Suvankar Ghorai

Genome segment 6 of Antheraea mylitta cypovirus encodes a structural protein with ATPase activity

The genome segment 6 (S6) of the 11 double stranded RNA genomes from Antheraea mylitta cypovirus was converted into cDNA, cloned and sequenced. S6 consisted of 1944 nucleotides with an ORF of 607 amino acids and could encode a protein of 68 kDa, termed P68. Motif scan and molecular docking analysis of P68 showed the presence of two cystathionine beta synthase (CBS) domains and ATP binding sites. The ORF of AmCPV S6 was expressed in E. coli as His-tag fusion protein and polyclonal antibody was raised. Immunoblot analysis of virus infected gut cells and purified polyhedra using raised anti-p68 polyclonal antibody showed that S6 encodes a viral structural protein. Fluorescence and ATPase assay of soluble P68 produced in Sf-9 cells via baculovirus expression system showed its ability to bind and cleave ATP. These results suggest that P68 may bind viral RNA through CBS domains and help in replication and transcription through ATP binding and hydrolysis.

Fitness-balanced escape determines resolution of dynamic founder virus escape processes in HIV-1 infection

ABSTRACT To understand the interplay between host cytotoxic T-lymphocyte (CTL) responses and the mechanisms by which HIV-1 evades them, we studied viral evolutionary patterns associated with host CTL responses in six linked transmission pairs. HIV-1 sequences corresponding to full-length p17 and p24 gag were generated by 454 pyrosequencing for all pairs near the time of transmission, and seroconverting partners were followed for a median of 847 days postinfection. T-cell responses were screened by gamma interferon/interleukin-2 (IFN-γ/IL-2) FluoroSpot using autologous peptide sets reflecting any Gag variant present in at least 5% of sequence reads in the individuals viral population. While we found little evidence for the occurrence of CTL reversions, CTL escape processes were found to be highly dynamic, with multiple epitope variants emerging simultaneously. We found a correlation between epitope entropy and the number of epitope variants per response (r = 0.43; P = 0.05). In cases in which multiple escape mutations developed within a targeted epitope, a variant with no fitness cost became fixed in the viral population. When multiple mutations within an epitope achieved fitness-balanced escape, these escape mutants were each maintained in the viral population. Additional mutations found to confer escape but undetected in viral populations incurred high fitness costs, suggesting that functional constraints limit the available sites tolerable to escape mutations. These results further our understanding of the impact of CTL escape and reversion from the founder virus in HIV infection and contribute to the identification of immunogenic Gag regions most vulnerable to a targeted T-cell attack. IMPORTANCE Rapid diversification of the viral population is a hallmark of HIV-1 infection, and understanding the selective forces driving the emergence of viral variants can provide critical insight into the interplay between host immune responses and viral evolution. We used deep sequencing to comprehensively follow viral evolution over time in six linked HIV transmission pairs. We then mapped T-cell responses to explore if mutations arose due to adaption to the host and found that escape processes were often highly dynamic, with multiple mutations arising within targeted epitopes. When we explored the impact of these mutations on replicative capacity, we found that dynamic escape processes only resolve with the selection of mutations that conferred escape with no fitness cost to the virus. These results provide further understanding of the complicated viral-host interactions that occur during early HIV-1 infection and may help inform the design of future vaccine immunogens.

Molecular characterization of genome segment 2 encoding RNA dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus

Abstract Genome segment 2 (S2) from Antheraea mylitta cypovirus (AmCPV) was converted into cDNA, cloned and sequenced. S2 consisted of 3798 nucleotides with a long ORF encoding a 1116 amino acid long protein (123kDa). BLAST and phylogenetic analysis showed 29% sequence identity and close relatedness of AmCPV S2 with RNA dependent RNA polymerase (RdRp) of other insect cypoviruses, suggesting a common origin of all insect cypoviruses. The ORF of S2 was expressed as 123kDa soluble His-tagged fusion protein in insect cells via baculovirus recombinants which exhibited RdRp activity in an in vitro RNA polymerase assay without any intrinsic terminal transferase activity. Maximum activity was observed at 37°C at pH 6.0 in the presence of 3mM MgCl2. Site directed mutagenesis confirmed the importance of the conserved GDD motif. This is the first report of functional characterization of a cypoviral RdRp which may lead to the development of anti-viral agents.

Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylitta cytoplasmic polyhedrosis virus

