The small noncoding RNAs (sncRNAs) of murine gammaherpesvirus 68 (MHV-68) are involved in regulating the latent-to-lytic switch in vivo

The human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), which are associated with a variety of diseases including tumors, produce various small noncoding RNAs (sncRNAs) such as microRNAs (miRNAs). Like all herpesviruses, they show two stages in their life cycle: lytic replication and latency. During latency, hardly any viral proteins are expressed to avoid recognition by the immune system. Thus, sncRNAs might be exploited since they are less likely to be recognized. Specifically, it has been proposed that sncRNAs might contribute to the maintenance of latency. This has already been shown in vitro, but the respective evidence in vivo is very limited. A natural model system to explore this question in vivo is infection of mice with murine gammaherpesvirus 68 (MHV-68). We used this model to analyze a MHV-68 mutant lacking the expression of all miRNAs. In the absence of the miRNAs, we observed a higher viral genomic load during late latency in the spleens of mice. We propose that this is due to a disturbed regulation of the latent-to-lytic switch, altering the balance between latent and lytic infection. Hence, we provide for the first time evidence that gammaherpesvirus sncRNAs contribute to the maintenance of latency in vivo

A Gammaherpesviral Internal Repeat Contributes to Latency Amplification

BACKGROUND: Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. The genomes of gammaherpesviruses contain variable numbers of internal repeats whose precise role for in vivo pathogenesis is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We used infection of laboratory mice with murine gammaherpesvirus 68 (MHV-68) to explore the biological role of the 40 bp internal repeat of MHV-68. We constructed several mutant viruses partially or completely lacking this repeat. Both in vitro and in vivo, the loss of the repeat did not substantially affect lytic replication of the mutant viruses. However, the extent of splenomegaly, which is associated with the establishment of latency, and the number of ex vivo reactivating and genome positive splenocytes were reduced. Since the 40 bp repeat is part of the hypothetical open reading frame (ORF) M6, it might function as part of M6 or as an independent structure. To differentiate between these two possibilities, we constructed an N-terminal M6STOP mutant, leaving the repeat structure intact but rendering ORF M6 unfunctional. Disruption of ORF M6 did neither affect lytic nor latent infection. In contrast to the situation in lytically infected NIH3T3 cells, the expression of the latency-associated genes K3 and ORF72 was reduced in the latently infected murine B cell line Ag8 in the absence of the 40 bp repeat. CONCLUSIONS/SIGNIFICANCE: These data suggest that the 40 bp repeat contributes to latency amplification and might be involved in the regulation of viral gene expression

A Gammaherpesvirus Complement Regulatory Protein Promotes Initiation of Infection by Activation of Protein Kinase Akt/PKB

BACKGROUND: Viruses have evolved to evade the host's complement system. The open reading frames 4 (ORF4) of gammaherpesviruses encode homologs of regulators of complement activation (RCA) proteins, which inhibit complement activation at the level of C3 and C4 deposition. Besides complement regulation, these proteins are involved in heparan sulfate and glycosaminoglycan binding, and in case of MHV-68, also in viral DNA synthesis in macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Here, we made use of MHV-68 to study the role of ORF4 during infection of fibroblasts. While attachment and penetration of virions lacking the RCA protein were not affected, we observed a delayed delivery of the viral genome to the nucleus of infected cells. Analysis of the phosphorylation status of a variety of kinases revealed a significant reduction in phosphorylation of the protein kinase Akt in cells infected with ORF4 mutant virus, when compared to cells infected with wt virus. Consistent with a role of Akt activation in initial stages of infection, inhibition of Akt signaling in wt virus infected cells resulted in a phenotype resembling the phenotype of the ORF4 mutant virus, and activation of Akt by addition of insulin partially reversed the phenotype of the ORF4 mutant virus. Importantly, the homologous ORF4 of KSHV was able to rescue the phenotype of the MHV-68 ORF4 mutant, indicating that ORF4 is functionally conserved and that ORF4 of KSHV might have a similar function in infection initiation. CONCLUSIONS/SIGNIFICANCE: In summary, our studies demonstrate that ORF4 contributes to efficient infection by activation of the protein kinase Akt and thus reveal a novel function of a gammaherpesvirus RCA protein

Murine Gammaherpesvirus 68 Contains Two Functional Lytic Origins of Replication▿

A 1.25-kbp DNA fragment from the right side of the genome containing the lytic origin of replication (oriLyt) of murine gammaherpesvirus 68 (MHV-68) has been identified by a plasmid replication assay. Here we show that a mutant MHV-68 with a deletion of an essential part of this oriLyt, generated by using an MHV-68 bacterial artificial chromosome, was only slightly attenuated and still able to replicate but that a mutant containing an additional deletion on the left side of the genome was replication deficient. The newly identified region was sufficient to support plasmid replication, thus providing evidence for a second oriLyt

Lytic replication in the lungs after i.n. inoculation.

