Akhil C. Banerjea

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.

Sequence specific cleavage of the HIV-1 coreceptor CCR5 gene by a hammer-head ribozyme and a DNA-enzyme: inhibition of the coreceptor function by DNA-enzyme

The chemokine receptor CCR5 is used as a major coreceptor for fusion and entry by non‐syncytia inducing macrophage tropic isolates of HIV‐1, which is mainly involved in transmission. Individuals who are homozygous for the Δ32 allele of CCR5 are usually resistant to HIV‐1 infection and continue to lead a normal healthy life. Thus this gene is dispensable and is, therefore, an attractive target in the host cell for interfering specifically with the virus‐host interaction. With the aim to develop a specific antiviral approach at the molecular level, we have synthesized a hammer‐head ribozyme and a DNA‐enzyme. Both ribozyme and DNA‐enzyme cleaved the CCR5 RNA in a sequence specific manner. This cleavage was protein independent but Mg2+ dependent. The extent of cleavage increased with increasing concentration of magnesium chloride. DNA‐enzyme was more effective in cleaving a full length (1376 bases) in vitro generated transcript of CCR5 gene. In this communication, we show that the DNA‐enzyme when introduced into a mammalian cell, results in decreased CD4‐CCR5‐gp160 mediated fusion of cell membranes. Potential applications of these trans acting molecules are discussed.

Molecular Epidemiology of HIV-1 Subtypes in India: Origin and Evolutionary History of the Predominant Subtype C

Background India has the third largest HIV-1 epidemic with 2.4 million infected individuals. Molecular epidemiological analysis has identified the predominance of HIV-1 subtype C (HIV-1C). However, the previous reports have been limited by sample size, and uneven geographical distribution. The introduction of HIV-1C in India remains uncertain due to this lack of structured studies. To fill the gap, we characterised the distribution pattern of HIV-1 subtypes in India based on data collection from nationwide clinical cohorts between 2007 and 2011. We also reconstructed the time to the most recent common ancestor (tMRCA) of the predominant HIV-1C strains. Methodology/Principal Findings Blood samples were collected from 168 HIV-1 seropositive subjects from 7 different states. HIV-1 subtypes were determined using two or three genes, gag, pol, and env using several methods. Bayesian coalescent-based approach was used to reconstruct the time of introduction and population growth patterns of the Indian HIV-1C. For the first time, a high prevalence (10%) of unique recombinant forms (BC and A1C) was observed when two or three genes were used instead of one gene (p<0.01; p = 0.02, respectively). The tMRCA of Indian HIV-1C was estimated using the three viral genes, ranged from 1967 (gag) to 1974 (env). Pol-gene analysis was considered to provide the most reliable estimate [1971, (95% CI: 1965–1976)]. The population growth pattern revealed an initial slow growth phase in the mid-1970s, an exponential phase through the 1980s, and a stationary phase since the early 1990s. Conclusions/Significance The Indian HIV-1C epidemic originated around 40 years ago from a single or few genetically related African lineages, and since then largely evolved independently. The effective population size in the country has been broadly stable since the 1990s. The evolving viral epidemic, as indicated by the increase of recombinant strains, warrants a need for continued molecular surveillance to guide efficient disease intervention strategies.

Inhibition of hepatitis B virus X gene expression by novel DNA enzymes.

Two mono- and a di-RNA-cleaving DNA enzymes with the 10-23 catalytic motif were synthesized that were targeted to cleave at the conserved site/sites of the X gene of the hepatitis B virus. In each case, protein-independent but Mg(2+)-dependent cleavage of in vitro-synthesized full-length X RNA was obtained. Specific cleavage products were obtained with two different mono- and a di-DNA enzyme, with the latter giving rise to multiple RNA fragments that retained the cleavage specificity of the mono-DNA enzymes. A relatively less efficient cleavage was also obtained under simulated physiological conditions by the two mono-DNA enzymes but the efficiency of the di-DNA enzyme was significantly reduced. A single nucleotide change (G to C) in the 10-23 catalytic motif of the DNA enzyme 307 abolished its ability to cleave target RNA completely. Both, mono- and di-DNA enzymes, when introduced into a mammalian cell, showed specific inhibition of X-gene-mediated transactivation of reporter-gene expression. This decrease was due to the ability of these DNA enzymes to cleave X RNA intracellularly, which was also reflected by significant reduction in the levels of X protein in a liver-specific cell line, HepG2. Ribonuclease protection assay confirmed the specific reduction of X RNA in DNA-enzyme-treated cells. Potential in vivo applications of mono- and di-DNA enzymes in interfering specifically with the X-gene-mediated pathology are discussed.

Targeted cleavage of HIV-1 envelope gene by a DNA enzyme and inhibition of HIV-1 envelope-CD4 mediated cell fusion

With the ultimate aim of developing an effective antiviral strategy against HIV‐1, a mono‐DNA enzyme possessing the 10–23 catalytic motif [Santoro and Joyce (1997) Proc. Natl. Acad. Sci. USA 94, 4264–4266] was synthesized against the HIV‐1 envelope gene. We tested the in vitro cleavage efficiency of the 178 bp long truncated HIV‐1 Env transcript by DNA enzyme 6339. Protein independent and Mg2+ dependent specific cleavage products were obtained. As soon as 5 min after mixing equimolar concentrations of DNA enzyme and substrate RNA, more than 50% cleavage was observed which increased steadily over a period of 4 h. Very little cleavage was obtained at 1 mM MgCl2 concentration which improved significantly when the concentration of MgCl2 was increased up to 20 mM. Specific inhibition of cell membrane fusion caused by the interaction of gp160 and CD4 in HeLa cells was observed when the above DNA enzyme was used. Thus, these chemically synthesized DNA enzymes could prove to be very useful for in vivo application.

