Vikas Sood

Role of RNA Interference (RNAi) in Dengue Virus Replication and Identification of NS4B as an RNAi Suppressor

ABSTRACT RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication.

Potent knock down of HIV-1 replication by targeting HIV-1 Tat/Rev RNA sequences synergistically with catalytic RNA and DNA.

Objective:Ribozymes (Rzs) and DNA-enzymes (Dzs) possess the ability to prevent gene expression by cleaving target RNA in a catalytic and sequence-specific manner. Although Rzs or Dzs have been used earlier for HIV-1 gene suppression, the present study explored the possibility of using catalytic RNA and DNA simultaneously in a synergistic manner with the hope that this novel approach will allow more potent inhibition for a longer duration. Methods:In order to achieve long-term inhibition of HIV-1 replication, a novel non-GUX hammerhead Rz was designed by standard recombinant DNA technology and cloned it under the powerful CMV promoter containing expression vector. A 10–23 catalytic motif containing Dz that was targeted against the conserved second exon of HIV-1 Tat/Rev region was also assembled. Results:Both Rz and Dz possessed sequence-specific cleavage activities individually and simultaneously cleaved target RNA in a synergistic manner under the same in vitro cleavage conditions. These catalytic molecules inhibited HIV-1 replication in macrophages individually and exhibited potent inhibitory effects when used in combination. Conclusions:The combination strategy described here can be widely used against any target RNA to achieve more effective gene inhibition that exploits the simultaneous sequence-specific cleavage potentials of catalytic RNA and DNA.

The 7a Accessory Protein of Severe Acute Respiratory Syndrome Coronavirus Acts as an RNA Silencing Suppressor

ABSTRACT RNA silencing suppressors (RSSs) are well studied for plant viruses but are not well defined to date for animal viruses. Here, we have identified an RSS from a medically important positive-sense mammalian virus, Severe acute respiratory syndrome coronavirus. The viral 7a accessory protein suppressed both transgene and virus-induced gene silencing by reducing the levels of small interfering RNA (siRNA). The suppression of silencing was analyzed by two independent assays, and the middle region (amino acids [aa] 32 to 89) of 7a was responsible for suppression. Finally, the RNA suppression property and the enhancement of heterologous replicon activity by the 7a protein were confirmed for animal cell lines.

Nucleic Acid-Mediated Cleavage of M1 Gene of Influenza A Virus Is Significantly Augmented by Antisense Molecules Targeted to Hybridize Close to the Cleavage Site

Influenza A virus genome segment 7 encodes protein M1, which is the matrix protein playing crucial role in the virus life cycle. Any antiviral strategy that aims at reducing, in particular, the expression of this genome segment should, in principle, reduce the infectivity of the virus. We developed a specific antiviral approach at the molecular level and designed several novel 10–23 DNAzymes (Dz) and hammerhead ribozymes (Rz), specifically targeted to cleave at the conserved domains of the influenza virus M1 RNA. We sought to use antisense molecules with the hope that it will facilitate the ribozyme-mediated cleavage. We observed that the Mg2+-dependent sequence-specific cleavage of M1 RNA was achieved by both the Dz and Rz in a dose-dependent manner. This combination of catalytic Dz and Rz with antisense molecules, in principle, resulted in more effective gene suppression, inhibited the whole virus replication in host cell, and thus could be exploited for therapeutic purposes.

Genetic analysis of HIV-1 Circulating Recombinant Form 02_AG, B and C subtype-specific envelope sequences from Northern India and their predicted co-receptor usage.

