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

Abstract

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 Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

“…The latter finding is contrary to that of the HSV-1 pUL32 orthologue, which influences HSV-1 pUL25 nuclear localization (72). This may be due to the recently described ability of pUL32 to affect the disulfide bond profile of pUL25 (73,74); however, it is not known whether HCMV pUL52 exerts a similar function. In any case, the results presented here indicate that pUL52 is neither a capsid constituent nor an interaction partner of pUL77.…”

Replication Compartments of DNA Viruses in the Nucleus: Location, Location, Location

“…In the case of herpesviruses, in particular HSV-1 and CMV, the assembly and packaging of viral particles is proposed to occur within VRCs [11,15,76]. Several HSV-1 capsid proteins can be detected in VRCs, including VP22, VP23, and VP5 hexamers; the latter are present on the surface of assembled capsids [169][170][171][172]. Furthermore, packaging protein UL32 is required for the localization of VP5 hexamers to VRCs, inferring that UL32 is required to localize assembled capsids at VRCs so that they can be packaged [170].…”

From structure to redox: The diverse functional roles of disulfides and implications in disease

“…This disulfide‐bond forming machinery is shared by several other nucleocytoplasmic large DNA viruses . Additionally, the herpes simplex virus 1 (HSV‐1) chaperone UL32 contains three CXXC motifs and is thought to regulate disulfide‐bond formation on capsid proteins to ensure structural integrity . Absence of UL32 resulted in the formation of non‐native disulfide bonds, likely due to spontaneous and uncontrolled disulfide‐bond formation during infection and capsid maturation .…”

“…Additionally, the herpes simplex virus 1 (HSV‐1) chaperone UL32 contains three CXXC motifs and is thought to regulate disulfide‐bond formation on capsid proteins to ensure structural integrity . Absence of UL32 resulted in the formation of non‐native disulfide bonds, likely due to spontaneous and uncontrolled disulfide‐bond formation during infection and capsid maturation . While disulfide‐bond formation in the cytoplasm is rare and only demonstrated for non‐host viral proteins, it serves to exemplify that even reducing compartments of the cell are capable of hosting oxidoreductases for controlled disulfide formation.…”