SINE RNA promotes IKKβ-mediated phosphorylation of RTA to potentiate viral transcription.

<p>(<b>A</b>) NIH3T3 cells were transfected with FLAG-RTA and either a control construct, B1 SINE, or B2 SINE expression plasmid. 24 h later cells were labeled with [³²P]-orthophosphoric acid for 6 h. Whole cell lysates were precipitated with anti-FLAG antibody and analyzed by autoradiography (top) or western blot using an anti-FLAG antibody (bottom). (<b>B</b>) FLAG-RTA was transfected in to NIH3T3 cells. 24 h later cells were transfected with indicated ASOs and subsequently infected with MHV68 for 6 h in the presence of [³²P]-orthophosphoric acid. Whole cell lysates were precipitated with anti-FLAG antibody and analyzed by autoradiography (top) or western blot using an anti-FLAG antibody (bottom). (<b>C</b>) NIH3T3 cells transfected with the indicated viral promoter luciferase plasmids were cotransfected with wild-type RTA or RTA TTS/A, and control, B1 SINE, or B2 SINE expression constructs. 48 h later luciferase levels were monitored.</p

SINE RNAs signal through MAVS to activate NF-κB.

<p>(<b>A</b>) Control, B1 SINE, or B2 SINE expression constructs were co-transfected with either the NF-κB or Sp1 reporter luciferase plasmid into WT and MAVS^-/- fibroblasts. 48 h later luciferase levels were determined. (<b>B</b>) MAVS^-/-, WT, and WT fibroblasts transfected with either B1 or B2 ASOs were infected with MHV68. At the indicated time points whole cell lysates were prepared and western blotted for phospho p65 (Ser468) and total p65. (<b>C</b>) WT, MAVS^-/-, and MAVS^-/- fibroblasts transfected with B2 ASO were infected with MHV68 and western blot analysis was performed at the indicated times for phospho p65 (Ser468) and total p65. (<b>D</b>) WT and MAVS^-/- fibroblasts that are transfected with either control of B2 ASOs infected with MHV68 at an MOI of 0.05. Infection was allowed to progress for 72 h. Infectious virus produced was quantified by plaque assay in NIH3T3 cells.</p

Infection-Induced Retrotransposon-Derived Noncoding RNAs Enhance Herpesviral Gene Expression via the NF-κB Pathway

<div><p>Short interspersed nuclear elements (SINEs) are highly abundant, RNA polymerase III-transcribed noncoding retrotransposons that are silenced in somatic cells but activated during certain stresses including viral infection. How these induced SINE RNAs impact the host-pathogen interaction is unknown. Here we reveal that during murine gammaherpesvirus 68 (MHV68) infection, rapidly induced SINE RNAs activate the antiviral NF-κB signaling pathway through both mitochondrial antiviral-signaling protein (MAVS)-dependent and independent mechanisms. However, SINE RNA-based signaling is hijacked by the virus to enhance viral gene expression and replication. B2 RNA expression stimulates IKKβ-dependent phosphorylation of the major viral lytic cycle transactivator protein RTA, thereby enhancing its activity and increasing progeny virion production. Collectively, these findings suggest that SINE RNAs participate in the innate pathogen response mechanism, but that herpesviruses have evolved to co-opt retrotransposon activation for viral benefit.</p></div

Model for SINE RNA enhancement of viral gene expression.

<p>SINE RNAs are robustly induced in response to MHV68 infection. Through both MAVS-dependent and independent mechanisms, they stimulate the activity of the IKKβ component of the NF-κB pathway. IKKβ is co-opted by the virus to promote phosphorylation of RelA/p65, blunting the NF-κB transcriptional response. Additionally, IKKβ phosphorylates RTA, thereby enhancing viral gene expression and replication.</p

Viral gene expression is required for sustained SINE expression.

