The Virion-Associated Open Reading Frame 49 of Murine Gammaherpesvirus 68 Promotes Viral Replication both In

(B) Genomic integrity of M3FL. At the outset of this study, 20 wood mice were tested both serologically for MHV-68-specific antibodies by ELISA and for the presence of viral DNA by PCR analysis of blood and spleen (3), and all were found to be MHV-68 negative. Although roles for ERK1/2 in MHV68 replication have not been described, ERK1/2 were previously implicated in facilitating KSHV replication [38], [39], [40]. The peak of latency is measured around 16 days post-infection in secondary lymphoid tissues such as the spleen. As detailed in Table S1 in the supplemental material, these sequences included the previously confirmed miRNA sequences: mghv-miR-M1-1-3p and mghv-miR-M1-10-5p (TMER1), mghv-miR-M1-3-3p (TMER2), mghv-miR-M1-4-5p (TMER3), mghv-miR-M1-5-5p, mghv-miR-M1-6-3p (TMER4), mghv-miR-M1-7-5p and mghv-miR-M1-7-3p (TMER5), mghv-miR-M1-13-5p (TMER6), mghv-miR-M1-14-3p (TMER7), and mghv-miR-M1-15-5p and mghv-miR-M1-9-3p (TMER8). 1A and plasmids expressing transactivator(s) RTA or RTA and Zebra. To assure that interferon effects were not due to effects on the indicator monolayer, IFN-αβγR−/− MEFs were used in all experiments.

No virus was detected in samples pooled from harvests at 1 to 3 days or 6 to 12 days. Subgroup A and C strains immortalize common marmoset lymphocytes, but none of the subgroup B strains scored positive in in vitro immortalization assays. Based on these data, we hypothesize that the unanticipated role of the viral cyclin and bcl-2 genes in acute pathogenesis is uniquely revealed in IFN-γ−/− hosts. Immunostained slides were counterstained with hematoxylin (Sigma, St. It is to be noted that pSTAT5 is predominantly activated in T cells in response to IL-2 [26] and in myeloid cells in response to GM-CSF [27]. Proteins were resolved by SDS-PAGE in 5%, 10%, or 12% polyacrylamide gels. NT-2 was initially isolated from a lung metastasis of a 22-year-old male with primary embryonal carcinoma of the testis (58).

Mechanical disruption kills >99% of cells but has at most a twofold effect on viral titer (81), thus enabling experimental distinction between reactivation from latency, which requires live cells, and persistent replication. The correct sequence, size, and expression of each protein were verified by Western blotting, using a monoclonal antibody against FLAG (Sigma) (Fig. The time point of 12 h postinfection was selected because no difference in viral titers between medium and IFN-γ treatment was observed at this time point (Fig. At 6 months p.i., two viruses containing wt v-cyclin, wt γHV68 and v-cyclin.MR, reactivated equivalently from latently infected peritoneal cells when the cells were cultured ex vivo (wt reactivation frequency, 1 in 81,900; Fig. Fifty nanograms of each was spotted in duplicate onto charged nylon membranes (Hybond N+; Amersham) with a 384-pin multiblotter (V & P Scientific). EcoRI and BamHI digests result in diagnostic fragments for the v-cyclin region, and theNsiI-NotI digestion is diagnostic for deletions at the left end of the viral genome (13). The dashed line shows the limit of assay sensitivity.

This mutant library was generated using in vitro Mu transposition, with an infectious BAC clone of MHV-68 as a target template. As such, our data provides novel and compelling evidence for a need to evaluate primary acute EBV infections as a potential risk factor in the development of non-cerebral severe malaria. Virol. A. The results were visualized with the software TreeView (9). The plasmid pEBG/M2 was generated to express a glutathione S-transferase (GST)-M2 fusion protein in mammalian cells. Thus, ORF11 was dispensable for viral lytic replication in both BHK-21 cells and murine embryo fibroblasts.

We used 8- to 12-week-old animals of mixed genders in groups of three to seven for most experiments. To generate viral stocks, it was necessary to remove BAC vector sequences (2). All other expression plasmids of MHV-68 ORFs except pCMV2-FLAG/RTA described in this study were prepared in pENTR and constructed into appropriate destination vectors using Gateway technology (Invitrogen) according to the manufacturer’s instructions. Vetted mice were bred at Baylor College of Medicine in accordance with all federal and university guidelines. Although EBV does not infect rodents, murine gamma herpesvirus-68 (γHV-68) has been a useful tool in studying the relationship between the immune system and latent γ-herpesvirus infection in mice [15]. Royal Soc. Briefly, spleen cells from DNA-vaccinated mice were restimulated in vitro with the M2-expressing S11 cell line (5, 24), which had been irradiated with 3,800 rads.

infection. LANA is transcribed with immediate early kinetics upon G2HV infection of host cells, which suggests a role in productive viral replication (26, 27). Successful recombination led to the replacement of the 40-bp repeat of MHV-68 with a Tet resistance gene. Several studies have recently identified the protein constituents of the virions of gammaherpesviruses, including murine gammaherpesvirus 68 (MHV-68), Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), and rhesus monkey rhadinovirus (RRV), by mass-spectrometric analyses (4, 6, 23, 31, 32, 37, 55). Both ORF24 of MHV-68 and its human cytomegalovirus homolog (UL87) have previously been identified as being essential for lytic replication by genome-wide mutagenesis (7, 19, 25).