HIV-1 epidemic in India is largely driven by subtype C but other subtypes or recombinants have also been reported from several states of India. This is mainly due to the co-circulation of other genetic subtypes that potentially can recombine to generate recombinant/mosaic genomes. In this study, we report detail genetic characterization of HIV-1 envelope sequences from North India (Delhi and neighboring regions). Six of 13 were related to subtype C, one B and the rest six showed relatedness with CRF02_AG strain. The subtype C possessed the highly conserved GPGQ motif but subtype B possessed the GPGR motif in the V3 loop as observed earlier. While most of the sequences suggested CCR5 co-receptor usage, one subtype C sample clearly indicated CXCR4 usage. A successful mother to child transmission was established in two pairs. Thus, co-circulation of multiple subtypes (B and C) and the recombinant CRF02_AG strains in North India suggests a rapidly evolving scenario of HIV-1 epidemic in this region with impact on vaccine formulation. Since this is the first report of CRF02_AG envelope from India, it will be important to monitor the spread of this strain and its impact on HIV-1 transmission in India.

Genetic and functional characterization of human immunodeficiency virus type 1 VprC variants from north India: Presence of unique recombinants with mosaic genomes from B, C and D subtypes within the open reading frame of Vpr

The human immunodeficiency virus type 1 (HIV-1) epidemic in India is predominantly caused by genetic subtype C, though other minor subtypes have also been reported. One of the major accessory proteins of HIV-1, namely Vpr, is known to influence key steps in viral replication, cell cycle progression, promoter activation, apoptosis and pathogenesis. Therefore, we carried out a genetic and functional analysis of the Vpr variants from eight HIV-1-infected individuals from north India. The sequence analyses revealed that six of eight samples clustered with ancestral subtype C. Remarkably, five of these showed a conserved and region-specific L64P mutation, located in the predicted third alpha-helix. This change adversely affected their ability to activate the HIV-1 long terminal repeat promoter without compromising their ability to cause apoptosis. Bootscan, phylogenetic and SimPlot analysis of the remaining two samples (VprS2 and A6) revealed very interesting mosaic genomes derived from B, C and D subtypes. The N-terminal half of the VprS2 gene consisted of genomic segments derived from subtypes B/D, C and D but the C-terminal half was derived predominantly from subtype C. Interestingly the N-terminal half of sample A6 also showed similar B/D, C and D inter-subtype recombinant structure but the C-terminal half was entirely derived from the consensus B subtype. Multiple breakpoints in a short stretch of 291 nt encoding the Vpr gene strongly suggest that this region is a potential hot-spot for the formation of inter-subtype recombinants and also highlight the importance of the rapidly evolving HIV-1 epidemic in the north Indian region due to multiple genetic subtypes.

Novel HIV-1 long terminal repeat (LTR) sequences of subtype B and mosaic intersubtype B/C recombinants in North India.

Although the HIV-1 epidemic in India is mainly due to subtype C, other subtypes have also been reported from different parts of India. HIV-1 LTR sequence analysis from six HIV-1 infected individuals from North India was carried out to determine the nature and extent of variations. Four out of six samples formed a unique phylogenetic cluster which was close to subtype B. The other two samples (A3 and S3) turned out to be novel mosaic recombinants showing resemblance to subtypes B, B/C-India and B/C-Myanmar gene segments. All four subtype B LTR samples and the two B/C recombinants showed conserved as well as unique polymorphisms in all of the putative transcription factor binding sites (TFBS). These changes may potentially alter basal as well as Tat-mediated HIV-1 LTR promoter activation. The two recombinants possessed three copies of the NF-κB TFBS as seen with the majority of subtype C and recombinant B/C isolates reported earlier, but the other four non-recombinant B-LTRs showed only two copies of the NF-κB site. This is the first study to show a dominance of unique subtype B-LTRs and strongly suggests that this region could also be a hot spot for the formation of highly complex inter subtype B/C recombinants.

