Evaluation of the role of heterogeneous nuclear ribonucleoprotein A1 as a host factor in murine coronavirus discontinuous transcription and genome replication

Viruses with RNA genomes often capture and redirect host cell components to assist in mechanisms particular to RNA-dependent RNA synthesis. The nidovi ruses are an order of positive-stranded RNA viruses, comprising coronavirus es and arteriviruses, that employ a unique strategy of discontinuous transc ription, producing a series of subgenomic mRNAs linking a 5' leader to dist al portions of the genome. For the prototype coronavirus mouse hepatitis vi rus (MHV), heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has been show n to be able to bind in vitro to the negative strand of the intergenic sequ ence, a cis-acting element found in the leader RNA and preceding each downs tream ORF in the genome. hnRNP A1 thus has been proposed as a host factor i n MHV transcription. To test this hypothesis genetically, we initially cons tructed MHV mutants with a very high-affinity hnRNP A1 binding site inserte d in place of, or adjacent to, an intergenic sequence in the MHV genome. Th is inserted hnRNP A1 binding site was not able to functionally replace, or enhance transcription from, the intergenic sequence. This finding led us to test more directly the role of hnRNP A1 by analysis of MHV replication and RNA synthesis in a murine cell line that does not express this protein. Th e cellular absence of hnRNP A1 had no detectable effect on the production o f infectious virus, the synthesis of genomic RNA, or the quantity or qualit y of subgenomic mRNAs. These results strongly suggest that hnRNP A1 is not a required host factor for MHV discontinuous transcription or genome replic ation.