaposed with the inner lamellae of the nuclear membranes, and that increased numbers of naked particles were present in the cytoplasm of cells infected with the mutant virus. There was a greater than 1,000-fold decrease in the amount of infectious extracellular virus released from Vero cells infected with the deletion mutant compared with that from cells infected with wild-type virus. Furthermore, the onset of release of infectious virus from cells infected with the UL11- mutant was significantly delayed: levels of extracellular UL11- virus increased 15-fold between 20 and 26 h after infection, while levels of wild-type extracellular virus increased 500-fold between 8 and 14 h after infection. A virus in which the UL11 gene was restored produced wild-type levels of total and extracellular virus and was indistinguishable from wild-type virus upon analysis by electron microscopy. Taken together, the data indicate that the absence of the UL11 gene causes a reduced capacity to envelope and transport virions into the extracellular space.

Numerous discrete lesions, which we have termed blebs, appeared in the nucleus of BHK cells 10 to 15 h after infection with herpes simplex virus type 1 (HSV-1). They were formed in the inner portion of the nuclear envelope by the apposition of two thickened lamellae overlying a vacuole. As demonstrated by electron microscopic studies, blebs were regular and associated with the peripheral lamina in the nucleus, averaging 3.5 blebs per micron2. They appeared to be associated with an enrichment of the 155K major capsid protein in the nuclear membrane subfractions as compared with the protein composition of nuclei and plasma membrane fractions. We propose that blebs represent the site of assembly of capsid proteins before DNA insertion and eventual envelopment.

During the maturation of two strains of herpes simplex virus type 1 (VR3 and Patton), intramembrane changes were detected with the freeze-fracture technique in the viral envelope and the infected cell plasma membrane, and these changes were compared with data obtained from thin sections. Regardless of the strain, the inner leaflet of the viral envelope of extracellular virions was characterized by a density of intramembrane particles (IMP) three times larger than the host nuclear and plasma membrane. Addition of IMP, which probably represent virus-coded proteins, was detected in the viral envelope only after budding from the nuclear membrane, whereas it occurred during envelopment of capsids at cytoplasmic vacuoles. Fused membranes also showed one of their fracture faces covered with a high density of IMP similar to that of the mature virion envelope. The internal side of the membrane leaflet bearing these numerous particles was always characterized by the presence of an electron-dense material in thin sections. In addition, the plasma membrane of fibroblasts and Vero cells showed strain-specific changes: patches of closely packed IMP were observed with the VR3 strain, whereas ridges almost devoid of IMP characterized the plasmalemma of cells infected with the Patton strain. These intramembrane changes, however, were not observed as early as herpes membrane antigens. Thus, application of the freeze-fracture technique to herpes simplex virus type 1-infected cells revealed striking structural differences between viral and uninfected cell membranes. These differences are probably related to insertion and clustering of virus-coded proteins in the hydrophobic part of the membrane bilayer.

In herpes simplex virus-infected cells, coreless capsids accumulate at the nuclear pores soon after infection, but subsequently disappear, suggesting that, as in adenovirus-infected cells (S. Dales and Y. Chardonnet, Virology 56:465-483, 1973), the release of viral DNA from nucleocapsids takes place at the nuclear pores. A nonlethal mutant, HSV-1(50B), produced by mutagenesis of HSV DNA fragments and selected for delayed production of plaques at 31 degrees C, accumulated coreless capsids at the nuclear pores late in infection in contrast to wild-type viruses. Recombinants selected for ability to produce plaques at 31 degrees C by marker rescue with digests of herpes simplex virus 2 DNA and selected clone fragments of HSV-1 DNA no longer accumulated empty capsids at nuclear pores late in infection. These results suggest that herpes simplex viruses encode a function which prevents accumulation of coreless capsids at nuclear pores, presumably by preventing uptake, unenvelopment, and DNA release from progeny virus, and indicate that the cold sensitivity of plaque formation and accumulation of coreless capsids might be related or comap in the S component of the genome.

Previous studies have shown that cells infected with the herpes simplex virus 1(HFEM) mutant tsB7 and maintained at the nonpermissive temperature fail to accumulate viral polypeptides. Analyses of intertypic recombinants generated by marker rescue of tsB7 with herpes simplex virus 2 DNA fragments localized the mutation between 0.46 and 0.52 map units on the viral genome (Knipe et al., J. Virol. 38:539-547, 1981). In this paper we report that the mutation in tsB7 affects several aspects of the reproductive cycle of the virus at the nonpermissive temperature. Thus, (i) viral capsids accumulate at the nuclear pores and do not release viral DNA for at least 6 h postinfection at 39 degrees C. The DNA was released within 30 min after a shift to the permissive temperature. (ii) Experiments involving shifts from the permissive to the nonpermissive temperature indicated that viral protein synthesis was not sustained in cells maintained at the permissive temperature for less than 4 h. (iii) Viral DNA synthesis was delayed at the permissive temperature for as long as 8 h. Once initiated, it continued at 39 degrees C. (iv) Marker rescue of tsB7 by transfection with herpes simplex virus 1(F) DNA fragments localized the mutation to between 0.501 and 0.503 map units on the viral genome. These results are consistent with the tsB7 lesion being in a gene coding for a virion component which affects release of viral DNA from capsids and onset of viral DNA synthesis.