Mechanism of tripartite RNA genome packaging in Rift Valley fever virus - PubMed (original) (raw)

Mechanism of tripartite RNA genome packaging in Rift Valley fever virus

Kaori Terasaki et al. Proc Natl Acad Sci U S A. 2011.

Abstract

The Bunyaviridae family includes pathogens of medical and veterinary importance. Rift Valley fever virus (RVFV), a member in the Phlebovirus genus of the family Bunyaviridae, is endemic to sub-Saharan Africa and causes a mosquito-borne disease in ruminants and humans. Viruses in the family Bunyaviridae carry a tripartite, single-stranded, negative-sense RNA genome composed of L, M, and S RNAs. Little is known about how the three genomic RNA segments are copackaged to generate infectious bunyaviruses. We explored the mechanism that governs the copackaging of the three genomic RNAs into RVFV particles. The expression of viral structural proteins along with replicating S and M RNAs resulted in the copackaging of both RNAs into RVFV-like particles, while replacing M RNA with M1 RNA, lacking a part of the M RNA 5' UTR, abrogated the RNA copackaging. L RNA was efficiently packaged into virus particles released from cells supporting the replication of L, M, and S RNAs, and replacing M RNA with M1 RNA abolished the packaging of L RNA. Detailed analyses using various combinations of replicating viral RNAs suggest that M RNA alone or a coordinated function of M and S RNAs exerted efficient L RNA packaging either directly or indirectly. Collectively, these data are consistent with the possibility that specific intermolecular interactions among the three viral RNAs drive the copackaging of these RNAs to produce infectious RVFV.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Replication competencies of M, M1, M2, and M3 RNAs. (A) Schematic diagram of antiviral-sense M, M1, M2, and M3 RNAs. The M gene ORF is shown in boxes. The numbers in M RNA represent the location of the 5′ UTR (in viral-sense RNA) from the 5′ end of antiviral-sense M RNA and those in each M RNA mutant represent the deletion site. (B) Cells were cotransfected with plasmids encoding N protein, L protein, and antiviral-sense M RNA or one of the M RNA mutants. The plasmid expressing N protein was omitted in samples shown in lanes 1 and 8. Intracellular RNAs were extracted at 24 h or 48 h posttransfection and subjected to Northern blot analysis using an RNA probe that selectively binds to viral-sense M RNA.

Fig. 2.

Characterization of VLPs carrying M RNA or M1 RNA. Cells were cotransfected with plasmids encoding N protein, L protein, M gene ORF, and antiviral-sense M RNA or M1 RNA. Intracellular proteins, intracellular RNAs, and VLPs were harvested at 3 d posttransfection and the released VLPs were purified by sucrose gradient centrifugation. (A) Intracellular proteins from mock-infected cells (lane 1), RVFV-infected cells (lane 2), cells expressing M RNA (lane 3), cells expressing M1 RNA (lane 4), purified VLPs from cells expressing M RNA (lane 5), and those from cells expressing M1 RNA (lane 6) were subjected to Western blot analysis using anti-RVFV mouse antibody. IC, intracellular samples. Gn/Gc, Gn/Gc proteins; NSs, NSs protein; and N, N protein. (B) RNA samples corresponding to the samples in A were subjected to Northern blot analysis using an RNA probe that hybridizes with viral-sense M or M1 RNA (lanes 1–6). Released VLPs carrying M RNA (lane 7) or M1 RNA (lane 8) were inoculated to cells coexpressing L and N proteins. Intracellular RNAs were extracted at 24 h postinoculation and subjected to Northern blot analysis.

Fig. 3.

