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.     

Mutations in the 5' trailer region of a respiratory syncytial virus minigenome which limit RNA replication to one step

The 3' termini of the genomic and antigenomic RNAs of human respiratory syncytial virus (RSV) are identical at 10 of the first 11 nucleotide positions and 21 of the first 26 positions. These conserved 3'-terminal sequences are thought to contain the genomic and antigenomic promoters. Furthermore, the complement of each conserved sequence (i.e., the 5' end of the RNA it encodes) might contain an encapsidation signal. Using an RSV minigenome system, we individually mutated each of the last seven nucleotides in the 5' trailer region of the genome. We analyzed effects of these mutations on encapsidation of the T7 polymerase-transcribed negative-sense genome, its ability to function as a template for RSV-driven synthesis of positive-sense antigenome and mRNA, and the ability of this antigenome to be encapsidated and to function as template for the synthesis of more genome. As a technical complication, mutations in the last five nucleotides of the trailer region were found to affect the efficiency of the adjoining T7 promoter over more than a 10-fold range, even though three nonviral G residues had been included between the core promoter and the trailer to maximize the efficiency of promoter activity. This was controlled in all experiments by monitoring the levels of total and encapsidated genome. The efficiency of encapsidation of the T7 polymerase-transcribed genome was not affected by any of the trailer mutations. Furthermore, neither the efficiency of positive-sense RNA synthesis from the genome nor the efficiency of encapsidation of the encoded antigenome was affected by the mutations. However, nucleotide substitution at positions 2, 3, 6, or 7 relative to the 5' end of the trailer blocked the production of progeny genome, whereas substitution at positions 1 and 5 allowed a low level of genome production and substitutions at position 4 were tolerated. Position 4 is the only one of the seven positions examined that is not conserved between the 3' ends of genomic and antigenomic RNA. The mutations that blocked the synthesis of progeny genome thus limited RNA replication to one step, namely, the synthesis and encapsidation of antigenome. Restoration of terminal complementarity for one of the trailer mutants by making a compensatory mutation in the leader region did not restore synthesis of genomic RNA, confirming that its loss was not due to reduced terminal complementarity. Interestingly, this leader mutation appeared to prevent antigenome synthesis with only a slight effect on mRNA synthesis, apparently providing a dissociation between these two synthetic activities. Genomes in which the terminal 24 or 325 nucleotides of the trailer have been deleted were competent for encapsidation and the synthesis of mRNA and antigenomic RNA, further confirming that terminal complementarity was not required for these functions.     

RNA replication from the simian virus 5 antigenomic promoter requires three sequence-dependent elements separated by sequence-independent spacer regions

We have previously shown for the paramyxovirus simian virus 5 (SV5) that a functional promoter for RNA replication requires proper spacing between two discontinuous elements: a 19-base segment at the 3' terminus (conserved region I [CRI]) and an 18-base internal region (CRII) that is contained within the coding region of the L protein gene. In the work described here, we have used a reverse-genetics system to determine if the 53-base segment between CRI and CRII contains additional sequence-specific signals required for optimal replication or if this segment functions solely as a sequence-independent spacer region. A series of copyback defective interfering minigenome analogs were constructed to contain substitutions of nonviral sequences in place of bases 21 to 72 of the antigenomic promoter, and the relative level of RNA replication was measured by Northern blot analysis. The results from our mutational analysis indicate that in addition to CRI and CRII, optimal replication from the SV5 antigenomic promoter requires a third sequence-dependent element located 51 to 66 bases from the 3' end of the RNA. Minigenome RNA replication was not affected by changes in the either the position of this element in relation to CRI and CRII or the predicted hexamer phase of NP encapsidation. Thus, optimal RNA replication from the SV5 antigenomic promoter requires three sequence-dependent elements, CRI, CRII and bases 51 to 66.     

Nucleolar targeting of hepatitis delta antigen abolishes its ability to initiate viral antigenomic RNA replication

Hepatitis delta virus (HDV) is a small RNA virus that contains one 1.7-kb single-stranded circular RNA of negative polarity. The HDV particle also contains two isoforms of hepatitis delta antigen (HDAg), small (SHDAg) and large HDAg. SHDAg is required for the replication of HDV, which is presumably carried out by host RNA-dependent RNA polymerases. The localization and the HDAg and host RNA polymerase responsible for HDV replication remain important issues to be addressed. In this study, using recombinant SHDAg fused with a heterologous nucleolar localization sequence (NoLS) to confine its subcellular localization in nucleoli, we aimed to study the effect of SHDAg subcellular localization on HDV RNA replication. The initiation of genomic RNA synthesis from antigenomic template was hardly detectable when SHDAg was fused with the NoLS motif and localized mainly in nucleoli. In contrast, the initiation of antigenomic RNA synthesis was not affected. Drug treatment to release a SHDAg-NoLS mutant from nucleoli could partially restore the replication of HDV genomic RNA from antigenomic RNA. This also recovered the cointeraction between SHDAg and RNA polymerase II. These data strongly suggest that nuclear polymerase (RNA polymerase II) is involved in the synthesis of genomic RNA and that the synthesis of antigenomic RNA can occur in nucleoli. Our results support the idea that the replication of HDV genomic RNA or antigenomic RNA is likely to be carried out by different machineries in different subcellular localizations.