A Gammaherpesvirus Cooperates with Interferon-alpha/beta-Induced IRF2 to Halt Viral Replication, Control Reactivation, and Minimize Host Lethality

The gammaherpesviruses, including Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), establish latency in memory B lymphocytes and promote lymphoproliferative disease in immunocompromised individuals. The precise immune mechanisms that prevent gammaherpesvirus reactivation and tumorigenesis are poorly defined. Murine gammaherpesvirus 68 (MHV68) is closely related to EBV and KSHV, and type I (alpha/beta) interferons (IFNαβ) regulate MHV68 reactivation from both B cells and macrophages by unknown mechanisms. Here we demonstrate that IFNβ is highly upregulated during latent infection, in the absence of detectable MHV68 replication. We identify an interferon-stimulated response element (ISRE) in the MHV68 M2 gene promoter that is bound by the IFNαβ-induced transcriptional repressor IRF2 during latency in vivo. The M2 protein regulates B cell signaling to promote establishment of latency and reactivation. Virus lacking the M2 ISRE (ISREΔ) overexpresses M2 mRNA and displays uncontrolled acute replication in vivo, higher latent viral load, and aberrantly high reactivation from latency. These phenotypes of the ISREΔ mutant are B-cell-specific, require IRF2, and correlate with a significant increase in virulence in a model of acute viral pneumonia. We therefore identify a mechanism by which a gammaherpesvirus subverts host IFNαβ signaling in a surprisingly cooperative manner, to directly repress viral replication and reactivation and enforce latency, thereby minimizing acute host disease. Since we find ISREs 5′ to the major lymphocyte latency genes of multiple rodent, primate, and human gammaherpesviruses, we propose that cooperative subversion of IFNαβ-induced IRFs to promote latent infection is an ancient strategy that ensures a stable, minimally-pathogenic virus-host relationship

Borrelia miyamotoi Infection in Patients from Upper Midwestern United States, 2014–2015

We confirmed Borrelia miyamotoi infection in 7 patients who had contracted an illness while near La Crosse, Wisconsin, USA, an area where Ixodes scapularis ticks are endemic. B. miyamatoi infection should now be considered among differential diagnoses for patients from the midwestern United States who have signs and symptoms suggestive of tickborne illness

Conditional mutagenesis in vivo reveals cell type- and infection stage-specific requirements for LANA in chronic MHV68 infection.

Gammaherpesvirus (GHV) pathogenesis is a complex process that involves productive viral replication, dissemination to tissues that harbor lifelong latent infection, and reactivation from latency back into a productive replication cycle. Traditional loss-of-function mutagenesis approaches in mice using murine gammaherpesvirus 68 (MHV68), a model that allows for examination of GHV pathogenesis in vivo, have been invaluable for defining requirements for specific viral gene products in GHV infection. But these approaches are insufficient to fully reveal how viral gene products contribute when the encoded protein facilitates multiple processes in the infectious cycle and when these functions vary over time and from one host tissue to another. To address this complexity, we developed an MHV68 genetic platform that enables cell-type-specific and inducible viral gene deletion in vivo. We employed this system to re-evaluate functions of the MHV68 latency-associated nuclear antigen (mLANA), a protein with roles in both viral replication and latency. Cre-mediated deletion in mice of loxP-flanked ORF73 demonstrated the necessity of mLANA in B cells for MHV68 latency establishment. Impaired latency during the transition from draining lymph nodes to blood following mLANA deletion also was observed, supporting the hypothesis that B cells are a major conduit for viral dissemination. Ablation of mLANA in infected germinal center (GC) B cells severely impaired viral latency, indicating the importance of viral passage through the GC for latency establishment. Finally, induced ablation of mLANA during latency resulted in complete loss of affected viral genomes, indicating that mLANA is critically important for maintenance of viral genomes during stable latency. Collectively, these experiments provide new insights into LANA homolog functions in GHV colonization of the host and highlight the potential of a new MHV68 genetic platform to foster a more complete understanding of viral gene functions at discrete stages of GHV pathogenesis

RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency

RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. Lipopolysaccharide (LPS)/toll-like receptor (TLR)4 engagement enhances rhadinovirus reactivation. We developed two new systems to examine the interaction of RTA with host NF-kappaB (NF-κB) signaling during murine gammaherpesvirus 68 (MHV68) infection: a latent B cell line (HE-RIT) inducible for RTA-Flag expression and virus reactivation; and a recombinant virus (MHV68-RTA-Bio) that enabled in vivo biotinylation of RTA in BirA transgenic mice. LPS acted as a second stimulus to drive virus reactivation from latency in the context of induced expression of RTA-Flag. ORF6, the gene encoding the single-stranded DNA binding protein, was one of many viral genes that were directly responsive to RTA induction; expression was further increased upon treatment with LPS. However, NF-κB sites in the promoter of ORF6 did not influence RTA transactivation in response to LPS in HE-RIT cells. We found no evidence for RTA occupancy of the minimal RTA-responsive region of the ORF6 promoter, yet RTA was found to complex with a portion of the right origin of lytic replication (oriLyt-R) that contains predicted RTA recognition elements. RTA occupancy of select regions of the MHV-68 genome was also evaluated in our novel in vivo RTA biotinylation system. Streptavidin isolation of RTA-Bio confirmed complex formation with oriLyt-R in LPS-treated primary splenocytes from BirA mice infected with MHV68 RTA-Bio. We demonstrate the utility of reactivation-inducible B cells coupled with in vivo RTA biotinylation for mechanistic investigations of the interplay of host signaling with RTA