Copackaging of M and S RNAs into VLPs. (A) Intracellular proteins, intracellular RNAs, and released VLPs were collected from cells expressing all of the viral structural proteins, M RNA, and S RNA (M+S) at 3 d posttransfection. M1+S represent samples in which M1 RNA was used in place of M RNA. Intracellular proteins (IC) and proteins in purified VLPs were subjected to Western blot analysis using anti-RVFV antibody (Left two panels). Intracellular RNAs (IC) and RNAs in purified VLPs were detected by Northern blot analysis using an RNA probe that hybridizes to viral-sense M RNA or M1 RNA (Right Top) or viral-sense S RNA (S) (Right Bottom). Lane 1 and lane 2 represent intracellular samples from mock-infected cells and RVFV-infected cells, respectively. (B) VLPs produced from cells supporting the replication of M and S RNAs (M+S) or from those supporting the replication of M1 and S RNAs (M1+S) were inoculated into cells coexpressing L and N proteins (lanes 1 and 2) or L protein-expressing cells (lanes 3 and 4). Intracellular RNAs were extracted at 24 h postinoculation and subjected to Northern blot analysis to detect viral-sense M RNA (Top, lanes 1 and 3), M1 RNA (Top, lanes 2 and 4), or viral-sense S RNA (Bottom). (C) Cells expressing L protein were inoculated with either M+S VLP sample, obtained from cells supporting the replication of M and S RNAs, or a mixture of M VLP sample and S VLP sample (M VLP+S VLP), obtained from cells supporting M RNA replication and S RNA replication, respectively. Both VLP samples carried the same amount of M RNA. Similarly, the amount of S RNA was also the same in both VLP samples. Intracellular RNAs were extracted at 24 h postinoculation and subjected to Northern blot analysis to detect viral-sense M RNA (Top) or viral-sense S RNA (Bottom). (D) VLPs produced from cells supporting S RNA replication were concentrated ∼10 times (S VLP). Cells coexpressing L and N proteins (lanes 1 and 2) and those expressing L protein (lanes 3 and 4) were coinoculated with VLPs carrying M RNA (M VLP) and S VLP (lanes 1 and 3) or those carrying M1 RNA (M1 VLP) and S VLP (lanes 2 and 4). Intracellular RNAs were collected at 24 h postinoculation and subjected to Northern blot analysis to detect viral-sense M or M1 RNA (Top) or viral-sense S RNA (Bottom).

Fig. 4.

Effect of M1 RNA on L RNA packaging. (A) VLPs, intracellular RNAs, and intracellular proteins were collected from cells supporting the replication of L, M, and S RNAs (lane 1) or from those supporting the replication of L, M1, and S RNAs (lane 2) at 3 d posttransfection. (Left) Western blot analysis of virus-specific intracellular (IC) and VLP proteins. Mock, mock-infected cells; RVFV, RVFV-infected cells. (Right) Northern blot analysis of virus-specific intracellular RNAs (IC) and viral RNAs in VLPs. Top, Middle, and Bottom represent viral-sense L RNA, viral-sense M RNA, and viral-sense S RNA, respectively. (B) Schematic diagram of antiviral-sense L and L2 RNAs. The numbers in L RNA represent the location of the 5′ UTR (in viral-sense) from the 5′ end of antiviral-sense L RNA and those in L2 RNA represents the deletion site. The boxed region represents the L gene ORF. (C) Experiments were performed as described in A except that L2 RNA was used in place of L RNA.

Fig. 5.

Analysis of L RNA packaging into VLPs from cells supporting the replication of various combinations of viral RNAs. (A) VLPs, intracellular RNAs, and intracellular proteins were collected from cells supporting the replication of various combinations of viral RNAs as shown at the Top of the panels at 3 d posttransfection. (Left two panels) Western blot analysis of virus-specific intracellular proteins (IC) and VLP proteins. Mock, mock-infected cells; RVFV, RVFV-infected cells. (Right two panels) Northern blot analysis of virus-specific intracellular RNAs (IC) and viral RNAs packaged into VLPs using RNA probes, each of which hybridizes with viral-sense L RNA (L), viral-sense M RNA (M), or viral-sense S RNA (S). (B) Experiments were performed as described in A except that L2 RNA was used in place of L RNA.

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Mechanism of tripartite RNA genome packaging in Rift Valley fever virus - PubMed (original) (raw)