The Herpes simplex virus type I alkaline nuclease, UL12, has 5' to 3' exonuclease activity and shares homology with nucleases from other members of the Herpesviridae family. We previously reported that a UL12 null virus exhibits a severe defect in viral growth. To determine whether the growth defect was a result of loss of nuclease activity or another function of UL12, we introduced an exonuclease-inactivating mutation into the viral genome. The recombinant virus, UL12-D340E (D340E), behaved identically to the null virus (AN-1) in virus yield experiments, exhibiting a 4-log decrease in the production of infectious virus. Furthermore, both viruses were severely defective in cell-to-cell spread and produced fewer DNA containing capsids and more empty capsids than wild type virus. In addition, DNA packaged by the viral mutants was aberrant as determined by infectivity assays and pulsed-field gel electrophoresis. We conclude that UL12 exonuclease activity is essential for the production of viral DNA that can be packaged to produce infectious virus. This conclusion was bolstered by experiments showing that a series of natural and synthetic α-hydroxytropolones recently reported to inhibit HSV replication also inhibit the nuclease activity of UL12. Taken together, our results demonstrate that the exonuclease activity of UL12 is essential for the production of infectious virus and may be considered as a target for development of antiviral agents. Herpes Simplex Virus is a major pathogen, and although nucleoside analogs such as acyclovir are highly effective in controlling HSV1/2 infections in immunocompetent individuals, their use in immunocompromised patients is complicated by the development of resistance. Identification of additional proteins essential for viral replication is necessary to develop improved therapies. In this communication, we confirm that the exonuclease activity of UL12 is essential for viral replication through the analysis of a nuclease-deficient viral mutant. We demonstrate that the exonuclease activity of UL12 is essential for the production of viral progeny and thus provides an attractive, druggable enzymatic target.

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...

The herpes simplex virus type 1 UL6 protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for encapsidation of the viral genome. To characterize UL6 protein domains that are involved in intersubunit interactions and interactions with other capsid proteins, we engineered a set of deletion mutants spanning the entire gene. Three deletion constructs, D-5 (⌬198-295), D-6 (⌬322-416), and D-LZ (⌬409-473, in which a putative leucine zipper was removed), were introduced into the viral genome. All three mutant viruses produced only B capsids, indicating a defect in encapsidation. Western blot analysis showed that the UL6 protein was present in the capsids isolated from two mutants, D-6 and D-LZ. The protein encoded by D-5, on the other hand, was not associated with capsids and was instead localized in the cytoplasm of the infected cells, indicating that this deletion affected the nuclear transport of the portal protein. The UL6 protein from the KOS strain (wild type) and the D-6 mutant were purified from insect cells infected with recombinant baculoviruses and shown to form ring structures as assessed by sucrose gradient centrifugation and electron microscopy. In contrast, the D-LZ mutant protein formed aggregates that sedimented throughout the sucrose gradient as a heterogeneous mixture and did not yield stable ring structures. A mutant (L429E L436E) in which two of the heptad leucines of the putative zipper were replaced with glutamate residues also failed to form stable rings. Our results suggest that the integrity of the leucine zipper region is important for oligomer interactions and stable ring formation, which in turn are required for genome encapsidation.The formation of mature capsids containing the linear double-stranded herpes simplex virus type 1 (HSV-1) genome is a complex process involving a preassembled protein shell (procapsid), concatemeric viral DNA, and seven other viral proteins believed to play a role in cleavage and packaging of the genome (1, 10). The procapsid contains three shell proteins, VP5, VP19C, and VP23, and the viral scaffolding proteins (encoded by UL26 and UL26.5). During wild-type (WT) infection, three capsid forms are observed: "A" capsids, which have initiated the encapsidation process and have lost both scaffold and DNA; "B" capsids, which contain the scaffold but no DNA; and DNA-containing mature "C" capsids, which have lost scaffold during the process of taking up DNA. The cleavage and packaging machinery consists of the UL6 portal ring, a two-subunit terminase (UL15 and UL28), a protein which seals the capsid after DNA uptake has occurred (UL25), and three other proteins which play less well defined roles in the process (UL17, UL32, and UL33). Defects in any of these genes except the UL25 gene result in the accumulation of B capsids.The protein encoded by the UL6 gene is present in all three forms of viral capsids (19) and is believed to form a ring structure at a unique vertex which serves as the portal for the entry of viral DNA into t...

The herpes simplex virus 1 (HSV-1) UL6 portal protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for the encapsidation of the viral genome. We have demonstrated previously that the leucine zipper region of UL6 is important for intersubunit interactions and stable ring formation (J. K. Nellissery, R. Szczepaniak, C. Lamberti, and S. K. Weller, J. Virol. 81:8868-8877, 2007). We now demonstrate that intersubunit disulfide bonds exist between monomeric subunits and contribute to portal ring formation and/or stability. Intersubunit disulfide bonds were detected in purified portal rings by SDS-PAGE under nonreducing conditions. Furthermore, the treatment of purified portal rings with dithiothreitol (DTT) resulted in the disruption of the rings, suggesting that disulfide bonds confer stability to this complex structure. The UL6 protein contains nine cysteines that were individually mutated to alanine. Two of these mutants, C166A and C254A, failed to complement a UL6 null mutant in a transient complementation assay. Furthermore, viral mutants bearing the C166A and C254A mutations failed to produce infectious progeny and were unable to cleave or package viral DNA. In cells infected with C166A or C254A, B capsids were produced which contained UL6 at reduced levels compared to those seen in wild-type capsids. In addition, C166A and C254A mutant proteins expressed in insect cells infected with recombinant baculovirus failed to form ring structures. Cysteines at positions 166 and 254 thus appear to be required for intersubunit disulfide bond formation. Taken together, these results indicate that disulfide bond formation is required for portal ring formation and/or stability and for the production of procapsids that are capable of encapsidation.The products of herpes simplex virus 1 (HSV-1) DNA replication are head-to-tail concatemers which are resolved into monomeric genomic units and packaged into a preformed capsid shell in the nucleus of the infected cell (reviewed in references 2, 6, and 10). The HSV-1 capsid shell is composed of the major capsid protein (VP5), two triplex proteins (VP19C and VP23), and VP26. Minor capsid proteins include UL6, UL15, UL17, UL25, UL28, and UL33. The process of cleavage and DNA packaging requires the six minor capsid proteins as well as UL32, which is not found associated with capsids (2, 6, 10, 21).HSV capsid formation and genome encapsidation are reminiscent of the double-stranded DNA bacteriophages, in that a procapsid shell is preassembled around a scaffolding protein that is not present in the mature virion (3, 37, 38). Bacteriophage and herpesviruses share an important structural element, a dodecameric portal ring located at a unique capsid vertex (8,9,28,40). During HSV genome encapsidation, the portal ring provides a docking site for the terminase, an ATPdriven molecular motor that facilitates the uptake of viral DNA (34,42,45,46). Terminase is responsible not only for viral DNA uptake but also for the specific cleavage of viral genomes ...

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...