RESUMO
Several proteins and RNAs expressed by mammalian viruses have been reported to interfere with RNA interference (RNAi) activity. We investigated the ability of the HIV-1-encoded RNA elements Trans-Activation Response (TAR) and Rev-Response Element (RRE) to alter RNAi. MicroRNA let7-based assays showed that RRE is a potent suppressor of RNAi activity, while TAR displayed moderate RNAi suppression. We demonstrate that RRE binds to TAR-RNA Binding Protein (TRBP), an essential component of the RNA Induced Silencing Complex (RISC). The binding of TAR and RRE to TRBP displaces small interfering (si)RNAs from binding to TRBP. Several stem-deleted RRE mutants lost their ability to suppress RNAi activity, which correlated with a reduced ability to compete with siRNA-TRBP binding. A lentiviral vector expressing TAR and RRE restricted RNAi, but RNAi was restored when Rev or GagPol were coexpressed. Adenoviruses are restricted by RNAi and encode their own suppressors of RNAi, the Virus-Associated (VA) RNA elements. RRE enhanced the replication of wild-type and VA-deficient adenovirus. Our work describes RRE as a novel suppressor of RNAi that acts by competing with siRNAs rather than by disrupting the RISC. This function is masked in lentiviral vectors co-expressed with viral proteins and thus will not affect their use in gene therapy. The potent RNAi suppressive effects of RRE identified in this study could be used to enhance the expression of RNAi restricted viruses used in oncolysis such as adenoviruses.
Assuntos
Genes env , Repetição Terminal Longa de HIV , HIV-1/genética , Interferência de RNA , Proteínas de Ligação a RNA/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Ligação Competitiva , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , HIV-1/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Células Jurkat , Lentivirus/genética , Lentivirus/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genética , Produtos do Gene pol do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The hepatitis C virus (HCV) genome contains structured elements thought to play important regulatory roles in viral RNA translation and replication processes. We used in vitro RNA binding assays to map interactions involving the HCV 5'UTR and distal sequences in NS5B to examine their impact on viral RNA replication. The data revealed that 5'UTR nucleotides (nt) 95â»110 in the internal ribosome entry site (IRES) domain IIa and matching nt sequence 8528â»8543 located in the RNA-dependent RNA polymerase coding region NS5B, form a high-affinity RNA-RNA complex in vitro. This duplex is composed of both wobble and Watson-Crick base-pairings, with the latter shown to be essential to the formation of the high-affinity duplex. HCV genomic RNA constructs containing mutations in domain IIa nt 95â»110 or within the genomic RNA location comprising nt 8528â»8543 displayed, on average, 5-fold less intracellular HCV RNA and 6-fold less infectious progeny virus. HCV genomic constructs containing complementary mutations for IRES domain IIa nt 95â»110 and NS5B nt 8528â»8543 restored intracellular HCV RNA and progeny virus titers to levels obtained for parental virus RNA. We conclude that this long-range duplex interaction between the IRES domain IIa and NS5B nt 8528â»8543 is essential for optimal virus replication.
Assuntos
Regiões 5' não Traduzidas , Genoma Viral , Hepacivirus/genética , RNA Polimerase Dependente de RNA/genética , Replicação Viral/genética , Linhagem Celular Tumoral , Genômica , Hepacivirus/fisiologia , Humanos , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Ligação Proteica , Biossíntese de Proteínas , RNA Viral/genéticaRESUMO
Zika virus (ZIKV) is an emerging pathogen from the Flaviviridae family. It represents a significant threat to global health due to its neurological and fetal pathogenesis (including microcephaly and congenital malformations), and its rapid dissemination across Latin America in recent years. The virus has spread from Africa to Asia, the Pacific islands and the Americas with limited knowledge about the pathogenesis associated with infection in recent years. Herein, we compared the ability of the Canadian-imported Thai strain PLCal_ZV and the Brazilian isolate HS-2015-BA-01 from Bahia to produce infectious ZIKV particles and cytopathic effects in a cell proliferation assay. We also compared the intracellular viral RNA accumulation of the two strains by quantitative RT-PCR (reverse transcription polymerase chain reaction) analyses. Our observations show that HS-2015-BA-01 is more cytopathic than PLCal_ZV in proliferation assays in Vero, Human Embryonic Kidney HEK 293T and neuroblastoma SH-SY5Y cells. Quantitative RT-PCR shows that the level of viral RNA is higher with HS-2015-BA-01 than with PLCal_ZV in two cell lines, but similar in a neuroblastoma cell line. The two strains have 13 amino acids polymorphisms and we analyzed their predicted protein secondary structure. The increased cytopathicity and RNA accumulation of the Brazilian ZIKV isolate compared to the Thai isolate could contribute to the increased pathogenicity observed during the Brazilian epidemic.