Cooperation of an RNA packaging signal and a viral envelope protein in coronavirus RNA packaging

Abstract

Murine coronavirus mouse hepatitis virus (MHV) produces a genome-length mRNA, mRNA 1, and six or seven species of subgenomic mRNAs in infected cells. Among these mRNAs, only mRNA 1 is efficiently packaged into MHV particles. MHV N protein binds to all MHV mRNAs, whereas envelope M protein interacts only with mRNA 1. This M protein-mRNA 1 interaction most probably determines the selective packaging of mRNA 1 into MHV particles. A short cis-acting MHV RNA packaging signal is necessary and sufficient for packaging RNA into MHV particles. The present study tested the possibility that the selective M protein-mRNA 1 interaction is due to the packaging signal in mRNA 1. Regardless of the presence or absence of the packaging signal, N protein bound to MHV defective interfering RNAs and intracellularly expressed non-MHV RNA transcripts to form ribonucleoprotein complexes; M protein, however, interacted selectively with RNAs containing the packaging signal. Moreover, only the RNA that interacted selectively with M protein was efficiently packaged into MHV particles. Thus, it was the packaging signal that mediated the selective interaction between M protein and viral RNA to drive the specific packaging of RNA into virus particles. This is the first example for any RNA virus in which a viral envelope protein and a known viral RNA packaging signal have been shown to determine the specificity and selectivity of RNA packaging into virions.

FIG. 1

Schematic diagrams of the structures of MHV genomic RNA and DI RNAs. The five domains of DF1-2 (domains I through V) are indicated below the diagram of DF1-2; the locations of these domains on MHV genomic RNA are shown as shaded boxes. The numbers 1 through 7 represent the seven genes of MHV. The deleted regions in DI RNAs are shown as dashed lines. The exact locations of the deleted regions are shown as nucleotides numbered from the 5′ end of DF1-2. The locations of the 190-nt packaging signal and the 69-nt packaging signal in DI RNAs are also indicated. DFΔPS and ΔFA2 both had a deletion of the 69-nt packaging signal within the 190-nt packaging signal. The packaging efficiency for a given RNA species was calculated as the ratio of amount of that RNA from virions divided by the amount of that RNA from cells. The packaging efficiencies of different DI RNAs are reported as approximate percentages of the packaging efficiency of MHV genomic RNA.

FIG. 2

Comparison of packaging efficiencies of MHV DI RNAs containing the 190-nt packaging signal (FA1, FA992A, and DF1-2) with the efficiencies of those containing the 69-nt packaging signal (FA4+PS and FB1) and those lacking the packaging signal (FA4 and DFΔPS). The same amount of in vitro-synthesized RNA of each DI clone was independently transfected into MHV-infected cells. Released virus particles were harvested at 12 h p.i. and purified by sucrose gradient centrifugation. Viral RNAs were extracted from purified virus particles. Intracellular (i.c.) RNAs were also extracted at 12 h p.i. from cytoplasmic protein lysates. Intracellular RNAs and virion RNAs were analyzed using Northern blot analysis with a probe that binds to MHV genomic RNA (or mRNA 1) and DI RNAs. The arrowheads indicate MHV genomic RNA (mRNA 1). The arrows indicate DI RNAs of expected sizes. Panels A to D represent separate experiments, each of which was repeated in triplicate.

FIG. 3

Specific binding of M protein to replicating DI RNAs containing the packaging signal in MHV-infected cells. Cell lysates were prepared from DI RNA-transfected, MHV-infected cells at 12 h p.i. Anti-N protein monoclonal antibody (anti-N) and anti-M protein monoclonal antibody (anti-M) were independently added to equal volumes of cell lysates, and immunoprecipitation was performed. RNA was extracted from the immunoprecipitated samples. Intracellular (i.c.) RNAs and virion RNAs were extracted as described in the legend to Fig. 2. Extracted RNAs were analyzed on Northern blots using a probe that binds to DI RNAs. Only the section of the autoradiogram with the DI RNAs is shown. Each panel shows representative data from triplicate experiments.

FIG. 4

Specific binding of M protein to non-MHV RNA transcripts containing the packaging signal. (A) Schematic diagrams of the structures of plasmids PS5A and PS5B190. T7 Pr, T7 promoter; T7 Ter; T7 terminator. (B) Northern blot analysis of expressed RNA transcripts in RNA-expressing, MHV-infected cells (intracellular [i.c.] RNA) and packaged RNA transcripts in MHV particles (virion RNAs). PS5A RNA transcripts or PS5B190 RNA transcripts were independently expressed in MHV-infected cells. Intracellular RNAs and virion RNAs were analyzed on Northern blots using a probe that binds to the CAT sequence. The arrows indicate expressed RNA transcripts. (C) Specific binding of M protein to the expressed PS5B190 RNA transcripts in MHV-infected cells. Cell lysates were prepared from MHV-infected cells expressing non-MHV RNA transcripts at 12 h post-MHV infection. Anti-N protein monoclonal antibody (anti-N) and anti-M protein monoclonal antibody (anti-M) were independently added to equal volumes of cell lysates, and immunoprecipitation was performed. RNA was extracted from the immunoprecipitated samples. Intracellular (i.c.) RNAs and coimmunoprecipitated RNAs were analyzed using Northern blot analysis with a probe that binds to the CAT sequence. The arrows indicate expressed RNA transcripts.