The capacity of herpes simplex virus type 1 (HSV-1) to replicate in vitro decreases tremendously when animal cell cultures are exposed to ligands of both the alpha/beta interferon (IFN-a/b) receptor and IFN-c receptor prior to inoculation with low m.o.i.s of HSV-1. However, the available evidence provides no insight into the possible mechanisms by which co-activation of the IFN-a/b-and IFN-c-signalling pathways produces this effect. Therefore, it has not been possible to differentiate between whether these observations represent an important in vitro model of host immunological suppression of HSV-1 infection or an irrelevant laboratory phenomenon. Therefore, the current study was initiated to determine whether co-activation of the host cell's IFN-a/b and IFN-c pathways either (i) induced death of HSV-1-infected cells such that virus replication was unable to occur; or (ii) disrupted one or more steps in the process of HSV-1 replication. To this end, multiple steps in HSV-1 infection were compared in populations of Vero cells infected with HSV-1 strain KOS (m.o.i. of 2?5) and exposed to ligands of the IFN-a/b receptor, the IFN-c receptor or both. The results demonstrated that IFN-b and IFN-c interact in a synergistic manner to block the efficient synthesis of viral DNA and nucleocapsid formation in HSV-1-infected cells and do so without compromising host-cell viability. It was inferred that IFN-mediated suppression of HSV-1 replication may be a central mechanism by which the host immune system limits the spread of HSV-1 infection in vivo.

During DNA encapsidation, herpes simplex virus 1 (HSV-1) procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins, and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28, and UL33) and one viral protein, UL32, not found to be associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional hemagglutinin-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 to 9 h postinfection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell, while at 24 hpi, it was additionally found in the cytoplasm. A newly generated UL32-null mutant was used to confirm that although B capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation. IMPORTANCEAlthough functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of those in proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32-null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.T he products of herpes simplex virus 1 (HSV-1) DNA replication are head-to-tail concatemers that are resolved into monomeric genomic units and packaged into a procapsid shell in the nucleus of the infected cell (reviewed in references 1 to 3). The procapsid is comprised of the major capsid protein (VP5), triplex proteins (VP19C and VP23), and a dodecameric UL6 portal ring. The precursor of the viral protease (UL26) and the scaffolding protein (UL26.5) form a scaffold around which the capsid shell assembles (3, 4). During DNA encapsidation, the viral protease (V...

Disulfide bonds reportedly stabilize the capsids of several viruses, including papillomavirus, polyomavirus, and simian virus 40, and have been detected in herpes simplex virus (HSV) capsids. In this study, we show that in mature HSV-1 virions, capsid proteins VP5, VP23, VP19C, UL17, and UL25 participate in covalent crosslinks, and that these are susceptible to dithiothreitol (DTT). In addition, several tegument proteins were found in high-molecular-weight complexes, including VP22, UL36, and UL37. Cross-linked capsid complexes can be detected in virions isolated in the presence and absence of N-ethylmaleimide (NEM), a chemical that reacts irreversibly with free cysteines to block disulfide formation. Intracellular capsids isolated in the absence of NEM contain disulfide cross-linked species; however, intracellular capsids isolated from cells pretreated with NEM did not. Thus, the free cysteines in intracellular capsids appear to be positioned such that disulfide bond formation can occur readily if they are exposed to an oxidizing environment. These results indicate that disulfide cross-links are normally present in extracellular virions but not in intracellular capsids. Interestingly, intracellular capsids isolated in the presence of NEM are unstable; B and C capsids are converted to a novel form that resembles A capsids, indicating that scaffold and DNA are lost. Furthermore, these capsids also have lost pentons and peripentonal triplexes as visualized by cryoelectron microscopy. These data indicate that capsid stability, and especially the retention of pentons, is regulated by the formation of disulfide bonds in the capsid.Virus capsids have evolved mechanisms to protect viral genomes from the extracellular environment while maintaining the ability to release the viral genome upon entry into a new host cell during the next round of infection. The formation of capsids containing the herpes simplex virus type 1 (HSV-1) double-stranded DNA (dsDNA) genome is a complex process involving a preassembled protein shell (procapsid) containing the viral protease and scaffolding proteins (encoded by UL26 and UL26.5), concatemeric viral DNA, and seven cleavage and packaging proteins (4,13,24). The capsid shell is composed primarily of the major capsid protein (VP5), two triplex proteins (VP19C and VP23), and VP26, as well as a dodecameric UL6 portal ring located at a unique vertex through which DNA most likely is packaged and released.During wild-type (WT) infection, three capsid forms are observed: A capsids, which have participated in an abortive encapsidation process and have lost both scaffold proteins and DNA; B capsids, which contain proteolytically processed forms of the internal scaffold proteins but no DNA; and DNA-containing mature C capsids, which have lost scaffold during the process of taking up DNA (18). DNA-containing C capsids undergo structural alterations which result in the acquisition of increased amounts of a heterodimer made up of UL25 and UL17 (81) believed to stabilize DNA-containing capsids (10,12...