Journal: mBio

Article Title: Virus-Encoded MicroRNAs Facilitate Gammaherpesvirus Latency and Pathogenesis In Vivo

doi: 10.1128/mBio.00981-14

Figure Lengend Snippet: Expression kinetics of MHV68-encoded miRNAs during lytic infection. Stem-loop qRT-PCR was used to determine the level of expression of MHV68-encoded mature miRNAs during lytic replication. NIH 3T12 fibroblasts were infected with MHV68 at MOI 5 and then harvested at the indicated time points. Stem-loop qRT-PCR was performed using TaqMan primers and probe sets (see Table S2 in the supplemental material). (A) Expression time course for all tested viral miRNAs relative (10 2 ) to the endogenous noncoding RNA control snoRNA234. (B) Time course for all tested viral miRNAs expressed as approximate miRNA copy number per cell, based on the synthetic miR-2-5p standard curve. (C) Viral miRNAs that peak between 16 and 48 hpi, expressed as miRNA copy number per cell. (D) Viral miRNAs that peak at 48 hpi. (E) Viral miRNAs with consistent low-level expression throughout the time course, depicted on an enhanced scale. All data are means ± standard deviations (SD) from 3 experiments.

Article Snippet: NIH 3T12 murine fibroblasts (ATCC CCL-164) and murine embryonic fibroblasts (MEFs) from C57BL/6J mice (Global Stem GSC-6002) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml of penicillin, 100 mg/ml streptomycin, and 2 mM l -glutamine.

Techniques: Expressing, Infection, Quantitative RT-PCR, Control

Journal: mBio

Article Title: Virus-Encoded MicroRNAs Facilitate Gammaherpesvirus Latency and Pathogenesis In Vivo

doi: 10.1128/mBio.00981-14

Figure Lengend Snippet: miRNA mutations incorporated in MHV68.Zt6 and corresponding expression of miRNAs and surrounding genes. (A) Depiction of MHV68.Zt6 mutations, including deletion of TMER1 to TMER5, TMER7, and TMER8 miRNA stem-loops (red X) and insertion of the PolIII stop site (red line) following vtRNA6 in TMER6. (B) Expression of viral miRNAs from wild-type MHV68 marker virus or MHV68.Zt6, relative (10 2 ) to the endogenous noncoding RNA control snoRNA202. NIH 3T12 fibroblasts were infected with MHV68.ORF73βla or MHV68.Zt6 at an MOI of 5 and then harvested at 48 hpi. Stem-loop qRT-PCR was performed as described for Fig. 1 . Values are means ± SD from 2 experiments. (C) Expression of viral coding genes surrounding miRNA mutations. NIH 3T12 fibroblasts were infected with MHV68.ORF73βla or MHV68.Zt6 at an MOI of 5 for 24 h. M1, M2, and M3 expression was quantified using qRT-PCR. Values are relative (10 3 ) to endogenous GAPDH expression and are means ± SD from 3 experiments.

Article Snippet: NIH 3T12 murine fibroblasts (ATCC CCL-164) and murine embryonic fibroblasts (MEFs) from C57BL/6J mice (Global Stem GSC-6002) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml of penicillin, 100 mg/ml streptomycin, and 2 mM l -glutamine.

Techniques: Expressing, Marker, Virus, Control, Infection, Quantitative RT-PCR

Journal: mBio

Article Title: Virus-Encoded MicroRNAs Facilitate Gammaherpesvirus Latency and Pathogenesis In Vivo

doi: 10.1128/mBio.00981-14

Figure Lengend Snippet: MHV68 miRNAs are dispensable for lytic replication. Plaque assays were used to determine the titer of wild-type or miRNA deletion mutant viruses during acute replication in vitro and in vivo . (A) Lytic replication in fibroblasts in vitro . Single-step and multistep growth curves were generated following infection of NIH 3T12 fibroblasts with MHV68.ORF73βla or MHV68.Zt6 at an MOI of 5 or 0.05, respectively. At the specified time points, cells and supernatant fluid were collected, and titers were determined by plaque assay. Data are means ± SD of 3 experiments. (B) Lytic replication in lungs in vivo . C57BL6/J mice were infected i.n. with 10 4 PFU MHV68.ORF73βla or MHV68.Zt6. At 5 or 8 dpi, lungs were harvested and viral titers were determined by plaque assay. Lines represent the mean titers for eight individual mice. For all experiments, statistical significance was determined by Student’s t test. *, P < 0.05; **, P < 0.01.

Article Snippet: NIH 3T12 murine fibroblasts (ATCC CCL-164) and murine embryonic fibroblasts (MEFs) from C57BL/6J mice (Global Stem GSC-6002) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml of penicillin, 100 mg/ml streptomycin, and 2 mM l -glutamine.

Techniques: Mutagenesis, In Vitro, In Vivo, Generated, Infection, Plaque Assay