Effects of S-acetylglutathione in cell and animal model of herpes simplex virus type 1 infection

UL16 is a tegument protein of herpes simplex virus (HSV) that is conserved among all members of the Herpesviridae, but its function is poorly understood. Previous studies revealed that UL16 is associated with capsids in the cytoplasm and interacts with the membrane protein UL11, which suggested a "bridging" function during cytoplasmic envelopment, but this conjecture has not been tested. To gain further insight, cells infected with UL16-null mutants were examined by electron microscopy. No defects in the transport of capsids to cytoplasmic membranes were observed, but the wrapping of capsids with membranes was delayed. Moreover, clusters of cytoplasmic capsids were often observed, but only near membranes, where they were wrapped to produce multiple capsids within a single envelope. Normal virion production was restored when UL16 was expressed either by complementing cells or from a novel position in the HSV genome. When the composition of the UL16-null viruses was analyzed, a reduction in the packaging of glycoprotein E (gE) was observed, which was not surprising, since it has been reported that UL16 interacts with this glycoprotein. However, levels of the tegument protein VP22 were also dramatically reduced in virions, even though this gE-binding protein has been shown not to depend on its membrane partner for packaging. Cotransfection experiments revealed that UL16 and VP22 can interact in the absence of other viral proteins. These results extend the UL16 interaction network beyond its previously identified binding partners to include VP22 and provide evidence that UL16 plays an important function at the membrane during virion production. Infectious herpesviruses contain approximately 40 viral proteins and are produced when their DNA-containing capsids are wrapped with a cell-derived membrane in the cytoplasm (1). This envelopment process is driven by complex interactions that are still poorly understood but is known to involve bridging interactions provided by a growing list of tegument proteins, which provide linkages between the capsid and viral membrane proteins (1-3). The UL16 tegument protein of herpes simplex virus (HSV) is remarkable for its numerous interactions with several other viral proteins, namely, tegument protein UL21 (4, 5), membrane protein UL11 (4, 6-8), membrane glycoprotein E (gE) (4, 9), and an unidentified protein(s) that is associated with the capsid (10-12). UL16 is conserved among all the alpha-, beta-, and gammaherpesvirinae (2, 13, 14), but its actual function remains unknown.There are several reasons for suggesting a role for UL16 in HSV envelopment. The earliest study showed that a U L 16-null mutant (here named the ⌬U L 16B mutant) produces infectious virions at a level only one-tenth that of the wild-type virus (15). Also, UL16 has been shown to be bound in some manner to cytoplasmic capsids (10, 16, 17), and thus, its direct interactions with membrane proteins UL11 (8) and gE (9) suggest that UL16 might provide bridging functions that help drive virion production, as first pro...

Section: Discussionmentioning

confidence: 99%

Elucidation of the Block to Herpes Simplex Virus Egress in the Absence of Tegument Protein UL16 Reveals a Novel Interaction with VP22

Starkey

Han

Chadha

et al. 2014

“…It is becoming increasingly clear that oxidative stress and cellular responses to it play critical roles in modulating a large number of biological processes, including pathogenesis by bacterial and viral infections. Many viruses, including HSV, have been shown to induce oxidative stress, and antioxidants have shown promise in the treatment of viral infection (54)(55)(56)(59)(60)(61)(62)(63). Viral proteins that play roles in sensing and responding to changes in redox status may thus provide targets for therapeutic intervention.…”

Section: Discussionmentioning

confidence: 99%

The Putative Herpes Simplex Virus 1 Chaperone Protein UL32 Modulates Disulfide Bond Formation during Infection

Albright

Kosinski

Szczepaniak

et al. 2015

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...

“…We hypothesize that the C-terminal regulatory domain of UL16 may also contain a switch that is activated in response to oxidation. In fact, it has been shown that HSV-infected cells undergo oxidative stress (17,21,33,49,56,59,65,70). Hence, it is possible that UL16 utilizes this change to modulate its function.…”

Section: Discussionmentioning

confidence: 99%

Regulated Interaction of Tegument Proteins UL16 and UL11 from Herpes Simplex Virus

Chadha

Han

Starkey

et al. 2012

It is well known that proteins in the tegument (located between the viral capsid and envelope proteins) play critical roles in the assembly and budding of herpesviruses. Tegument proteins UL16 and UL11 of herpes simplex virus (HSV) are conserved among all the Herpesviridae. Although these proteins directly interact in vitro, UL16 was found to colocalize poorly with UL11 in cotransfected cells. To explain this discrepancy, we hypothesized that UL16 is initially made in an inactive form and is artificially transformed to the binding-competent state when cells are disrupted. Consistent with a regulated interaction, UL16 was able to fully colocalize with UL11 when a large C-terminal segment of UL16 was removed, creating mutant UL16 (1-155). Moreover, membrane flotation assays revealed a massive movement of this mutant to the top of sucrose gradients in the presence of UL11, whereas both the full-length UL16 and the C-terminal fragment (residues 156 to 373) remained at the bottom. Further evidence for the presence of a C-terminal regulatory domain was provided by single-amino-acid substitutions at conserved cysteines (C269S, C271S, and C357S), which enabled the efficient interaction of full-length UL16 with UL11. Lastly, the binding site for UL11 was further mapped to residues 81 to 155, and to our surprise, the 5 Cys residues within UL16(1-155) are not required, even though the modification of free cysteines in UL16 with N-ethylmaleimide does in fact prevent binding. Collectively, these results reveal a regulatory function within the C-terminal region of UL16 that controls an N-terminal UL11-binding activity.