The herpes simplex virus 2 UL21 protein is essential for virus propagation.

Herpes simplex virus 2 (HSV-2) is an important human pathogen that is the major cause of genital herpes infections and a significant contributor to the epidemic spread of human immunodeficiency virus infections. The UL21 gene is conserved throughout the Alphaherpesvirinae subfamily and encodes a tegument protein that is dispensable for HSV-1 and pseudorabies virus replication in cultured cells; however, its precise functions have not been determined. To investigate the role of UL21 in the HSV-2 replicative cycle, we constructed a UL21 deletion virus (HSV-2 ΔUL21) using an HSV-2 bacterial artificial chromosome, pYEbac373. HSV-2 ΔUL21 was unable to direct the production of infectious virus in noncomplementing cells, whereas the repaired HSV-2 ΔUL21 strain grew to wild-type (WT) titers, indicating that UL21 is essential for virus propagation. Cells infected with HSV-2 ΔUL21 demonstrated a 2-h delay in the kinetics of immediate early viral gene expression. However, this delay in gene expression was not responsible for the inability of cells infected with HSV-2 ΔUL21 to produce virus insofar as late viral gene products accumulated to WT levels by 24 h postinfection (hpi). Electron and fluorescence microscopy studies indicated that DNA-containing capsids formed in the nuclei of ΔUL21-infected cells, while significantly reduced numbers of capsids were located in the cytoplasm late in infection. Taken together, these data indicate that HSV-2 UL21 has an early function that facilitates viral gene expression as well as a late essential function that promotes the egress of capsids from the nucleus.

A mutation in UL15 of herpes simplex virus 1 that reduces packaging of cleaved genomes.

Herpesvirus genomic DNA is cleaved from concatemers that accumulate in infected cell nuclei. Genomic DNA is inserted into preassembled capsids through a unique portal vertex. Extensive analyses of viral mutants have indicated that intact capsids, the portal vertex, and all components of a tripartite terminase enzyme are required to both cleave and package viral DNA, suggesting that DNA cleavage and packaging are inextricably linked. Because the processes have not been functionally separable, it has been difficult to parse the roles of individual proteins in the DNA cleavage/packaging reaction. In the present study, a virus bearing the deletion of codons 400 to 420 of U(L)15, encoding a terminase component, was analyzed. This virus, designated vJB27, failed to replicate on noncomplementing cells but cleaved concatemeric DNA to ca. 35 to 98% of wild-type levels. No DNA cleavage was detected in cells infected with a U(L)15-null virus or a virus lacking U(L)15 codons 383 to 385, comprising a motif proposed to couple ATP hydrolysis to DNA translocation. The amount of vJB27 DNA protected from DNase I digestion was reduced compared to the wild-type virus by 6.5- to 200-fold, depending on the DNA fragment analyzed, thus indicating a profound defect in DNA packaging. Capsids containing viral DNA were not detected in vJB27-infected cells, as determined by electron microscopy. These data suggest that pU(L)15 plays an essential role in DNA translocation into the capsid and indicate that this function is separable from its role in DNA cleavage.

Effects of lamin A/C, lamin B1, and viral US3 kinase activity on viral infectivity, virion egress, and the targeting of herpes simplex virus U(L)34-encoded protein to the inner nuclear membrane.

Previous results indicated that the U(L)34 protein (pU(L)34) of herpes simplex virus 1 (HSV-1) is targeted to the nuclear membrane and is essential for nuclear egress of nucleocapsids. The normal localization of pU(L)34 and virions requires the U(S)3-encoded kinase that phosphorylates U(L)34 and lamin A/C. Moreover, pU(L)34 was shown to interact with lamin A in vitro. In the present study, glutathione S-transferase/pU(L)34 was shown to specifically pull down lamin A and lamin B1 from cellular lysates. To determine the role of these interactions on viral infectivity and pU(L)34 targeting to the inner nuclear membrane (INM), the localization of pU(L)34 was determined in LmnA(-/-) and LmnB1(-/-) mouse embryonic fibroblasts (MEFs) by indirect immunofluorescence and immunogold electron microscopy in the presence or absence of U(S)3 kinase activity. While pU(L)34 INM targeting was not affected by the absence of lamin B1 in MEFs infected with wild-type HSV as viewed by indirect immunofluorescence, it localized in densely staining scalloped-shaped distortions of the nuclear membrane in lamin B1 knockout cells infected with a U(S)3 kinase-dead virus. Lamin B1 knockout cells were relatively less permissive for viral replication than wild-type MEFs, with viral titers decreased at least 10-fold. The absence of lamin A (i) caused clustering of pU(L)34 in the nuclear rim of cells infected with wild-type virus, (ii) produced extensions of the INM bearing pU(L)34 protein in cells infected with a U(S)3 kinase-dead mutant, (iii) precluded accumulation of virions in the perinuclear space of cells infected with this mutant, and (iv) partially restored replication of this virus. The latter observation suggests that lamin A normally impedes viral infectivity and that U(S)3 kinase activity partially alleviates this impediment. On the other hand, lamin B1 is necessary for optimal viral replication, probably through its well-documented effects on many cellular pathways. Finally, neither lamin A nor B1 was absolutely required for targeting pU(L)34 to the INM, suggesting that this targeting is mediated by redundant functions or can be mediated by other proteins.

