A Gammaherpesviral Internal Repeat Contributes to Latency Amplification

Abstract

BackgroundGammaherpesviruses 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 FindingsWe 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/SignificanceThese data suggest that the 40 bp repeat contributes to latency amplification and might be involved in the regulation of viral gene expression.

A comparative epigenome analysis of gammaherpesviruses suggests cis-acting sequence features as critical mediators of rapid polycomb recruitment

“…While the lower CpG frequency of MHV-68 genomes does not support rapid recruitment of PRC complexes in a genome-wide fashion, our data suggest that the internal repeat regions may nevertheless serve as seed regions for polycomb acquisition. This hypothesis is also supported by the fact that the left internal repeat region has been previously found to be important for latency amplification in vivo [41]. In the right panel of Fig 11 we have therefore depicted a scenario in which PRC1.1 binds initially at CpG-rich (repeat) regions of the MHV-68 genome in a delayed manner (or as the result of a stochastically rare event), followed by spreading of PRCs and associated histone modifications via canonical complexes.…”

Gammaherpesvirus 68 Infection of Endothelial Cells Requires both Host Autophagy Genes and Viral Oncogenes for Optimal Survival and Persistence

“…To determine whether the v-cyclin was uniquely able to promote EC survival following ␥HV68 infection, we tested additional candidate genes for their contribution to EC survival: M11, M3, mK3, M1, M2, v-GPCR, and the viral polymerase III transcripts. Importantly, these viral genes have previously been identified as being dispensable for lytic replication in vitro (3,4,7,66,71). The viability of ECs (MB114 cells) infected with wt ␥HV68 or gene-specific mutant viruses was examined at 6 days postinfection by staining with annexin V and PI ( Thus, our viability screen identified several additional candidate genes that play a part in EC survival and persistence during ␥HV68 infection.…”

Chemokine Receptor CCR7 Contributes to a Rapid and Efficient Clearance of Lytic Murine γ-Herpes Virus 68 from the Lung, Whereas Bronchus-Associated Lymphoid Tissue Harbors Virus during Latency

“…Viral load in the spleens of infected mice was quantified by real-time PCR using the ABI 7300 Real Time PCR System (Applied Biosystems), as described (20). Briefly, amplification was performed with Taqman universal PCR master mix and universal cycling conditions (Applied Biosystems).…”