Murine Gammaherpesvirus 68 Open Reading Frame 31 Is Required for Viral Replication

Abstract

Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) and Epstein-Barr virus (EBV). It has been proposed as a model for gammaherpesvirus infection and pathogenesis. Open reading frame 31 (ORF31) is conserved among the Beta-and Gammaherpesvirinae subfamily, and there is no known mammalian homologue of this protein.The function of MHV-68 ORF31 and its viral homologues has not yet been determined. We described here a primary characterization of this protein and its requirement for lytic replication. The native MHV-68 ORF31 was detected at peak levels by 24 h postinfection, and the FLAG-tagged and green fluorescent protein fusion ORF31 were localized in the cytoplasm and nucleus in a diffuse pattern. Two independent experimental approaches were then utilized to demonstrate that ORF31 was required for lytic replication. First, small interfering RNA generated against ORF31 expression blocked protein expression and virus production in transfected cells. Then, two-independent bacterial artificial chromosome-derived ORF31-null MHV-68 mutants (31STOP) were generated and found to be defective in virus production in fibroblast cells. This defect can be rescued in trans by MHV-68 ORF31 and importantly by its KSHV homologue. A repair virus of 31STOP was also generated by homologous recombination in fibroblast cells. Finally, we showed that the defect in ORF31 blocked late lytic protein expression. Our results demonstrate that MHV-68 ORF31 is required for viral lytic replication, and its function is conserved in its KSHV homologue.

A Replication-Deficient Murine γ-Herpesvirus Blocked in Late Viral Gene Expression Can Establish Latency and Elicit Protective Cellular Immunity

“…Wild-type ␥HV68 and ORF31STOP virus titers were determined by plaque assay on NIH-3T3 and T-Rex cells, respectively. The absence of reversion either in vivo or during in vitro propagation of the virus was confirmed by the presence of the unique BamHI restriction site integrated into the mutant virus (13,22), the inability of mutant virus to grow on NIH-3T3 cells, and the absolute dependence on the T-Rex complementary cell line for plaque formation. Female C57BL/6 and BALB/c mice were anesthetized with 2,2,2-tribromoethanol (200 mg/kg) before intranasal (i.n.)…”

“…The generation of the replication-deficient ORF31STOP ␥HV68 viruses, BAC ϩ ORF31STOP, and BAC Ϫ ORF31STOP have been described (22). The mutant viruses were propagated in the complementary T-Rex mouse fibroblast cell line in DMEM medium supplemented with 10% FBS, penicillin, streptomycin (Sigma-Aldrich), and doxycyline (BD Biosciences).…”

“…We have previously characterized infection with a replication-deficient mutant of ␥HV68 (ORF31STOP) (22) and shown that following i.p. inoculation, cells harboring viral genome could be detected in the spleen, whereas following i.n.…”

“…ORF31 is a protein of unknown function that is conserved between the ␤-and ␥-herpesviruses but for which there is no known mammalian homologue. The mutant virus (ORF31STOP) expressed immediate early and early genes but was deficient in late viral protein expression and failed to produce infectious viral particles in the absence of trans rescue (13,22). The data show that i. p. infection with ORF31STOP virus established latency in the spleen, was highly immunogenic, and conferred substantial protection against challenge with wild-type virus, although sterilizing immunity was not achieved.…”

“…Therefore, in the current study, we used a replication-deficient virus generated by insertion of triple stop codons into the N terminus of ORF31 (22). ORF31 is a protein of unknown function that is conserved between the ␤-and ␥-herpesviruses but for which there is no known mammalian homologue.…”