ABSTRACT
The Herpesviridae contain a group of highly conserved proteins designated the Herpes UL33 Superfamily (pfam03581). The Varicella-zoster virus (VZV) homolog, encoded by the ORF25 gene, was used to generate a GST-ORF25 fusion protein. Purified GST-ORF25 was used to generate a polyclonal rabbit antiserum that detected the 17.5 kDa ORF25 protein (pORF25) in VZV infected cells. In pull-down assays, GST-ORF25 interacted with a number of encapsidation proteins including ORF30, ORF42 (the second exon of ORF45/42) and itself. The self-interaction was confirmed via a yeast two-hybrid assay. Additionally, pORF25 and pORF30 were shown to co-immunoprecipitate from VZV infected cells. Our results suggest that pORF25 is part of the trimeric terminase complex for VZV. However, combined with data from previous studies on HSV-1 and Kaposi's sarcoma associated herpesvirus (KSVH), we hypothesize that VZV pORF25 and the Herpes UL33 Superfamily homologs are not encapsidation proteins per se but instead work to bring viral proteins together to form functional complexes.
Subject(s)
Herpesvirus 3, Human/physiology , Protein Interaction Mapping , Viral Proteins/metabolism , Virus Assembly , Amino Acid Sequence , Antibodies, Viral/immunology , Cell Line , Herpesvirus 3, Human/genetics , Humans , Immunoprecipitation , Molecular Sequence Data , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Sequence Alignment , Viral Proteins/geneticsABSTRACT
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.
Subject(s)
Endodeoxyribonucleases/metabolism , Genes, Viral , Herpesvirus 3, Human/genetics , Herpesvirus 3, Human/metabolism , Animals , Cell Line , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/isolation & purification , Humans , Open Reading Frames , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
The 230-kbp murine cytomegalovirus (MCMV) genome is predicted to encode 182 open reading frames (orfs). One gene whose functional role is not known is encoded by the 762-bp m136 orf. Sequence analysis of rat cytomegalovirus (RCMV) strains Maastricht and English revealed homologous orfs, pr136, and ORF HJ4, respectively. Conservation of these orfs suggested that m136 and the RCMV homologs might play a role during virus replication. Expression of an epitope tagged form of m136 (m136-V5) yielded a polypeptide of 34 kDa that localized to the perinuclear region of transfected mouse 3T3 fibroblasts. Three independently generated MCMV m136 mutants were isolated and characterized. Mutations were introduced into the m136 orf by inserting either a beta-glucuronidase (m136-beta-gluc) or a guanosine phosphoribosyl transferase (m136-gpt) expression cassette into a unique BglII site, or by inserting a gpt cassette into a deleted region (Deltam136) of m136. No differences were observed in viral yield, plaque size, and plaque morphology between the parental strain and any of the m136 mutant viruses. In vivo analysis using a SCID mouse virulence model showed a consistently measurable attenuated phenotype for all three m136 mutants. The results showed that although the m136 gene was not essential for replication in vitro or in vivo, an intact m136 gene was necessary to yield wild type virulence during infection of the host.