<p><b>(A)</b> Temporal progression of the MHV68 productive replication cycle. Upon MHV68 infection, the virus enters cells and the dsDNA viral genome is delivered to the nucleus. The viral gene expression program commences with transcription of immediate early (IE) genes, which in turn activate transcription of early (E) genes. This is followed by viral DNA synthesis, which activates transcription of late (L) viral genes, and culminates in infectious progeny virus production. The point at which different treatments (UV-irradiated virus, or PAA) arrest the viral life cycle is shown. (<b>B</b>) RNA isolated from NIH3T3 cells incubated with mock- or U.V.-treated MHV68 at the indicated time points was subjected to RT-qPCR for the indicated viral genes to demonstrate that UV treatment prevents viral gene expression. (<b>C</b>) RNA described in (B) was used to monitor the levels of B1, B2, and 7SK RNA by primer extension. (<b>D</b>) NIH3T3 cells were infected with MHV68 at an MOI of 5 in the presence of 200 μg/mL of PAA. 24 hpi total RNA was used to monitor by RT-qPCR the expression of ORF65, a viral late gene whose transcription is dependent on viral DNA replication. (<b>E</b>) RNA described in (D) was used to monitor the levels of B1, B2, and 7SK RNA by primer extension.</p

SINE RNAs activate the NF-κB pathway.

<p>(<b>A</b>) Schematic of the experimental design. B1 and B2 SINE expression constructs were co-transfected with reporter plasmids containing various promoter elements cloned upstream of the luciferase gene. (<b>B</b>) The indicated plasmids were co-transfected in to NIH3T3 cells and 48 h later luciferase levels were determined. (<b>C</b>) NIH3T3 cells were transfected with B1 or B2 expression constructs, or a control construct. 48 h later protein extracts were analyzed by western blot analysis. (D) The indicated plasmids were co-transfected in to NIH3T3 cells and 48 h later luciferase levels were determined.</p

B2 RNA enhances viral replication and gene expression.

<p>(<b>A</b>) NIH3T3 cells transfected with indicated ASOs were infected with MHV68 at an MOI of 0.05. Infection was allowed to progress for 72 h. Primer extension was performed at 24 hpi infection to monitor the levels of B1, B2, and 7SK RNA. (<b>B</b>) Infectious virus produced in (A) was quantified by plaque assay in NIH3T3 cells. (<b>C</b>) RNA isolated at 72 hpi was used to monitor the levels of viral and cellular RNAs by RT-qPCR. (<b>D</b>) ChIRP was performed on NIH3T3 cells infected with MHV68 for 24 h. Antisense LacZ oligos were used as a negative control. RNAs isolated from the ChIRP experiment were analyzed by small RNA northern blotting for B2 SINE and 5S rRNA. (<b>E</b>) DNA isolated from the ChIRP experiment described in (D) was used for qPCR.</p

Failure to degrade viral mRNAs leads to enhanced lytic cycle entry.

<p>(A) Shown is the percent of lytic-expressing infected NIH 3T3 or MEF cells. Cells were infected at an MOI of 5 with GFP-BAC MHV68 MR or ΔHS and were analyzed at 18 hpi for GFP and M9 expression by immunofluorescence using anti-M9 antibodies. 5 fields of view from three independent experiments were counted and the percentage of GFP+M9+ cells calculated. ** Indicates p-value<0.01, determined by student t-test. (B–D) To measure levels of RTA-responsive transcripts after infection with MR or ΔHS MHV68, NIH 3T3 cells were infected at an MOI of 5 and RNA harvested at indicated times post infection. RT-qPCR was used to quantify relative levels of ORF 50 (B), ORF 57 (C), and ORF 6 (D). (E) GAPDH levels were measured to show that host shutoff had not yet initiated at the 8 hpi time point.</p

Normalization strategy for mass spectrometry results.

<p>Top portion of the table are proteins identified in both ΔHS and MR samples for the first mass spectrometry (MS) run. Lower portion of the table is from the second MS run. The peptide count was normalized to major capsid protein ORF 25, and subsequently normalized to genome number, as determined by qPCR.</p><p>^a Genome number for first MS run.</p><p>^b Genome number for second MS run.</p

muSOX-induced mRNA degradation is important for viral amplification in a cell type specific manner in vitro and in vivo.

<p>(A–C) Multi-step growth curves were done in (A) NIH 3T3 cells, (B) murine embryonic fibroblasts, or (C) DC2.4 cells. Cells were infected at an MOI of 0.05 with MR or ΔHS MHV68, cells and supernatant collected at the indicated times post infection, and the titer was determined by plaque assay. At least three independent experiments were performed for each cell type. (D) C57BL/6 mice were infected by the intraperitoneal route with 1×10³ pfu of MR or ΔHS MHV68. At 10 dpi spleens were harvested, homogenized, DNA extracted, and qPCR used to quantify viral particles. Each dot represents the relative value from a single spleen, and the bar indicates the mean value for each virus. * Indicates p-value<0.05.</p