BackgroundAntheraea mylitta cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of Reoviridae family, infects Indian non-mulberry silkworm, Antheraea mylitta, and contains 11 segmented double stranded RNA (S1-S11) in its genome. Some of its genome segments (S2 and S6-S11) have been previously characterized but genome segments encoding viral capsid have not been characterized.ResultsIn this study genome segments 1 (S1) and 3 (S3) of AmCPV were converted to cDNA, cloned and sequenced. S1 consisted of 3852 nucleotides, with one long ORF of 3735 nucleotides and could encode a protein of 1245 amino acids with molecular mass of ~141 kDa. Similarly, S3 consisted of 3784 nucleotides having a long ORF of 3630 nucleotides and could encode a protein of 1210 amino acids with molecular mass of ~137 kDa. BLAST analysis showed 20-22% homology of S1 and S3 sequence with spike and capsid proteins, respectively, of other closely related cypoviruses like Bombyx mori CPV (BmCPV), Lymantria dispar CPV (LdCPV), and Dendrolimus punctatus CPV (DpCPV). The ORFs of S1 and S3 were expressed as 141 kDa and 137 kDa insoluble His-tagged fusion proteins, respectively, in Escherichia coli M15 cells via pQE-30 vector, purified through Ni-NTA chromatography and polyclonal antibodies were raised. Immunoblot analysis of purified polyhedra, virion particles and virus infected mid-gut cells with the raised anti-p137 and anti-p141 antibodies showed specific immunoreactive bands and suggest that S1 and S3 may code for viral structural proteins. Expression of S1 and S3 ORFs in insect cells via baculovirus recombinants showed to produce viral like particles (VLPs) by transmission electron microscopy. Immunogold staining showed that S3 encoded proteins self assembled to form viral outer capsid and VLPs maintained their stability at different pH in presence of S1 encoded protein.ConclusionOur results of cloning, sequencing and functional analysis of AmCPV S1 and S3 indicate that S3 encoded viral structural proteins can self assemble to form viral outer capsid and S1 encoded protein remains associated with it as inner capsid to maintain the stability. Further studies will help to understand the molecular mechanism of capsid formation during cypovirus replication.

Nuclear matrix protein SMAR1 control regulatory T-cell fate during inflammatory bowel disease (IBD).

Regulatory T (Treg) cells are essential for self-tolerance and immune homeostasis. Transcription factor Foxp3, a positive regulator of Treg cell differentiation, has been studied to some extent. Signal transducer and activator of transcription factor 3 (STAT3) is known to negatively regulate Foxp3. It is not clear how STAT3 is regulated during Treg differentiation. We show that SMAR1, a known transcription factor and tumor suppressor, is directly involved in maintaining Treg cell fate decision. T-cell-specific conditional knockdown of SMAR1 exhibits increased susceptibility towards inflammatory disorders, such as colitis. The suppressive function of Treg cells is compromised in the absence of SMAR1 leading to increased T helper type 17 (Th17) differentiation and inflammation. Compared with wild-type, the SMAR1−/− Treg cells showed increased susceptibility of inflammatory bowel disease in Rag1−/− mice, indicating the role of SMAR1 in compromising Treg cell differentiation resulting in severe colitis. We show that SMAR1 negatively regulate STAT3 expression favoring Foxp3 expression and Treg cell differentiation. SMAR1 binds to the MAR element of STAT3 promoter, present adjacent to interleukin-6 response elements. Thus Foxp3, a major driver of Treg cell differentiation, is regulated by SMAR1 via STAT3 and a fine-tune balance between Treg and Th17 phenotype is maintained.

Analysis of Transcripts Expressed in One-Day-Old Larvae and Fifth Instar Silk Glands of Tasar Silkworm, Antheraea mylitta

Antheraea mylitta is one of the wild nonmulberry silkworms, which produces tasar silk. An EST project has been undertaken to understand the gene expression profile of A. mylitta silk gland. Two cDNA libraries, one from the whole bodies of one-day-old larvae and the other from the silkglands of fifth instar larvae, were constructed and sequenced. A total of 2476 good-quality ESTs (1239 clones) were obtained and grouped into 648 clusters containing 390 contigs and 258 singletons to represent 467 potential unigenes. Forty-five sequences contained putative coding region, and represented potentially novel genes. Among the 648 clusters, 241 were categorized according to Gene Ontology hierarchy and showed presence of several silk and immune-related genes. The A. mylitta ESTs have been organized into a freely available online database “AmyBASE”. These data provide an initial insight into the A. mylitta transcriptome and help to understand the molecular mechanism of silk protein production in a Lepidopteran species.

Oxidant mediated one-step complete conversion of multi-walled carbon nanotubes to graphene quantum dots and their bioactivity against mammalian and bacterial cells

It is essential for any antibacterial agent (for clinical applications) that it should have high and selective toxicity towards bacterial cells only, and should not affect the human cells at the concentration used. Graphene quantum dots (GQDs) have emerged as a potential candidate for biomedical applications. However, a simple, low cost, safe, easy to execute, one-step synthesis of uniform and monodispersed GQDs with selective toxicity towards bacterial cells rather than mammalian cells is difficult to achieve. Herein, we have reported a one-step, low-cost, aqueous-phase, simple approach for the complete conversion of multi-walled carbon nanotubes into water-dispersible GQDs with an average size of ∼3 nm using sodium bismuthate (NaBiO3) as a strong oxidant. The cyclic voltammetry and X-ray photoelectron spectroscopy results indicated that the as-synthesized GQDs suspension possess almost negligible amounts of metallic impurities. The cytotoxicity studies of GQDs against mammalian NIH 3T3 (mouse embryo fibroblast cells) and HEK 293T (human embryonic kidney cells) cells showed that the as-synthesized GQDs were non-cytotoxic up to the concentration of ∼200 μg mL-1. The antimicrobial study shows that the synthesized GQDs have high and selective toxicity towards bacterial cells with a minimum inhibitory concentration of ∼256 μg mL-1 for E. coli and B. subtilis and ∼512 μg mL-1 for P. aeruginosa and S. aureus. The scanning electron microscopy and atomic force microscopy images show extensive cell damage via the perturbation of bacterial cell walls, which was consistent with the enhancement of reactive oxygen species production by almost two times in the bacterial cells upon incubation with ∼256 μg mL-1 GQDs. Our study suggested that the as-synthesized GQDs can be used as a potential candidate for clinical applications as they possess high toxicity to bacterial cells and low toxicity to mammalian cells.