Release of the herpes simplex virus 1 protease by self cleavage is required for proper conformation of the portal vertex.

We identify an NLS within herpes simplex virus scaffold proteins that is required for optimal nuclear import of these proteins into infected or uninfected nuclei, and is sufficient to mediate nuclear import of GFP. A virus lacking this NLS replicated to titers reduced by 1000-fold, but was able to make capsids containing both scaffold and portal proteins suggesting that other functions can complement the NLS in infected cells. We also show that Vp22a, the major scaffold protein, is sufficient to mediate the incorporation of portal protein into capsids, whereas proper portal immunoreactivity in the capsid requires the larger scaffold protein pU(L)26. Finally, capsid angularization in infected cells did not require the HSV-1 protease unless full length pU(L)26 was expressed. These data suggest that the HSV-1 portal undergoes conformational changes during capsid maturation, and reveal that full length pU(L)26 is required for this conformational change.

Characterization of a UL49-null mutant: VP22 of herpes simplex virus type 1 facilitates viral spread in cultured cells and the mouse cornea.

Herpes simplex virus type 1 (HSV-1) virions, like those of all herpesviruses, contain a proteinaceous layer termed the tegument that lies between the nucleocapsid and viral envelope. The HSV-1 tegument is composed of at least 20 different viral proteins of various stoichiometries. VP22, the product of the U(L)49 gene, is one of the most abundant tegument proteins and is conserved among the alphaherpesviruses. Although a number of interesting biological properties have been attributed to VP22, its role in HSV-1 infection is not well understood. In the present study we have generated both a U(L)49-null virus and its genetic repair and characterized their growth in both cultured cells and the mouse cornea. While single-step growth analyses indicated that VP22 is dispensable for virus replication at high multiplicities of infection (MOIs), analyses of plaque morphology and intra- and extracellular multistep growth identified a role for VP22 in viral spread during HSV-1 infection at low MOIs. Specifically, VP22 was not required for either virion infectivity or cell-cell spread but was required for accumulation of extracellular virus to wild-type levels. We found that the absence of VP22 also affected virion composition. Intracellular virions generated by the U(L)49-null virus contained reduced amounts of ICP0 and glycoproteins E and D compared to those generated by the wild-type and U(L)49-repaired viruses. In addition, viral spread in the mouse cornea was significantly reduced upon infection with the U(L)49-null virus compared to infection with the wild-type and U(L)49-repaired viruses, identifying a role for VP22 in viral spread in vivo as well as in vitro.

Entry of influenza viruses into cells is inhibited by a highly specific protein kinase C inhibitor.

Following binding to cell surface sialic acid, entry of influenza viruses into cells is mediated by endocytosis. Productive entry of influenza virus requires the low-pH environment of the late endosome for fusion and release of the virus into the cytoplasm and transport of the virus genome into the nucleus. We investigated novel mechanisms to inhibit influenza virus infection using highly specific inhibitors of protein kinase C. We found that one inhibitor, bisindolylmaleimide I, prevented replication of influenza A virus in a dose-dependent manner when added at the time of infection, but had little specific effect when added 2 h after infection had commenced. Virus yields dropped by more than 3 log units in the presence of micromolar levels of bisindolylmaleimide I. Influenza B virus replication was also inhibited by bisindolylmaleimide at micromolar concentrations. We carried out experiments to determine the point in infection that was blocked by bisindolylmaleimide I, and determined that entry of viral ribonucleoproteins (vRNPs) into the nucleus was prevented. Upon drug washout vRNP nuclear entry resumed, showing that bisindolylmaleimide I is reversible. Bisindolylmaleimide I did not affect virus binding and was apparently not acting as a weak base, because its effects were independent of the pH of the external growth medium. These experiments show that bisindolylmaleimide I blocks replication of different types of influenza virus in a dose-dependent and reversible manner, and that virus entry into the cell is inhibited.

Ultrastructural localization of the herpes simplex virus type 1 UL31, UL34, and US3 proteins suggests specific roles in primary envelopment and egress of nucleocapsids.

The wild-type UL31, UL34, and US3 proteins localized on nuclear membranes and perinuclear virions; the US3 protein was also on cytoplasmic membranes and extranuclear virions. The UL31 and UL34 proteins were not detected in extracellular virions. US3 deletion caused (i) virion accumulation in nuclear membrane invaginations, (ii) delayed virus production onset, and (iii) reduced peak virus titers. These data support the herpes simplex virus type 1 deenvelopment-reenvelopment model of virion egress and suggest that the US3 protein plays an important, but nonessential, role in the egress pathway.