Cre-mediated deletion of <i>ORF73</i> yields a reduction in previously established MHV68 latency.

<p>(A) Schematic depicting the working model and experimental strategy for selective Cre-mediated deletion of ORF73 during MHV68 latency maintenance. (B) WT or Hemizygous Cre-ERT2 transgenic mice were infected IN with 1000 PFU of the indicated virus. On days 23–28 post-infection, mice were injected IP with vehicle or tamoxifen (2 mg/dose; one dose per day for 5 consecutive days) to induce Cre-ERT2 nuclear translocation. Mice were sacrificed on day 42 post-infection. Single-cell suspensions of splenocytes were serially diluted, and frequencies of cells harboring MHV68 genomes were determined using a limiting-dilution PCR analysis to detect either <i>ORF50</i> or <i>ORF73</i> genomic loci. Note: <i>ORF73</i> primer pairs overlap the deletion junction. Therefore, the target sequence is lost upon Cre-mediated deletion of <i>ORF73</i>. Results are means of 2 independent infections. Error bars represent standard error of the means. CTRL = mice treated with corn oil; TAM = mice treated with tamoxifen.</p

O73.loxP exhibits efficient acute replication in lungs of mice.

<p>C57BL/6 (A) or CD19^Cre/+ (B) mice were infected IN with 1000 PFU of the indicated viruses. Mice were sacrificed on day 7 post-infection, and viral titers in lung homogenates were determined by plaque assay. Each dot represents one mouse. Error bars represent standard error of the means. * denotes p < 0.05 in a two-tailed student’s t-test.</p

Reciprocal frequencies of ORF50⁺ and ORF73⁺ cells for vehicle or tamoxifen-treated Cre-ERT2 or WT mice infected with O73.loxP or FRT MHV68.

<p>Reciprocal frequencies of ORF50⁺ and ORF73⁺ cells for vehicle or tamoxifen-treated Cre-ERT2 or WT mice infected with O73.loxP or FRT MHV68.</p

O73.loxP establishes latency in spleens of C57BL/6 mice following IN or IP inoculation.

<p>C57BL/6 mice were infected IN (A and B) or IP (C and D) with 1000 PFU of the indicated virus. Mice were sacrificed on days 16–18 post-infection. (A and C) Single-cell suspensions of spleen cells were serially diluted, and frequencies of cells harboring MHV68 genomes were determined using a limiting-dilution PCR analysis. (B and D) Reactivation frequencies were determined by <i>ex vivo</i> plating of serially diluted cells on an indicator monolayer. Cytopathic effect was scored 2–3 weeks post-plating. Groups of 3–5 mice were pooled for each infection and analysis. Results are means of 2–3 independent infections. Error bars represent standard error of the means.</p

Frequency of viral-genome positive cells.

<p>Frequency of viral-genome positive cells.</p

Derivation and validation of O73.loxP MHV68.

<p>(A) Schematic depicting the insertion of <i>loxP</i> sites flanking <i>ORF73</i> in the MHV68 genome and its deletion in the presence of Cre recombinase. (B) 3T3 fibroblasts were infected with WT MHV68 or O73.loxP at an MOI of 5 PFU/cell (single-step, left panel) or 0.05 PFU/cell (multi-step, right panel). Viral titers were determined by plaque assay at the indicated times post-infection. Results are means of triplicate samples. Error bars represent standard deviations. (C and D) 3T3 fibroblasts that encode Cre-ERT2 were treated with vehicle (NOT induced) or 4-hydroxytamoxifen (Cre induced) to induce Cre activity 24 h prior to infection. Treated cells were infected with the indicated viruses at an MOI of 0.05 PFU/cell. (C) Total DNA was isolated on day 4 post-infection, and PCR was performed as illustrated in the schematic to detect the intact or deleted <i>ORF73</i> locus or the distal <i>ORF59</i> locus as a control. (D) Cells were lysed on day 4 post-infection, and proteins were resolved by SDS-PAGE. Immunoblot analyses were preformed using antibodies to detect the indicated proteins. Cellular β-actin serves as a loading control.</p