Inhibition of β-TrcP–dependent ubiquitination of p53 by HIV-1 Vpu promotes p53–mediated apoptosis in human T cells

HIV-1 viral protein U (Vpu) is involved in ubiquitination and degradation of BM stromal cell Ag 2 and surface receptor CD4 through their recruitment to SCF(β-TrcP) (Skp1/Cul1/F-box) ubiquitin ligase (SCF) complex. Here, we show that specific interaction of wild-type Vpu protein with SCF complex leads to inhibition of ubiquitination and proteasomal degradation of p53 protein in a β-TrcP-dependent manner. Successful interaction of SCF(β-TrcP) complex with β-TrcP binding motif (DS(52)GNES(56)) present in Vpu is essential because mutant Vpu possessing specific alanine substitutions (DA(52)GNEA(56)) in the β-TrcP binding motif not only failed to stabilize p53 protein but was also unable to inhibit ubiquitination of p53 protein. Furthermore, Vpu competes efficiently with the interaction of p53 protein with the β-TrcP subunit of the SCF complex and inhibits subsequent ubiquitination of p53 proteins in a dose-dependent manner. We also observed potent apoptotic activity in a p53 null cell line (H-1299) that was cotransfected with p53 and Vpu-expressing plasmids. Furthermore, MOLT-3 (human T-lymphoblast) cells when infected with vesicular stomatitis virus glycoprotein-pseudotypic HIV-1 possessing wild-type vpu gene exhibited maximum activation of p53/Bax proteins and p53-mediated cell death. These findings establish a novel function of Vpu in modulating the stability of p53 protein that correlates positively with apoptosis during late stages of HIV-1 infection.

Immunogenicity and efficacy of single antigen Gp63, polytope and polytopeHSP70 DNA vaccines against visceral Leishmaniasis in experimental mouse model.

Polytope approach of genetic immunization is a promising strategy for the prevention of infectious disease as it is capable of generating effective cell mediated immunity by delivering the T cell epitopes assembled in series. Leishmaniasis is a significant world wide health problem for which no vaccine exists. In this study we have compared immunogenicity and efficacy of three types of DNA vaccines: single antigen Gp63 (Gp63/pcDNA), polytope (Poly/pcDNA) and Polytope fused with hsp70 (Poly/hsp/pcDNA) against visceral leishmaniasis in susceptible BALB/c mice. Mice vaccinated with these plasmids generated strong Th1 immune response as seen by dominating IFN-γ over IL-10 cytokine. Interestingly, cytotoxic responses generated by polytope DNA plasmid fused with hsp70 of Leishmania donovani were significantly higher when compared to polytope and single antigen Gp63 vaccine. Challenge studies revealed that the parasite load in liver and spleen was significantly lower with Poly/hsp/pcDNA vaccination compared to other vaccines. Therefore, our study indicates that polytope DNA vaccine is a feasible, practical and effective approach for visceral leishmaniasis.

Injecting drug users in Bangladesh: prevalence of syphilis hepatitis HIV and HIV subtypes.

Injecting drug users (IDU) were enrolled from two detoxification clinics and two needle/syringe exchange programmes (NEP) in central and northwest Bangladesh. Syphilis, hepatitis C and HIV rates were highest in IDU from the NEP of central Bangladesh (23, 66.5 and 1.4%, respectively), whereas current hepatitis B infection rates were highest in IDU from the NEP of northwest Bangladesh (12%). Five HIV strains were subtype C and one E/B. The 32 base pair (bp) deletion of the CCR5 gene was not detected.

Targeted cleavage of HIV-1 coreceptor-CXCR-4 by RNA-cleaving DNA-enzyme: inhibition of coreceptor function

HIV needs the chemokine receptors (HIV-1 coreceptors) to initiate infection and gain entry into a susceptible cell. CCR5 receptor is used by macrophage tropic viruses to establish infection, and CXCR-4 is used by T lymphocyte tropic virus which are usually found at the terminal stages of the disease. These chemokine receptors are, therefore, attractive targets to interfere with the entry as well as spread of HIV-1 in the host. As our antiviral approach, we have earlier assembled a DNA-enzyme-916 against CCR5 (Goila and Banerjea, 1998). We have now designed against the CXCR-4 gene a mono-DNA-enzyme, which showed sequence specific cleavage activity. When CXCR-4-DNA-enzyme was placed in tandem with CCR5-DNA-enzyme, specific cleavage of their respective target sites were observed using a 60 bases long synthetic target RNA which possessed the target sites for both the DNA-enzymes. The cleavage by the CXCR-4 DNA-enzyme was found to be significantly more efficient than by the CCR5-DNA-enzyme. Analyses of the cleaved fragments by mono- and di-DNA-enzyme indicated strongly that hybridization of the CCR5-DNA-enzyme with its cognate target RNA, actually facilitated the cleavage by the CXCR-4 DNA-enzyme. Furthermore, the di-DNA-enzyme was able to cleave the substrate RNA to completion. These DNA-enzymes, when introduced into a mammalian cell line expressing the appropriate chemokine receptor, interfered specifically with the HIV-1 coreceptor functions. Using this strategy, it may be possible to interfere with the infection and spread of R5 as well as X4 viruses.

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.