Genetic architecture of HIV-1 genes circulating in north India & their functional implications

This review presents data on genetic and functional analysis of some of the HIV-1 genes derived from HIV-1 infected individuals from north India (Delhi, Punjab and Chandigarh). We found evidence of novel B/C recombinants in HIV-1 LTR region showing relatedness to China/Mynmar with 3 copies of Nfκb sites; B/C/D mosaic genomes for HIV-1 Vpr and novel B/C Tat. We reported appearance of a complex recombinant form CRF_02AG of HIV-1 envelope sequences which is predominantly found in Central/Western Africa. Also one Indian HIV-1 envelope subtype C sequence suggested exclusive CXCR4 co-receptor usage. This extensive recombination, which is observed in about 10 per cent HIV-1 infected individuals in the Vpr genes, resulted in remarkably altered functions when compared with prototype subtype B Vpr. The Vpu C was found to be more potent in causing apoptosis when compared with Vpu B when analyzed for subG1 DNA content. The functional implications of these changes as well as in other genes of HIV-1 are discussed in detail with possible implications for subtype-specific pathogenesis highlighted.

HIV-1 Rev downregulates Tat expression and viral replication via modulation of NAD(P)H:quinine oxidoreductase 1 (NQO1)

UNLABELLED HIV-1 gene expression and replication largely depend on the regulatory proteins Tat and Rev, but it is unclear how the intracellular levels of these viral proteins are regulated after infection. Here we report that HIV-1 Rev causes specific degradation of cytoplasmic Tat, which results in inhibition of HIV-1 replication. The nuclear export signal (NES) region of Rev is crucial for this activity but is not involved in direct interactions with Tat. Rev reduces the levels of ubiquitinated forms of Tat, which have previously been reported to be important for its transcriptional properties. Tat is stabilized in the presence of NAD(P)H quinine oxidoreductase 1 (NQO1), and potent degradation of Tat is induced by dicoumarol, an NQO1 inhibitor. Furthermore, Rev causes specific reduction in the levels of endogenous NQO1. Thus, we propose that Rev is able to induce degradation of Tat indirectly by downregulating NQO1 levels. Our findings have implications in HIV-1 gene expression and latency.

Risk for HIV-1 infection is not associated with Repeat-Region polymorphism in the DC-SIGN neck domain and Novel Genetic DC-SIGN Variants among North Indians

Abstract Background Several genetic factors have been related to HIV-1 resistance, the homozygosity for a mutation in CCR5 gene (CCR5Δ 32 allele) is presently considered the most relevant one. The C-type lectin, DC-SIGN efficiently binds and transmits HIV-1 to susceptible cell in trans thereby augmenting the infection. A potential association of the DC-SIGN neck domain repeats polymorphism and risk of HIV-1 infection is currently under debate. Methods Genetic risk association study was conducted in HIV-1 exposed seronegative (HES; n =50) individuals, HIV-1 seronegative (HSN; n =314) healthy control and HIV-1 infected seropositive patients (HSP; n =190) for polymorphism in neck domain of DC-SIGN gene. The DC-SIGN genotypes were identified by PCR from DNA extracted from peripheral blood and confirmed by sequencing. Fisher exact or χ2 test was used for statistical analysis. Results One HSN and HSP individual who were heterozygous (7/8) with respect to DC-SIGN repeat regions were found. The DC-SIGN neck repeat polymorphism among North Indian individuals was not associated with susceptibility to HIV-1 infection. Furthermore, inheritance study of heterozygous mutation (7/8) in HSN individuals family showed that one parent, two brothers, one sister and one daughter were heterozygous (7/8) for DC-SIGN mutant allele. Sequence analyses of DC-SIGN exon 4 repeat region of randomly selected 25 North Indian individuals from HSP, HSN and HES revealed four conserved intronic mutations. These mutations were at nucleotide position 1283, 1306, 1308 upstream and 1906 downstream of the DC-SIGN exon 4 repeat region when compared with the wild type sequence (NCBI Acc. No. AF209479). Conclusion The polymorphism in DC-SIGN neck repeats region was rare and not associated with HIV-1 susceptibility among North Indians. Sequencing analysis of DC-SIGN gene confirmed four novel genetic variants in intronic region flanking exon 4 coding region.