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. 2001 Apr 24;98(9):5246-51.
doi: 10.1073/pnas.091000398.

RNA is a structural element in retrovirus particles

D Muriaux  1 , J Mirro, D Harvin, A Rein
Affiliations

RNA is a structural element in retrovirus particles

D Muriaux et al. Proc Natl Acad Sci U S A. .

Abstract

A single retroviral protein, Gag, is sufficient for virus particle assembly. While Gag is capable of specifically packaging the genomic RNA into the particle, this RNA species is unnecessary for particle assembly in vivo. In vitro, nucleic acids profoundly enhance the efficiency of assembly by recombinant Gag proteins, apparently by acting as "scaffolding" in the particle. To address the participation of RNA in retrovirus assembly in vivo, we analyzed murine leukemia virus particles that lack genomic RNA because of a deletion in the packaging signal of the viral RNA. We found that these particles contain cellular mRNA in place of genomic RNA. This result was particularly evident when Gag was expressed by using a Semliki Forest virus-derived vector: under these conditions, the Semliki Forest virus vector-directed mRNA became very abundant in the cells and was readily identified in the retroviral virus-like particles. Furthermore, we found that the retroviral cores were disrupted by treatment with RNase. Taken together, the data strongly suggest that RNA is a structural element in retrovirus particles.

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Figures

Figure 1
Figure 1
Analysis of wild-type and mutant virion RNA content by RNA end-labeling. RNA extracted from virions was end-labeled with T4 RNA ligase and [32P]pCp, and was analyzed on a denaturing 1% agarose gel. (A) RNAs extracted from the same number of wild-type (lane 1) and Ψ− (lane 2) virion particles or from a mock preparation (lane 3). (B) RNAs extracted from the same number of PR− (lane 4) and Ψ−PR− (lane 5) virion particles or from a mock preparation (lane 6). Lanes 1, 2, and 3 of B represent 2, 1, and 0.5 pmol of yeast tRNA, respectively, which were labeled by the same technique as the other RNA samples.
Figure 2
Figure 2
RT-PCR analysis of human β-actin and G3PDH mRNAs packaged in wild-type and Ψ− mutant particles. Lanes 1–6, 2-fold serial dilutions of total RNA extracted from wild-type virus; lanes 7–12, 2-fold dilutions of RNA from Ψ− virions; lanes 13–15, RNA from dilutions of mock supernatant; lanes 16–21, 2-fold dilutions starting from 200 ng of purified cellular poly(A)+ RNA from 293T cells; lane 22, yeast tRNA (50 ng). Starting material represented the same amount of wild-type and Ψ− virions. As controls for DNA contamination, lanes 6, 12, 15, and 21 do not contain any RT.
Figure 3
Figure 3
RNA content of SFV-derived MuLV Gag VLPs. (A) Schematic representation of the two (+) strand RNAs produced by the SFV vector expressing MuLV Gag: the SFV genomic and subgenomic mRNAs. CSFV represents the sequence of SFV CA, which is removed from Gag cotranslationally (30). (B) RNA end-labeling analysis of VLPs and cell lysates. RNAs were analyzed as in Fig. 1. Lanes 1 and 2, RNAs were obtained from the same amount of PR− (lane 1) and Ψ−PR− (lane 2) virions; lane 3, “VLP” preparations from BHK 21 cells electroporated with pSFV1 RNA (“mock”); lane 4, VLPs produced by BHK21 cells electroporated with pSFVC-Pr65gag RNA (Gag VLPs); and lane 5, cellular RNA from the same electroporated culture as in lane 4. Lanes 1, 2, and 4 contain the same number of particles. (C) Identification of Pr65Gag mRNA by Northern blotting using a full-length proviral MuLV probe. RNA from the same amount of PR− (lane 1) and Ψ−PR− (lane 2) virions, and Gag VLPs (lane 4) were analyzed. Lane 3 is a mock preparation as in B. MuLV genomic RNA (8.33 kb), the SFV-Pr65Gag genomic RNA (13 kb), and the SFV subgenomic Pr65Gag mRNA (2.4 kb) are indicated, as are 28S (≈5 kb) and 18S (≈1.5 kb) ribosomal RNAs.
Figure 4
Figure 4
Effects of RNase A on virion core stability. PR− (A) and Ψ−PR− (B) virions were incubated with or without detergent as indicated. The samples were then incubated in the presence or absence of RNase A before fractionation by centrifugation. The Gag proteins present in the supernatant (S) and pellet (P) were fractionated by SDS/PAGE and analyzed by immunoblotting with rabbit anti-MuLV CA antiserum. Lanes 1–4, virion cores in 1% Nonidet P-40; lanes 7–10, no detergent; lanes 5 and 6, virions lysed in 1% SDS; lanes 3, 4, 9, and 10, + RNase A.
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