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, 89 (15), 7707-21

A Rhesus Rhadinovirus Viral Interferon (IFN) Regulatory Factor Is Virion Associated and Inhibits the Early IFN Antiviral Response

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A Rhesus Rhadinovirus Viral Interferon (IFN) Regulatory Factor Is Virion Associated and Inhibits the Early IFN Antiviral Response

Gabriela Morin et al. J Virol.

Abstract

The interferon (IFN) response is the earliest host immune response dedicated to combating viral infection. As such, viruses have evolved strategies to subvert this potent antiviral response. Two closely related gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and rhesus macaque rhadinovirus (RRV), are unique in that they express viral homologues to cellular interferon regulatory factors (IRFs), termed viral IRFs (vIRFs). Cellular IRFs are a family of transcription factors that are particularly important for the transcription of type I IFNs. Here, we demonstrate a strategy employed by RRV to ensure rapid inhibition of virus-induced type I IFN induction. We found that RRV vIRF R6, when expressed ectopically, interacts with a transcriptional coactivator, CREB-binding protein (CBP), in the nucleus. As a result, phosphorylated IRF3, an important transcriptional regulator in beta interferon (IFN-β) transcription, fails to effectively bind to the IFN-β promoter, thus inhibiting the activation of IFN-β genes. In addition, we found R6 within RRV virion particles via immunoelectron microscopy and, furthermore, that virion-associated R6 is capable of inhibiting the type I IFN response by preventing efficient binding of IRF3/CBP complexes to the IFN-β promoter in the context of infection. The work shown here is the first example of a vIRF being associated with either the KSHV or RRV virion. The presence of this immunomodulatory protein in the RRV virion provides the virus with an immediate mechanism to evade the host IFN response, thus enabling the virus to effectively establish an infection within the host.

Importance: Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus macaque rhadinovirus (RRV) are the only viruses known to encode viral homologues to cellular interferon regulatory factors (IRFs), known as vIRFs. In KSHV, these proteins have been shown to play major roles in a variety of cellular processes and are particularly important in the evasion of the host type I interferon (IFN) response. In this study, we delineate the immunomodulatory mechanism of an RRV vIRF and its ability to assist the virus in rapid immune evasion by being prepackaged within the virion, thus providing evidence, for the first time, of a virion-associated vIRF. This work further contributes to our understanding of the mechanisms behind immunomodulation by the RRV vIRFs during infection.

