THE NS1 PROTEIN OF HUMAN RESPIRATORY SYNCYTIAL VIRUS IS A POTENT INHIBITOR OF MINIGENOME TRANSCRIPTION AND RNA REPLICATION

The NS1 protein (139 amino acids) is one of the two nonstructural prot eins of human respiratory syncytial virus (RSV) and is encoded by a ve ry abundant mRNA transcribed from the promoter-proximal RSV gene, The function of NS1 was unknown and was investigated here by using a recon stituted transcription and RNA replication system that involves a mini replicon and viral proteins (N, P, L and M2-1) expressed from separate cotransfected plasmids, Coexpression of the NS1 cDNA strongly inhibit ed transcription and RNA replication mediated by the RSV polymerase, e ven when the level of expressed NS1 protein was substantially below th at observed in RSV-infected cells, The effect depended on synthesis of NS1 protein rather than NS1 RNA alone, Transcription and both steps o f RNA replication, namely, synthesis of the antigenome and the genome, appeared to be equally sensitive to inhibition, The efficiency of enc apsidation of the plasmid-derived minigenome was not altered by coexpr ession of NS1, indicating that the inhibition occurs at a later step, In two different dicistronic minigenomes, transcription of each gene v t as equally sensitive to inhibition by NS1. This suggested that the g radient of transcriptional polarity was unaffected and that the effect of NS1 instead probably involves an early event such as polymerase en try on the genome, NS1-mediated inhibition of transcription and RNA re plication was not affected by eoexpression of the M2 mRNA which has tw o open reading frames encoding the transcriptional elongation factor M 2-1 and the putative negative regulatory factor M2-2. The potent natur e of the NS1-mediated inhibition suggests that negative regulation is an authentic function of the NS1 protein, albeit not necessarily the o nly one.