Sequence and translation of the murine coronavirus 5'-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase

Abstract

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.

Immune response to a murine coronavirus: Identification of a homing receptor-negative CD4+ T cell subset that responds to viral glycoproteins

“…A clone containing ~28, the 5'terminal portion of the putative polymerase gene, was constructed using published sequences and PCR technology (Soe et a/., 1987). To construct the p28 clone, a 5' primer (GCATAmGCAAAGATGG) and a 3' primer (TAAGG-TCGCCITAGTCTTC)…”

“…Since p28 is normally cleaved from a large precursor, a T nucleotide was substituted for an A present in the original sequence (underlined above) in the 3' primer. This change introduced a termination codon into the construct at a position (amino acid 272) corresponding to a potential proteolytic cleavage site (Soe et al, 1987).…”

“…To determine the antigen specificity of T cell proliferation in vitro, VV recombinants expressing the MHV structural proteins, S, M, and N and the nonstructural protein, ~28, were constructed as described under Materials and Methods. P28, a basic protein encoded by the putative MHV polymerase gene (Denison and Perlman, 1986;Soe et al, 1987) studies since analysis of its structure indicated the presence of several possible T cell epitopes (Margalit et al, 1987).…”

“…With the goal of understanding the immune response in these persistently infected mice, we have compared the proliferative T cell response in these mice and that in immunized adult C57BU6 mice. For these experi-ments, we have used recombinant vaccinia viruses (VV) which expressed the three structural proteins (surface glycoprotein (S), the transmembrane glycoprotein (M) and the nucleocapsid protein (N)) and one nonstructural protein, ~28, a basic protein encoded by the putative polymerase gene (Denison and Perlman, 1986;Soe et al, 1987). The results indicate that the cells from immunized adult mice which specifically proliferate in response to antigen are CD4+ and do not express the lymphocyte surface homing receptor, recognized by the monoclonal antibody gp90).…”

Identification of the polymerase polyprotein products p72 and p65 of the murine coronavirus MHV-JHM

“…Denison and Perlman showed that in vitro translation of viral genomic RNA produced p28 and p220, and that production of p28 was inhibited by the addition of a proteinase inhibitor, zinc chloride (Denison and Perlman, 1986). Using an antiserum generated against a synthetic peptide representing a portion of p28, the p28 protein was detected in MHV-infected cells and shown to be the amino-terminal protein of the polymerase polyprotein (Denison and Perlman, 1987;Soe et al, 1987). Translation of RNAs representing the 5' end of the viral genome revealed that the PCP-1 domain was required for the autoproteolytic cleavage of p28 (Baker et al, 1989).…”