Cdc20 directs proteasome-mediated degradation of the tumor suppressor SMAR1 in higher grades of cancer through the anaphase promoting complex

The Tumor suppressor SMAR1 (scaffold matrix attachment region binding protein 1) has a crucial role in maintaining genomic stability, cell cycle progression and apoptosis.Our previous finding showed that it is highly suppressed in higher grade of cancer. However, the underlying mechanism of this suppression was not well understood. In this study, we show that SMAR1 expression levels are controlled at the proteasomal level by five RING finger E3 ubiquitin ligases including, Cdc20, a substrate receptor of ubiquitin ligase APC/C complex. We found that Cdc20 binds and promotes proteasomal degradation of SMAR1 in a D-box motif dependent manner. Further, our results demonstrated that Cdc20 promotes proteasomal degradation of SMAR1 through K48-linked specific polyubiquitylation, and that short hairpin RNA mediated inactivation of Cdc20 leads to significant stabilization of SMAR1. These findings suggest that Cdc20 is responsible for maintaining the cellular levels of SMAR1. However, since Cdc20 fails to target SMAR1 upon exposure to genotoxic stresses, SMAR1 helps to maintain genomic stability under these conditions through its DNA damage repair activity. Interestingly, Cdc20-mediated degradation of SMAR1 promotes cell migration and invasion.The reciprocal relationship of the duo is evident in breast cancer cell lines as well as in patient samples, suggesting that Cdc20 functions as an important negative regulator of SMAR1 in higher grades of cancer. Our study reveals for the first time, the molecular mechanism associated with lower levels of expression of the important tumor suppressor SMAR1 in higher grades of breast cancer.

Patterns and rates of viral evolution in HIV-1 subtype B infected females and males

Biological sex differences affect the course of HIV infection, with untreated women having lower viral loads compared to their male counterparts but, for a given viral load, women have a higher rate of progression to AIDS. However, the vast majority of data on viral evolution, a process that is clearly impacted by host immunity and could be impacted by sex differences, has been derived from men. We conducted an intensive analysis of HIV-1 gag and env-gp120 evolution taken over the first 6–11 years of infection from 8 Women’s Interagency HIV Study (WIHS) participants who had not received combination antiretroviral therapy (ART). This was compared to similar data previously collected from men, with both groups infected with HIV-1 subtype B. Early virus populations in men and women were generally homogenous with no differences in diversity between sexes. No differences in ensuing nucleotide substitution rates were found between the female and male cohorts studied herein. As previously reported for men, time to peak diversity in env-gp120 in women was positively associated with time to CD4+ cell count below 200 (P = 0.017), and the number of predicted N-linked glycosylation sites generally increased over time, followed by a plateau or decline, with the majority of changes localized to the V1-V2 region. These findings strongly suggest that the sex differences in HIV-1 disease progression attributed to immune system composition and sensitivities are not revealed by, nor do they impact, global patterns of viral evolution, the latter of which proceeds similarly in women and men.

p21WAF1/CIP1 RNA Expression in Highly HIV-1 Exposed, Uninfected Individuals

Some individuals remain HIV-1 antibody and PCR negative after repeated exposures to the virus, and are referred to as HIV-exposed seronegatives (HESN). However, the causes of resistance to HIV-1 infection in cases other than those with a homozygous CCR5Δ32 deletion are unclear. We hypothesized that human p21WAF1/CIP1 (a cyclin-dependent kinase inhibitor) could play a role in resistance to HIV-1 infection in HESN, as p21 expression has been associated with suppression of HIV-1 in elite controllers and reported to block HIV-1 integration in cell culture. We measured p21 RNA expression in PBMC from 40 HESN and 40 low exposure HIV-1 seroconverters (LESC) prior to their infection using a real-time PCR assay. Comparing the 20 HESN with the highest exposure risk (median = 111 partners/2.5 years prior to the 20 LESC with the lowest exposure risk (median = 1 partner/2.5 years prior), p21 expression trended higher in HESN in only one of two experiments (P = 0.11 vs. P = 0.80). Additionally, comparison of p21 expression in the top 40 HESN (median = 73 partners/year) and lowest 40 LESC (median = 2 partners/year) showed no difference between the groups (P = 0.84). There was a weak linear trend between risk of infection after exposure and increasing p21 gene expression (R2 = 0.02, P = 0.12), but again only in one experiment. Hence, if p21 expression contributes to the resistance to viral infection in HESN, it likely plays a minor role evident only in those with extremely high levels of exposure to HIV-1.