Increased Expression of the N Protein of Respiratory Syncytial Virus Stimulates Minigenome Replication but Does Not Alter the Balance between the Synthesis of mRNA and Antigenome (original) (raw)
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Journal of Virology, 2000
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 anti- genome 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.
Replication of the genomic RNA of a positive-strand RNA animal virus from negative-sense transcripts
Proceedings of the National Academy of …, 1994
Studies of RNA replication among the positive-strand RNA animal viruses have been hindered by the apparent inability of their RNA-dependent RNA polymerases to initiate replication on the corresponding negative-sense RNAs. However, here I report that in the case of the nodavirus flock house virus (FHV), which has a bipartite positive-sense RNA genome, the viral RNA replicase can replicate a negativesense transcript of the genome segment that encodes the viral capsid proteins. For this work, the FHV replication cycle was experimentafly reconstructed in baby hamster kidney cells that were transfected with specialized transcription plasmids designed to direct the synthesis of RNAs which corresponded closely to the two genome segments of FHV. The RNA replicase encoded by the larger genome segment could utilize either the positive or the negative strand of the smaller segment as a template, and it catalyzed RNA replication to produce similar RNA products in the two situations. Surprisingly, studies of the nucleotide sequences that were required for replication showed that the 3' end of the negative-strand RNA contained only a minimal cis-acting signal. The success of these experiments will facilitate further studies of the cis-and transacting factors involved in the recognition and replication of negative-sense RNA in this system.
Quantification and kinetics of viral RNA transcripts produced in Orthohantavirus infected cells
Virology Journal
Background: Rodent borne viruses of the Orthohantavirus genus cause hemorrhagic fever with renal syndrome among people in Eurasia, and hantavirus cardiopulmonary syndrome in the Americas. At present, there are no specific treatments or efficient vaccines against these diseases. Improved understanding of viral transcription and replication may instigate targeted treatment of Orthohantavirus infections. For this purpose, we investigated the kinetics and levels of viral RNA transcription during an ongoing infection in-vitro. Methods: Vero E6 cells were infected with Puumala Orthohantavirus (strain Kazan) before cells and supernatants were collected at different time points post infection for the detection of viral RNAs. A plasmid containing primer binding sites of the three Orthohantavirus segments small (S), medium (M) and large (L) was constructed and standard curves were generated to calculate the copy numbers of the individual transcripts in the collected samples. Results: Our results indicated a rapid increase in the copy number of viral RNAs after 9 h post infection. At peak days, 2-6 days after infection, the Sand M-segment transcripts became thousand and hundred-fold more abundant than the copy number of the L-segment RNA, respectively. The presence of viral RNA in the cell culture media was detected at later time-points. Conclusions: We have developed a method to follow RNA transcription in-vitro after synchronous infection of Vero cells. The obtained results may contribute to the understanding of the viral replication, and may have implications in the development of antiviral drugs targeting transcription or replication of negative stranded RNA viruses.
Journal of Virology, 2011
The RNA replication and transcription complex of coronaviruses is associated with an elaborate reticulovesicular network (RVN) of modified endoplasmic reticulum. Using cycloheximide and puromycin, we have studied the effect of translation inhibition on the RNA synthesis of severe acute respiratory syndrome coronavirus and mouse hepatitis virus. Both inhibitors prevented the usual exponential increase in viral RNA synthesis, with immunofluorescence and electron microscopy indicating that RVN development came to a standstill. Nevertheless, limited RNA synthesis was supported, implying that continued translation is not an absolute requirement and suggesting a direct link between RVN formation and accumulation of coronavirus proteins.
Journal of Virology, 1995
The RNA-dependent RNA polymerase of human respiratory syncytial (RS) virus was expressed in a functional form from a cDNA clone. Coexpression of the viral polymerase (L) protein, phosphoprotein (P), and nucleocapsid (N) protein allowed us to develop a system for expression and recovery of replicable RS virus RNA entirely from cDNA clones. cDNA clones of the N, P, and L genes were constructed in pGEM-based expression plasmids and shown to direct expression of the appropriate polypeptides. Two types of RS virus genomic RNA analogs were expressed from an intracellular transcription plasmid that directed the synthesis of RNAs with defined 5' and 3' ends. One analog included the authentic 5' and 3' termini of the genome, and the second contained the authentic 5' terminus and its complement at the 3' terminus as found in copyback defective interfering RNAs of other negative-strand RNA viruses. Both types of genomic analogs were encapsidated and replicated in cells ...
The Journal of biological chemistry, 1994
Amplifiable messenger RNAs (Wu, Y., Zhang, D. Y., and Kramer, F. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11769-11773) were used as templates in coupled replication-translation reactions. These amplifiable mRNAs contained a preselected messenger sequence embedded within the sequence of MDV-1 RNA, which is a small, naturally occurring template for Q beta replicase. When these recombinant mRNAs were incubated in vitro in reactions that contained both an Escherichia coli cell-free translation system and Q beta replicase, the encoded protein was synthesized more efficiently than in corresponding reactions that did not contain Q beta replicase. Moreover, when coupled replication-translation reactions were carried out in a continuous-flow format (Spirin, A. S., Baranov, V. I., Ryabova, L. A., Ovodov, S. Yu., and Alakhov, Yu. B. (1988) Science 242, 1162-1164), the synthesis of biologically active protein continued for a prolonged period. The results suggest that the mechanism of replica...
Effect of double-stranded RNA associated with viral messenger RNA on in vitro protein synthesis
Biochemistry, 1978
virions is translated about as efficiently as globin m R N A in the wheat germ cell-free system, but much less efficiently than globin mRNA in a reticulocyte cell free system. In the wheat germ system both mRNAs are optimally translated at the same salt concentration, whereas in the reticulocyte system vaccinia m R N A is optimally translated a t higher salt concentrations than globin mRNA. This pattern of translation is due to the presence of an inhibitor in the poly(A)-containing vaccinia mRNA preparations, which is more inhibitory at low salt concentrations than at high salt concentrations. A similar inhibitor is present in R N A transcribed in vitro by reovirus virions. The inhibitor is probably dsRNA since it inhibits protein synthesis in exactly the same manner as synthetic dsRNA.
RNA Signals in Entero- and Rhinovirus Genome Replication
Seminars in Virology, 1997
The VPg-linked, plus-stranded RNA genomes of entero-and rhinoviruses contain very different 5Ј and 3Ј terminal regions which harbor signals for RNA replication. The terminal cloverleaf-like structure of the 5Ј-nontranslated region (5ЈNTR) is known to be required for plus-strand RNA synthesis. Genetic evidence suggest that two stem-loop structures and the poly(A) tail of the 3ЈNTR have a function in minus-strand synthesis. All of the nonstructural viral proteins, and possibly also some cellular polypeptides, are believed to be involved in RNA replication. RNA synthesis is initiated on a poly(A) template and involves uridylylation of VPg to yield VPgpU(pU). This precursor is likely to serve as primer for the RNA polymerase 3D pol during both minus-and plus-strand RNA synthesis. 1997 Academic Press