Bunyamwera bunyavirus RNA synthesis requires cooperation of 3'- and 5'-terminal sequences

Bunyamwera virus (BUNV) is the prototype of both the Orthobunyavirus genus and the Bunyaviridae family of segmented negative-sense RNA viruses. The tripartite BUNV genome consists of small (S), medium (M), and large (L) segments that are each transcribed to yield a single mRNA and are replicated to generate an antigenome that acts as a template for synthesis of further genomic strands. As for all negative-sense RNA viruses, the 3'- and 5'-terminal nontranslated regions (NTRs) of the BUNV S, M, and L segments exhibit nucleotide complementarity and, except for one conserved U-G pairing, this complementarity extends for 15, 18, and 19 nucleotides, respectively. We investigated whether the complementarity of 3' and 5' NTRs reflected a functional requirement for terminal cooperation to promote BUNV RNA synthesis or, alternatively, was a consequence of genomic and antigenomic NTRs having similar functions requiring sequence conservation. We show that cooperation between 3'- and 5'-NTR sequences is required for BUNV RNA synthesis, and our results suggest that this cooperation is due to nucleotide complementarity allowing 3' and 5' NTRs to associate through base-pairing interactions. To examine the importance of complementarity in promoting BUNV RNA synthesis, we utilized a competitive replication assay able to examine the replication ability of all possible combinations of interacting nucleotides within a defined region of BUNV 3' and 5' NTRs. We show here that maximal RNA replication was signaled when sequences exhibiting perfect complementarity within 3' and 5' NTRs were selected.     

5'-proximal hot spot for an inducible positive-to-negative-strand template switch by coronavirus RNA-dependent RNA polymerase

Coronaviruses have a positive-strand RNA genome and replicate through the use of a 3' nested set of subgenomic mRNAs each possessing a leader (65 to 90 nucleotides [nt] in length, depending on the viral species) identical to and derived from the genomic leader. One widely supported model for leader acquisition states that a template switch takes place during the generation of negative-strand antileader-containing templates used subsequently for subgenomic mRNA synthesis. In this process, the switch is largely driven by canonical heptameric donor sequences at intergenic sites on the genome that match an acceptor sequence at the 3' end of the genomic leader. With experimentally placed 22-nt-long donor sequences within a bovine coronavirus defective interfering (DI) RNA we have shown that matching sites occurring anywhere within a 65-nt-wide 5'-proximal genomic acceptor hot spot (nt 33 through 97) can be used for production of templates for subgenomic mRNA synthesis from the DI RNA. Here we report that with the same experimental approach, template switches can be induced in trans from an internal site in the DI RNA to the negative-strand antigenome of the helper virus. For these, a 3'-proximal 89-nt acceptor hot spot on the viral antigenome (nt 35 through 123), largely complementary to that described above, was found. Molecules resulting from these switches were not templates for subgenomic mRNA synthesis but, rather, ambisense chimeras potentially exceeding the viral genome in length. The results suggest the existence of a coronavirus 5'-proximal partially double-stranded template switch-facilitating structure of discrete width that contains both the viral genome and antigenome.     

RNA-protein interactions directed by the 3' end of human rhinovirus genomic RNA.

The replication of a picornavirus genomic RNA is a template-specific process involving the recognition of viral RNAs as target replication templates for the membrane-bound viral replication initiation complex. The virus-encoded RNA-dependent RNA polymerase, 3Dpol, is a major component of the replication complex; however, when supplied with a primed template, 3Dpol is capable of copying polyadenylated RNAs which are not of viral origin. Therefore, there must be some other molecular mechanism to direct the specific assembly of the replication initiation complex at the 3' end of viral genomic RNAs, presumably involving cis-acting binding determinants within the 3' noncoding region (3' NCR). This report describes the use of an in vitro UV cross-linking assay to identify proteins which interact with the 3' NCR of human rhinovirus 14 RNA. A cellular protein(s) was identified in cytoplasmic extracts from human rhinovirus 14-infected cells which had a marked binding preference for RNAs containing the rhinovirus 3' NCR sequence. This protein(s) showed reduced cross-linking efficiency for a 3' NCR with an engineered deletion. Virus recovered from RNA transfections with in vitro transcribed RNA containing the same 3' NCR deletion demonstrated a defective replication phenotype in vivo. Cross-linking experiments with RNAs containing the poliovirus 3' NCR and cytoplasmic extracts from poliovirus-infected cells produced an RNA-protein complex with indistinguishable electrophoretic properties, suggesting that the appearance of the cellular protein(s) may be a common phenomenon of picornavirus infection. We suggest that the observed cellular protein(s) is sequestered or modified as a result of rhinovirus or poliovirus infection and is utilized in viral RNA replication, perhaps by binding to the 3' NCR as a prerequisite for replication complex assembly at the 3' end of the viral genomic RNA.