The Varicella-zoster virus DNA encapsidation genes: Identification and characterization of the putative terminase subunits

Abstract

The putative DNA encapsidation genes encoded by open reading frames (ORFs) 25, 26, 30, 34, 43, 45/42 and 54 were cloned from Varicella-zoster virus (VZV) strain Ellen. Sequencing revealed that the Ellen ORFs were highly conserved at the amino acid level when compared to those of 19 previously published VZV isolates. Additionally, RT-PCR provided the first evidence that ORF45/42 was expressed as a spliced transcript in VZV-infected cells. All seven ORFs were expressed in vitro and full length products were identified using a C-terminal V5 epitope tag. The in vitro products of the putative VZV terminase subunits encoded by ORFs 30 and 45/42 proved useful in protein-protein interaction assays. Previous studies have reported the formation of a heterodimeric terminase complex involved in DNA encapsidation for both herpes simplex virus-type 1 (HSV-1) and human cytomegalovirus (HCMV). Here we report that the C-terminal portion of exon II of ORF45/42 (ORF42-C269) interacted in GST-pull down experiments with in vitro synthesized ORF30 and ORF45/42. The interactions were maintained in the presence of anionic detergents and in buffers of increasing ionic strength. Cells transiently transfected with epitope tagged ORF45/42 or ORF30 showed primarily cytoplasmic staining. In contrast, an antiserum directed to the N-terminal portion of ORF45 showed nearly exclusive nuclear localization of the ORF45/42 gene product in infected cells. An ORF30 specific antiserum detected an 87 kDa protein in both the cytoplasmic and nuclear fractions of VZV infected cells. The results were consistent with the localization and function of herpesviral terminase subunits. This is the first study aimed at the identification and characterization of the VZV DNA encapsidation gene products.

The Varicella-Zoster Virus Portal Protein Is Essential for Cleavage and Packaging of Viral DNA

“…5A). The 87-kDa protein is consistent with the size previously reported for the putative portal monomer, pORF54, encoded by the VZV ORF54 gene (19,35). The VZV LUC , ⌬54S, and 54R samples contained VZV gE (Fig.…”

“…When any of the seven genes were deleted from the viral genome, empty capsids accumulated in the nucleus. Few studies have been done on the VZV homologs-ORF54, -45/42, -43, -34, -30, -26, and -25 (19,21,(34)(35)(36). Studies of VZV encapsidation have lagged behind those of other alphaherpesviruses in part due to the highly cell-associated nature of VZV.…”

Intermolecular Complementation between Two Varicella-Zoster Virus pORF30 Terminase Domains Essential for DNA Encapsidation

“…The protein complex that docks or interacts with the capsid portal protein consists of the heterotrimeric viral terminase. Terminases of DNA bacteriophages have been well described (2, 3), and those of several members of the Herpesviridae, including herpes simplex virus 1 (HSV-1) (4-14), human cytomegalovirus (HCMV) (15-21), Epstein-Barr virus (EBV) (22), and varicella-zoster virus (VZV) (23)(24)(25), are under investigation.Members of the Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae share a core set of orthologous proteins required for DNA encapsidation. A heterotrimeric viral terminase complex consisting of three of these conserved core proteins was recently isolated from HSV-1-infected cells (4).…”

“…The protein complex that docks or interacts with the capsid portal protein consists of the heterotrimeric viral terminase. Terminases of DNA bacteriophages have been well described (2,3), and those of several members of the Herpesviridae, including herpes simplex virus 1 (HSV-1) (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), human cytomegalovirus (HCMV) (15)(16)(17)(18)(19)(20)(21), Epstein-Barr virus (EBV) (22), and varicella-zoster virus (VZV) (23)(24)(25), are under investigation.…”

“…Homologous terminase components have also been identified for HCMV (pUL89, pUL56, and pUL51) (16)(17)(18)(19)(20)(21)(26)(27)(28) and VZV (pORF45/42, pORF30, and pORF25) (23,24). The pUL15, pUL89, and pORF45/42 homologs are encoded by spliced gene products (12,24,29) derived from two exons, and pUL51 and pORF25 are the HSV-1 pUL33 family homologs (5,21,23,25,30).…”