RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA

Abstract

Previously, a cDNA was constructed so that transcription by T7 RNA polymerase yielded a ϳ1-kb negativesense analog of genomic RNA of human respiratory syncytial virus (RSV) containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative RSV transcription motifs and flanked by the RSV genomic termini. When transfected into RSV-infected cells, this minigenome was ''rescued,'' as evidenced by high levels of CAT expression and the production of transmissible particles which propagated and expressed high levels of CAT expression during serial passage (P. L. Collins, M. A. Mink, and D. S. Stec, Proc. Natl. Acad. Sci. USA, 88:9663-9667, 1991). Here, this cDNA, together with a second one designed to yield an exact-copy positive-sense RSV-CAT RNA antigenome, were each modified to contain a self-cleaving hammerhead ribozyme for the generation of a nearly exact 3 end. Each cDNA was transfected into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase, together with plasmids encoding the RSV N, P, and L proteins, each under the control of a T7 promoter. When the plasmid-supplied template was the mini-antigenome, the minigenome was produced. When the plasmid-supplied template was the minigenome, the products were mini-antigenome, subgenomic polyadenylated mRNA and progeny minigenome. Identification of progeny minigenome made from the plasmid-supplied minigenome template indicates that the full RSV RNA replication cycle occurred. RNA synthesis required all three RSV proteins, N, P, and L, and was ablated completely by the substitution of Asn for Asp at position 989 in the L protein. Thus, the N, P, and L proteins were sufficient for the synthesis of correct minigenome and antigenome, but this was not the case for subgenomic mRNA, indicating that the requirements for RNA replication and transcription are not identical. Complementation with N, P, and L alone yielded an mRNA pattern containing a large fraction of molecules of incomplete, heterogeneous size. In contrast, complementation with RSV (supplying all of the RSV gene products) yielded a single discrete mRNA band. Superinfection with RSV of cells staging N/P/L-based RNA synthesis yielded the single discrete mRNA species. Some additional factor supplied by RSV superinfection appeared to be involved in transcription, the most obvious possibility being one or more additional RSV gene products.

The structurally diverse intergenic regions of respiratory syncytial virus do not modulate sequential transcription by a dicistronic minigenome

“…Total cellular RNA was purified with Trizol reagent (Life Technologies) (15). Oligo(dT) chromatography was performed with oligo(dT)-cellulose by a minibatch method involving differential centrifugation in a microcentrifuge tube (15). RNA was denatured with glyoxal, electrophoresed through a 1.5% agarose gel, and blotted to nylon membrane (see Fig.…”

“…Consensus GS and GE motifs have been identified at the beginning and end of each gene and confirmed to be self-contained transcription signals (2,21). The N, P, and L proteins are necessary and sufficient for RSV minigenome RNA replication (15,30). On the other hand, RSV differs from the prototypic viruses in requiring a fourth protein, M2, for processive, sequential transcription (3,4).…”

Recombinant Newcastle Disease Virus Expressing a Foreign Viral Antigen Is Attenuated and Highly Immunogenic in Primates

“…Twentyfour hours later, the cells were harvested and used for isolation of total intracellular RNA with the RNeasy mini kit (QIAGEN, Inc., Valencia, CA) according to the manufacturer's recommendations. The RNA was analyzed by Northern blot hybridization (7) with a double-stranded DNA (dsDNA) probe prepared from a cDNA of the HN gene using the Megaprime DNA Labeling system (Amersham Biosciences Corp., Piscataway, NJ) and [ 32 P]dCTP. Alternatively, cells were harvested 24 h postinfection and lysed under denaturing and reducing conditions, and the clarified supernatant was subjected to gel electrophoresis on 4 to 12% bis-Tris acrylamide gels (NuPage protein electrophoresis system; Invitrogen, Mountain View, CA) according to the manufacturer's recommendations.…”

Engineering of Live Chimeric Vaccines against Human Metapneumovirus

“…The technique is based on transfecting permissive cells with a plasmid coding for the viral genome and satellite plasmids coding for all the proteins necessary for the formation of a ribonucleoprotein complex (RNP) that initiates the transcription of viral genes ( Figure 1). The proteins indispensable to form the RNP in paramyxoviruses and pneumoviruses are N, P, and L proteins [127][128][129], yet the addition of M2-1 protein facilitates the recovery HMPV from cDNA [130]. The elaboration of a polyvalent vaccine can be accomplished by either cloning additional genes into the plasmid coding for viral genome or replacing protective antigens of a vector with the ones of another pathogen.…”