Figures

FIG 1
FIG 1
R6 inhibits the IFN response. (A) tRF-ISREs were transfected for 40 h with pRL-SV40 and either pcDNA3.1-R6HA or empty pcDNA3.1. The cells were then transfected with poly(I · C) and assayed at the indicated time points (bottom). Firefly luciferase levels were normalized to constitutively expressed Renilla luciferase levels in each well, and all samples were normalized to the positive control [empty vector plus poly(I · C) at 8 h post-poly(I · C) transfection]. The data are averages (and standard errors of the mean [SEM]) from 3 independent experiments and are represented as RLU. (B) Telomerized RFs were transfected with pcDNA3.1-R6HA or mock transfected for 40 h and subsequently transfected with poly(I · C) for 8 h. RNA was extracted and analyzed by RT-PCR. The values are relative to GAPDH levels. The data were analyzed using a paired t test. P values of less than 0.05 were considered significant, and values greater than 0.05 were not significant (NS).
FIG 2
FIG 2
R6 localizes to the nucleus and affects nuclear accumulation of pIRF3. (A, B, and C) Telomerized RFs were transfected with pcDNA3.1-R6HA, pcDNA3.1-R7HA, or empty pcDNA3.1 for 40 h and subsequently transfected with poly(I · C) for the indicated times. (A) Transfected cells were fixed and analyzed by immunofluorescence for the detection of R6 (anti-HA) (green) and cellular IRF3 (red) and stained with Hoechst (blue) for the detection of nuclei. (B) Nuclear lysates prepared from cells 8 h post-poly(I · C) transfection were immunoprecipitated with anti-IRF3 antibody and then subjected to SDS-PAGE and probed with anti-HA antibody. Nuclear lysates were probed for HA expression, with PARP as a loading control and as a control for purity of nuclear fractionation. IB, immunoblotting. (C) Nuclear lysates were immunoprecipitated with CBP antibody and then subjected to SDS-PAGE and probed with anti-HA antibody. The nuclear lysates were probed for HA expression, with PARP as a loading control and as a control for purity of nuclear fractionation.
FIG 3
FIG 3
R6 competes with IRF3 for binding to CBP. (A) Telomerized RFs were transfected with pcDNA3.1-R6HA (5, 10, or 20 μg DNA) or empty pcDNA3.1 for 40 h and subsequently transfected with poly(I · C) for 6 h. Nuclear lysates were immunoprecipitated with anti-CBP antibody and then subjected to SDS-PAGE and probed with anti-HA antibody or anti-pIRF3 antibody. The nuclear lysates were probed for HA expression, with PARP as a loading control and a control for purity of nuclear fractionation. (B) EMSA was performed on whole-cell extracts (20 μg) derived from telomerized RFs transfected with pcDNA3.1-R6HA or empty pcDNA3.1. The biotin-labeled probe corresponds to the PRDI-PRDIII motif (5′-GAAAACTGAAAGGAGAACTGAAAGTG-3′) of the IFN-β promoter. Anti-CBP antibody and anti-IRF3 antibody were added as indicated to demonstrate the presence of CBP and IRF3 in the DNA-protein complexes. For oligonucleotide competition, a 500-fold molar excess of unlabeled PRDI-PRDIII probe was added as indicated. (C) Telomerized RFs were pretreated with MG132 or left untreated. The cells were then transfected with pcDNA3.1-R6HA or empty pcDNA3.1 for 40 h and then transfected with poly(I · C) for 6 h. Whole-cell lysates were immunoprecipitated with anti-IRF3 or anti-TBK and then subjected to SDS-PAGE and probed with anti-IRF3 or anti-TBK to gauge total levels of IRF3 and TBK within the cells. The nuclear lysates were subjected to SDS-PAGE and probed with anti-pIRF3 antibody or PARP, which served as a loading control and a control for purity of nuclear fractionation.
FIG 4
FIG 4
Molecular and in vitro characterization of R6HA RRV. (A) Comparative genome hybridization was used to directly compare viral DNA from the R6HA RRV recombinant to that from WTBACRRV. Alterations within the R6HA RRV genome resulted in incomplete hybridization to the array, depicted by the ratio of R6HA to WTBACRRV, and signaled a potential nucleotide mismatch between the two viral genomes. This comparison identified the HA tag located at the C-terminal end of R6. A second mismatch, indicated with an asterisk, was incorrectly identified, and the identified sequence was confirmed to be similar to the WT via PCR and DNA sequence analysis. (B) RFs were infected with either WTBACRRV or R6HA RRV at an MOI of 2.5 for single-step growth curve analysis. Infected RFs were harvested at the specified time points and subjected to a serial-dilution plaque assay on RFs to determine viral titers. The data from 4 separate experiments were averaged (±SEM). (C) RFs were infected with WTBACRRV or R6HA RRV or left uninfected, and at 48 h postinfection, total cellular RNA was harvested. Equivalent amounts of total RNA were analyzed by RT-PCR for ORF57, R7, and R6 transcripts, with cellular GAPDH as a loading control. Samples were run simultaneously with Taq polymerase only (−RT) to control for input and purity. Numbers at top indicate nucleotides.
