A viral nuclear noncoding RNA binds re-localized poly(A) binding protein and is required for late KSHV gene expression

Abstract

During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV) expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN) RNA avidly binds host poly(A)-binding protein C1 (PABPC1), which normally functions in the cytoplasm to bind the poly(A) tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX) protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A) tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.

Figure 1. PAN RNA binds to PABPC1 in the nucleus of lytically infected BCBL1 TReX-RTA cells.

A. Scheme for biochemical purification of the PAN RNP. B. 10% SDS-PAGE gel silver-stained to show proteins co-purifying with PAN RNA from lysates of doxycycline-treated BCBL1 TReX-vector cells (left lane) or BCBL1 TReX-RTA cells (right lane). C. Dual immunofluorescence and in situ hybridization for PABPC1 (red) and PAN RNA (green) from BCBL1 TReX-vector cells (top panels) or BCBL1 TReX-RTA cells (bottom panels) treated with doxycycline.

Figure 2. SOX stimulates PAN RNA expression in transient transfection assays.

A. Northern blot analysis of total RNA extracted from 293 tet-on cells transfected with: 1) a GFP expression vector, 2) a PAN RNA or PAN/U7 3′-end (PAN RNA Δ-poly(A) tail) expression vector driven from a tetracycline-regulated promoter, and 3) either blank DNA (left lanes) or a SOX-expression vector (right lanes). The blot was sequentially probed for the RNAs listed on the right. B. Confocal images of dual immunofluorescence/in situ hybridization staining for PABPC1 (red) and PAN RNA (green) in transiently transfected 293T cells. Cells were transfected with: 1) empty plasmid (top panels), 2) a PAN RNA expression vector driven from the endogenous RTA-responsive PAN promoter and an RTA expression vector (middle panels), or 3) a PAN RNA expression vector driven from the endogenous RTA-responsive PAN promoter, an RTA expression vector and a SOX-expression vector (bottom panels). C. Top two panels: northern blot analysis for 18S or PAN RNA in total RNA extracted from 293T cells transfected with: 1) PAN RNA driven from its endogenous promoter, 2) an RTA expression vector, and 3) either empty plasmid, an expression vector for wild-type SOX, the P176S mutant SOX or the Q129H mutant SOX. Bottom two panels: western blots showing equal levels of expression of all three SOX proteins. D. Northern blot analysis of total RNA extracted from 293T cells transfected with an RTA expression vector, a PAN RNA expression vector driven from the endogenous PAN promoter and either blank DNA (left lane), a PABPC1-NRS expression vector (center lane) or a SOX expression vector (right lane). E. Confocal images of dual immunofluorescence/in situ hybridization staining for FLAG-tagged PABPC1-NRS (red) and PAN RNA (green) in transiently transfected 293T cells.

Figure 3. Expression of PAN RNA is coincident with the host shutoff effect.

A. Northern blot showing host and viral RNA expression during progression of lytic reactivation by doxycycline treatment of BCBL1 TReX-vector (left) or BCBL1 TReX-RTA (right) cells. B. qRT-PCR data from a representative experiment showing increased expression of PAN RNA versus the decline in host GAPDH mRNA. C. qRT-PCR data from a representative experiment showing that host MALAT1 and NEAT1 RNAs decline during lytic infection. qRT-PCR signals were normalized to endogenous 18S rRNA.

Figure 4. PAN RNA and K7 mRNA levels are reduced by transfection of RNase H-targeting oligonucleotides.

A. Region of the KSHV genome from which K7 mRNA and PAN RNA are transcribed. Alternative transcriptional start sites for the K7 mRNA isoforms , regions targeted by anti-K7 (α-K7) and anti-PAN RNA (α-PAN) 1 and 2 modified oligonucleotides, as well as the oligonucleotide probe used to detect PAN RNA by northern blot, are indicated. The K7 ORF, ENE and poly(A) signal shared by all four RNA species are shown. B. K7 mRNA levels at 0, 10.5 or 24 hours post-induction of BCBL1 TReX-RTA cells after recovery from electroporation with α-GFP, α-K7 or α-PAN RNA 1 and 2 oligonucleotides. qRT-PCR signals were normalized to endogenous 18S rRNA. C. Northern blot analysis of total RNA extracted from BCBL1 TReX-RTA cells electroporated with different concentrations of α-GFP, α-K7 or α-PAN RNA 1 and 2 oligonucleotides and at 0, 12 or 24 hours post-induction.

