RESUMO
Rift Valley fever virus (RVFV, Bunyaviridae, Phlebovirus) is a mosquito-transmitted arbovirus that causes human and animal diseases in sub-Saharan Africa and was introduced into the Arabian Peninsula in 2000. Here, we describe a method of reverse genetics to recover infectious RVFV from transfected plasmids based on the use of the cellular RNA polymerase I promoter to synthesize viral transcripts. We compared its efficiency with a system using T7 RNA polymerase and found that both are equally efficient for the rescue of RVFV generating titers of approx. 10(7) to 10(8) pfu/ml. We used the RNA polymerase I-based system to rescue both attenuated MP12 and virulent ZH548 strains as well as chimeric MP12-ZH548 viruses, and in addition RVFV expressing reporter proteins.
Assuntos
RNA Polimerase I/metabolismo , RNA Viral/biossíntese , Vírus da Febre do Vale do Rift/crescimento & desenvolvimento , Virologia/métodos , Replicação Viral , Animais , Chlorocebus aethiops , Camundongos , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/genética , Vírus da Febre do Vale do Rift/patogenicidade , Análise de Sobrevida , Células Vero , Ensaio de Placa Viral , VirulênciaRESUMO
Rift Valley fever virus (RVFV) (Phlebovirus, Bunyaviridae) possesses a genome composed of three negative-stranded RNA molecules. Each segment contains 3' and 5' noncoding regions with terminal complementary sequences forming a panhandle structure. We showed that transcription-replication of the L, M and S segments is regulated, and we established a minigenome rescue system expressing a CAT reporter to investigate the role of the noncoding regions in this process. The L, M and S segment-based minigenomes were shown to drive bona fide transcription and replication and to express variable levels of CAT reporter, indicating differential promoter activities within the noncoding sequences. In addition, we found a good correlation between the relative promoter strength and the abundance of viral RNA species in RVFV-infected cells. Altogether, these results show that RVFV minigenomes are powerful tools to study transcription and replication and constitute a valuable basis to rescue infectious virus from cDNAs.
Assuntos
DNA Intergênico/genética , Regulação Viral da Expressão Gênica , RNA Viral/biossíntese , RNA Viral/genética , Vírus da Febre do Vale do Rift/genética , Transcrição Gênica/genética , Animais , Linhagem Celular , Cricetinae , Genoma ViralRESUMO
Rift Valley fever virus (RVFV) is a Phlebovirus in the Bunyaviridae family. The nucleoprotein N is the most abundant component of the virion; numerous copies of N associate with the viral RNA genome and form pseudohelicoidal ribonucleoproteins (RNPs) circularized by a panhandle structure formed by the base-paired RNA sequences at the 3' and 5' termini. These structures play a central role in transcription and replication. We investigated the intermolecular interactions of the RVFV N protein and found that after chemical cross-linking treatment, the nucleoprotein from purified RNPs migrates mainly as dimers. The N-N interaction was studied using the yeast two-hybrid system, the GST pull-down method, and mutational analysis. We demonstrated that the N terminus from residue 1 to 71, and particularly Tyr 4 and Phe 11, which are conserved among phlebovirus N sequences, are involved in the interaction. The C-terminal region did not seem to be essential for the N-N interaction. Moreover, we showed that N(TOS), the N protein of the related Toscana phlebovirus, interacts with itself and forms heterodimers with N(RVF), suggesting that the dimeric form of N may be a conserved feature in phlebovirus RNPs.