FIG 5
FIG 5
R6 is associated with RRV virions. (A) Primary RFs were pretreated with CHX and subsequently infected with R6HA-RRV at an MOI of 2.5. CHX was removed, and the cells were fixed at the indicated time points and analyzed by immunofluorescence for the detection of R6-HA (anti-HA) (green) and stained with Hoechst (blue) for the detection of nuclei. (B) 1 × 105 PFU of gradient-purified virus samples (extracellular R6HA-RRV, intracellular R6HA-RRV, WTBACRRV, and vIRFko-RRV) was subjected to SDS-PAGE and probed with anti-HA antibody and anti-MCP antibody as a control. (C) (i) Gradient-purified R6HA-RRV was fixed, pelleted, and sectioned. The sections were immunogold stained with anti-HA antibody and 10-nm gold-conjugated secondary antibody. (ii) Gradient-purified WTBACRRV was sectioned and immunogold stained with anti-HA antibody and 10-nm gold-conjugated secondary antibody as a control. (iii) R6HA-RRV sections were stained with 10-nm gold-conjugated secondary antibody alone as a control. Virus particles with gold particles are indicated by the white arrowhead, and virus particles with no gold particles are indicated by black arrowheads.
FIG 6
FIG 6
Virion-associated R6 is functional. (A) Telomerized RF-rtTAs stably transduced with R6-HA were treated with Dox for the indicated times. The nuclear and cytoplasmic lysates were subjected to SDS-PAGE and probed with anti-HA antibody. GAPDH and PARP served as loading controls and as controls for purity of fractionation for the cytoplasmic and nuclear extracts, respectively. (B) Telomerized RF-rtTAs were treated with Dox and infected with vIRFko-RRV at an MOI of 0.01 per cell. The resultant virus (vIRFkoR6-RRV) was gradient purified from cell supernatants. Five micrograms of gradient-purified virus (WTBACRRV, vIRFko-RRV, R6HA-RRV, and vIRFkoR6-RRV) was subjected to SDS-PAGE and probed with anti-HA antibody and anti-MCP antibody as a control. (C) tRF-ISREs were infected for 4 or 8 h with the indicated virus at an MOI of 2.5 PFU per cell. The cells were then assayed for firefly luciferase activity. Firefly luciferase levels were normalized to constitutively expressed Renilla luciferase levels in each well. The data are averages (and SEM) from the results of 3 independent experiments. (D) tRF-rtTA:R6HAs were infected with vIRFko-RRV at an MOI of 0.01 PFU per cell after pretreatment with the indicated amounts of Dox. Virus was then harvested and gradient purified. The resultant virus was used to infect tRF-ISREs at an MOI of 2.5 PFU per cell for 8 h. The cells were assayed for firefly luciferase activity. Firefly luciferase levels were normalized to constitutively expressed Renilla luciferase levels in each well. The data are averages (and SEM) from the results of 3 independent experiments. The total levels of MCP and R6HA in virion preparations were assessed by Western blotting with anti-MCP and anti-HA antibodies. (E) Telomerized RFs were infected with the indicated virus at an MOI of 2.5 PFU per cell for 8 h in the presence or absence of CHX. EMSA was performed on nuclear extracts (20 μg). The biotin-labeled probe corresponded to the PRDI-PRDIII motif (5′-GAAAACTGAAAGGAGAACTGAAAGTG-3′) of the IFN-β promoter. The data were analyzed using a paired t test. P values of ≤0.05 were considered significant, and values greater than 0.05 were not significant.
FIG 7
FIG 7
Potential model of IFN inhibition by R6. Upon detection of RRV infection by TLR3, RIG-I, or MDA-5, TBK1 is activated and subsequently phosphorylates IRF3. pIRF3 then dimerizes and translocates to the nucleus. Within the nucleus, R6 binds to the transcriptional coactivator CBP. This interaction prevents pIRF3 from binding to CBP, and pIRF3 is exported from the nucleus and degraded by the proteasome. Nuclear R6 interferes with complex formation between pIRF3 and CBP and, as a result, decreases pIRF3/CBP complex binding to the IFN-β promoter and inhibits IFN-β production.

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