Biomedical subjects

Andrea V Gamarnik

Publications and source records attributed to Andrea V Gamarnik.

5 recordsLinked to original sources

PubMed2006

A 5' RNA element promotes dengue virus RNA synthesis on a circular genome.

The mechanisms of RNA replication of plus-strand RNA viruses are still unclear. Here, we identified the first promoter element for RNA synthesis described in a flavivirus. Using dengue virus as a model, we found that the viral RdRp discriminates the viral RNA by specific recognition of a 5' element named SLA. We demonstrated that RNA-RNA interactions between 5' and 3' end sequences of the viral genome enhance dengue virus RNA synthesis only in the presence of an intact SLA. We propose a novel mechanism for minus-strand RNA synthesis in which the viral polymerase binds SLA at the 5' end of the genome and reaches the site of initiation at the 3' end via long-range RNA-RNA interactions. These findings provide an explanation for the strict requirement of dengue virus genome cyclization during viral replication.

Dengue Virus↗

PubMed2006

Exploring RNA virus replication in Xenopus oocytes.

Microinjection of poliovirus RNA in Xenopus oocytes initiates a complete and authentic viral replication cycle that yields newly synthesized infectious virus. This system can be used to study the molecular mechanism of the different steps involved in virus replication. Interestingly, viral replication only occurs if poliovirus RNA is coinjected with factors present in HeLa extracts. We have determined that two HeLa cell factors are required for viral replication in oocytes, one involved in initiation of translation (polio translation factor) and the other in RNA synthesis. Thus, microinjection in oocytes provides a strategy to identify and further analyze the function of these host cell factors and to biochemically dissect the mechanism of initiation of poliovirus translation and RNA synthesis. Here, we review protocols, approaches, and potential issues that can be addressed using the oocyte system.

Animals↗

PubMed2005

Role of RNA structures present at the 3'UTR of dengue virus on translation, RNA synthesis, and viral replication.

We have developed a dengue virus replicon system that can be used to discriminate between translation and RNA replication. Using this system, we analyzed the functional role of well-defined RNA elements present at the 3'UTR of dengue virus in mammalian and mosquito cells. Our results show that deletion of individual domains of the 3'UTR did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. We demonstrated that complementarity between sequences present at the 5' and 3' ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3'UTR resulted in replicons with decreased RNA amplification. We also characterized the vaccine candidate rDEN2Delta30 in the replicon system and found that viral attenuation is caused by inefficient RNA synthesis. Furthermore, using both the replicon system and recombinant viruses, we identified an RNA region of the 3'UTR that enhances dengue virus replication in BHK cells while is dispensable in mosquito cells.

3' Untranslated Regions↗

PubMed2005

Characterization of internal ribosomal entry sites of Triatoma virus.

Triatoma virus (TrV) belongs to a new family of RNA viruses known as Dicistroviridae. Nucleotide sequence comparisons between different dicistroviruses allowed two putative internal ribosomal entry sites (IRESs) in the TrV RNA to be defined: the 5'UTR IRES of 548 nt and the intergenic region (IGR) IRES of 172 nt. Using monocistronic and bicistronic RNAs, it was shown that the TrV genome contains two functional IRESs that mediate translation initiation in a cap-independent manner. In addition, it was found that the two TrV IRESs were able to direct efficient translation of reporter genes in microinjected Xenopus oocytes, suggesting minimum requirements for host factors. The IGR IRES begins with a non-canonical CUC; however, mutations of this triplet to AUG or CCU did not impair IRES function, indicating that the CUC is not essential for the initiation process. Furthermore, translation efficiency from two TrV IRESs was differentially modulated by IFN-alpha and viral infection.

5' Untranslated Regions↗

PubMed2005

Long-range RNA-RNA interactions circularize the dengue virus genome.

Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5' and 3' ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy revealed that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5' and 3' CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5' and 3' untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5' and 3' UAR (upstream AUG region). In order to investigate the functional role of 5'-3' UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5' or 3' UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.

Animals↗