Figure 5. PAN RNA does not contribute to the host shutoff effect.

Northern blot of total RNA harvested from BCBL1 TReX-RTA cells electroporated with increasing concentrations of α-PAN RNA 1 and 2 oligonucleotides at various times after doxycycline-mediated induction, probed for the RNAs indicated on the right. The faster migrating band in lanes 3 and 6 of the GAPDH panel is residual ³²P-labeled anti-PAN RNA probe.

Figure 6. Knockdown of PAN RNA expression adversely affects late gene expression in BCBL1 TReX-RTA cells.

A. BCBL1 TReX-RTA cells were electroporated with the indicated modified oligonucleotides and induced with doxycycline 12–16 hours later. After various times, lysates from equivalent numbers of living cells were loaded in each lane, and blotted proteins were probed sequentially with antibodies against the indicated proteins. B. Densitometric analysis of immunoblot signals (see Fig. S6 legend) for RTA (immediate early protein), vIL-6 (early protein) and K8.1 (late protein) after knockdown with control or α-PAN RNA oligonucleotides. Standard error of the mean from 9 experiments (7 experiments for α-K7 knockdown) is shown. C. Cell viability was measured by trypan blue staining 24 hours post-induction. Averages and standard error of the mean from 7 independent experiments are shown.

Figure 7. Knockdown of PAN RNA adversely affects gene expression in iSLK.219 cells, with a more pronounced effect on late genes.

A. Procedures were the same as in Fig. 6 B. Densitometric analyses of immunoblot signals of RTA (immediate early protein), vIL-6 (early protein) and K8.1 (late protein) after knockdown with control or α-PAN RNA oligonucleotides. Standard error of the mean from 4 experiments is shown. C. GFP expression in target 293 DC-SIGN cells after inoculation with media from iSLK.219 cells treated with no oligonucleotide (mock), α-K7 oligonucleotide or α-PAN RNA oligonucleotides 48 hours after induction with doxycycline. FACS sorting of cells indicated mean fluorescence intensities (in arbitrary units) of 0.07+/− 0.04 for uninfected cells, 1.00 for cells infected with supernatant from mock-transfected iSLK.219 cells, 0.14+/− 0.08 for cells infected with supernatant from anti-K7-transfected iSLK.219 cells and 0.05+/− 0.03 for cells infected with supernatant from anti-PAN-transfected iSLK.219 cells. Standard error of the mean for 3 independent experiments is given.

Figure 8. Knockdown of PAN RNA reduces production of encapsulated viral DNA without affecting levels of intracellular viral DNA.

A. Knockdown of PAN RNA lowers viral yield in cell culture supernatant. BCBL1 TReX-RTA cells were electroporated with the indicated modified oligonucleotides, allowed to recover overnight, and then induced with doxycycline. 8 days later, encapsulated viral DNA was harvested from the supernatant and quantified by qPCR, normalizing to control plasmid exogenously added at onset of purification. Values are the average of 4 independent experiments. Standard error of the mean is shown. Note that electroporation of all oligonucleotides decreased virus production to some degree, most probably because of cell death experienced upon electroporation of nucleic acids followed by lytic induction. B. Knockdown of PAN RNA does not decrease intracellular viral DNA. BCBL1 TReX-RTA cells were treated as above and intracellular DNA was collected 3 days post-induction and quantified by qPCR, normalizing to the amount of host DNA, as measured by GAPDH DNA signal. Values are the average of 3 independent experiments. Standard error of the mean is shown.

Figure 9. Model for PAN RNA sequestering nuclear re-localized PABPC1.

A. mRNA transcription, export and translation during latent infection. Host mRNAs (blue) are exported and bound by initiation factors and PABPC1. B. Viral infection in the absence of PAN RNA. SOX action mediates host shutoff and PABPC1 nuclear re-localization. Host and viral mRNAs are bound by PABPC1, leading to inefficient processing and/or export and resulting degradation. C. Viral infection in the presence of PAN RNA. As in B, PABPC1 is re-localized but bound by PAN RNA. Viral, but not host, mRNAs are bound by Orf57, which mediates their efficient export into the cytoplasm.