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

Generation of MHV-68 mutants.

<p>A) Schematic presentation of viral mutants. B) Scheme of the expected fragments after digestion of viral DNA with the restriction enzyme <i>EcoR</i>I. Digestion of DNA from both parental virus and the 40 bp revertant with <i>EcoR</i>I results in a 5.2 kb wildtype fragment. Deletion of the 40 bp repeat results in the loss of the 5.2 kb fragment and in the generation of a new 2.8 kb fragment. Partial loss of repeat units results in a shift of the 5.2 kb fragment to 4.6 kb and 4.2 kb fragments, respectively. “P” indicates the probe used for Southern blot analysis, corresponding to nucleotides 25889-26711. C) Structural analysis of reconstituted virus genomes by ethidium bromide-stained agarose gel analysis of viral DNA digested with <i>EcoR</i>I. Lane 1: Parental virus; Lane 2: 40 bp revertant; Lane 3: Delta 40 bp mutant; Lane 4: 40 bp moderate mutant; Lane 5: 40 bp low mutant; Lane 6: M6STOP mutant. D) Southern blot analysis of the gel shown in panel C using probe “P” indicated in panel B. The expected fragments are indicated by dots (panel C) or by arrows (panel D). Marker (M) sizes (in kilobase pairs) are indicated on the left.</p

The 40 bp internal repeat is important for virus-induced splenomegaly and for <i>ex vivo</i> reactivation of latently infected splenocytes.

<p>C57BL/6 mice were i.n. infected with 5×10⁴ PFU. A) Extent of splenomegaly. At the indicated time points after infection, spleens were harvested and the spleen weight was determined. Shown are means±SD of the number of mice indicated in brackets. The asterisks indicate a statistically significant difference (p = 0.043 at day 13 and p = 1.02×10⁻⁸ at day 17; Student's t-test). The extent of <i>ex vivo</i> reactivation was determined 10 (B), 13 (C), 17 (D) and 23 (E) days after infection. Data shown are from a representative experiment with splenocytes pooled from 3 mice per group. F) Summary of independent <i>ex vivo</i> reactivation experiments performed with splenocytes at day 17 after infection. In each individual experiment, splenocytes from 3 mice per group were pooled. Shown are the reciprocal frequencies of reactivation (mean±SD; n = 4 for parental virus and Delta 40 bp mutant, n = 2 for revertant), calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000733#s2" target="_blank">materials and methods</a>. The asterisk indicates a statistically significant difference (p = 0.040; Student's t-test).</p