Nucleocapsid-independent specific viral RNA packaging via viral envelope protein and viral RNA signal

Abstract

For any of the enveloped RNA viruses studied to date, recognition of a specific RNA packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the packaging signal, determines the selective RNA packaging into virus particles. In this report we show that expressed coronavirus envelope protein M specifically interacted with coexpressed noncoronavirus RNA transcripts containing the short viral packaging signal in the absence of coronavirus N protein. Furthermore, this M protein-packaging signal interaction led to specific packaging of the packaging signal-containing RNA transcripts into coronavirus-like particles in the absence of N protein. These findings not only highlight a novel RNA packaging mechanism for an enveloped virus, where the specific RNA packaging can occur without the core or N protein, but also point to a new, biologically important general model of precise and selective interaction between transmembrane proteins and specific RNA elements.

FIG. 1.

Specific interaction of M protein with the PS-containing RNA transcripts in the absence of N protein. (A) Schematic diagrams of the structures of plasmids PS5A (PS−) and PS5B190 (PS+). T7 pr, T7 promoter; T7 ter, T7 terminator. (B and C) Northern blot analysis of expressed RNA transcripts from RNA-expressing cells coinfected with SinM and SinN pseudovirions (M protein + N protein) (B) or infected with either SinM pseudovirion (M protein) or SinLacZ pseudovirion (β-galactosidase protein) (C). Equal volumes of cell lysates were immunoprecipitated with anti-M protein MAb (anti-M) and a control MAb (anti-H2K). Intracellular (i.c.) RNAs and coimmunoprecipitated RNAs were analyzed by Northern blot analysis with a probe that binds to the CAT gene sequence. The arrows indicate expressed RNA transcripts. Each panel shows representative data from triplicate experiments. RNAs extracted from 10⁵ cells and 10⁶ cells were analyzed for the intracellular RNA lanes and the anti-M and anti-H2K lanes, respectively. The anti-M and anti-H2K lanes were exposed eight times longer than the intracellular RNA lanes.

FIG. 2.

Nucleocapsid-independent RNA packaging into VLPs. [³⁵S]methionine-cysteine-labeled VLPs were purified from culture fluid of the cells expressing PS5B190 (PS+) or PS5A (PS−) RNA transcripts and the MHV-specific proteins. (A) Intracellular (i.c.) RNAs were extracted from the cytoplasmic lysates and analyzed by Northern blot analysis as described in the legend to Fig. 1. The intracellular RNAs extracted from 3 × 10⁵ cells were applied to each lane. (B) Release of M protein in VLPs. A part of the purified VLP lysate was immunoprecipitated with anti-M protein MAb and analyzed by SDS-PAGE. Only the section of the autoradiogram with M protein is shown. (C) Northern blot analysis of VLP RNAs. VLP RNA was extracted from purified VLPs and analyzed by Northern blot analysis as described above. VLPs released from 10⁷ cells were used for analysis of VLP RNAs. Panel C was exposed eight times longer than panel A. (D) Intracellular expression levels of M protein and E protein. Cytoplasmic lysates were immunoprecipitated with anti-M protein MAb and anti-E protein peptide-2 antibody and analyzed by SDS-PAGE. Only the sections of the autoradiogram with M protein and E protein are shown. Each panel shows representative data from triplicate experiments.

FIG. 3.

Northern blot analysis of VLP-associated RNAs after RNase A treatment. Partially purified VLPs, released from cells coexpressing PS5B190 (PS+) RNA transcripts, M protein, and E protein, were incubated in the presence (RNase +) or absence (RNase −) of RNase A and subsequently purified by ultracentrifugation. VLP-associated RNAs were extracted from purified VLPs. Intracellular (i.c.) RNAs were extracted from the cytoplasmic lysates of the same cells and incubated in the presence (RNase +) or absence (RNase −) of RNase A. Partially purified VLPs released from 2 × 10⁷ cells and 3 μg of intracellular RNA were used for RNase A digestion. Northern blot analysis was performed as described in the legend to Fig. 1 to examine the susceptibility of VLP-associated RNAs and intracellular RNAs to RNase A treatment. Each panel shows representative data from triplicate experiments.