<p>C57BL/6 mice were inoculated i.n. with 1x 10³ PFU of the indicated viruses. Lungs of mice were harvested at day 3 after infection (parental virus or mutant viruses) and at day 6 after infection (parental or mutant viruses and ectopic revertants). Virus titers were determined from organ homogenates by plaque assay. Each symbol represents an individual mouse, and the bars represent the median value. (A) The data are compiled from two (day 3) and five (day 6 mutant viruses) or six (day 6 parental virus) independent experiments. (B and C) The data are compiled from two independent experiments in each case. The asterisks indicate a statistically significant difference between the groups (* P < 0.05; ** P < 0.01; *** P < 0.001).</p

Proteins associated with the right oriLyt as identified by DNA-affinity purification and mass spectrometry analysis^a.

<p>Proteins associated with the right oriLyt as identified by DNA-affinity purification and mass spectrometry analysis<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005510#t001fn001" target="_blank">^a</a>.</p

Latent infection in PECs after i.p. inoculation.

<p>C57BL/6 mice were inoculated i.p. with 1x 10⁴ PFU of the indicated viruses. PECs were harvested at day 17 after infection to analyze latent infection in the ex vivo reactivation assay (A and B) or for DNA isolation and real-time PCR analysis of the viral genomic load (C and D). Data for parental virus and virus mutants are obtained from two independent experiments, each with five mice per group. Data for the ectopic revertants (ER Δright oriLyt and ER Δleft oriLyt) are obtained from a single experiment with five mice per group. For the ex vivo reactivation assay, cells from five mice per group were pooled in each experiment and data shown are the means ± SEM (parental virus or virus mutants) or values from a single experiment (ectopic revertants). The dashed line indicates the point of 63.2% Poisson distribution, determined by nonlinear regression, which was used to calculate the frequency of cells reactivating lytic replication. In panels C and D, each symbol represents an individual mouse, and the bars represent the median value. Asterisks indicate a statistically significant difference between the groups (* P < 0.05).</p

Virus replication in vitro after upregulation or downregulation of Hexim1.

<p>To upregulate Hexim1, NIH 3T3 cells were plated and after 4 hours stimulated with 5 mM or 10 mM HMBA or left untreated. 24 hours later, cells were infected with the indicated viruses at an MOI of 0.01 for 1 hour. After removing the inoculum, cells were incubated with fresh medium without HMBA (A), with 5 mM HMBA (B) or with 10 mM HMBA (C) at 37°C and 5% CO₂ until the supernatants together with the cells were harvested at different time points after infection. Virus titers were determined by plaque assay. For the “no treatment” control, the means ± SEM of duplicates from three independent experiments are shown. Data shown for the HMBA treated cells are from three independent experiments showing each single experiment. To downregulate Hexim1, TCMK-1 cells were stable transfected with two different shRNAs specific for Hexim1 or with a scrambled control shRNA, respectively (D). Cells were plated and after 24 hours infected with the indicated viruses at an MOI of 0.01 for 1 hour. After removing the inoculum, cells were incubated with fresh medium at 37°C and 5% CO₂ until the supernatants together with the cells were harvested at different time points after infection. Virus titers were determined by plaque assay. For the “scrambled shRNA” control, the means ± SEM of duplicates from two independent experiments are shown. For the shRNAs specific for Hexim1, the means ± SD of two independent experiments each are shown.</p

Confirmation of results from DNA-affinity purification and MassSpec.

<p>Formaldehyde cross-linking ChIP assays were performed to ensure that Hexim1 and Rbbp4 are associated with DNA of the right oriLyt of MHV-68. The immunoprecipitates isolated from TCMK-1 cells by a specific antibody against Hexim1 (A) or from NIH 3T3 cells by a specific antibody against Rbbp4 (B) were analyzed by quantitative PCR with primer pairs designed to amplify a part of the DNA sequence of the right oriLyt and the left oriLyt, respectively, or by a primer pair amplifying a non-relevant sequence located in the viral ORF23.</p