Phalguni Gupta, PhD

Molecular Mechanism of Sexual Transmission of HIV: A novel in vitro cervical tissue based organ culture has been developed to study heterosexual transmission of HIV-1. This model system allows the maintenance of the natural architecture of the tissue in the organ culture as evidenced by histology and quantitative immunostaining of immune and non-immune cellular proteins. Infectious virus is found to be transmitted from cell-free as well as cell-associated HIV-1 across the mucosal barrier. Using simultaneous in situ hybridization and immunophenotyping techniques, HIV-1 expressing cells are detected to the basal layer of the epithelium and identified as being activated CD4+ T lymphocytes and CD14+ macrophages. The model is being used to determine mechanism of sexual transmission of HIV-1, especially in elucidating viral and cellular factors that are involved in transmission through epithelial layer of cervical mucosa.

Development of anti-HIV Microbicides: An U19 Cooperative Agreement grant has been funded by NIH to perform studies of preclinical optimization of an antimicrobial peptide, retrocyclin in combination with and a nonnucloside reverse transcriptase inhibitor 5-chloro-3-phenylsulfonylindole-2-carboxamide (CSIC) to block HIV transmission and be non-toxic to human. Our multidisciplinary approach to microbicide development targets two different sites of HIV-1 replication (entry and reverse transcription) through a controlled-release ring formulated product or a self dissolving film formulation, utilizes a novel ex vivo organ culture to test toxicity, inflammation and antiviral activity across cervicovaginal mucosa, and applies macaque models to study toxicity and efficacy. In designing the scope of the proposed topical microbicide program, we have focused on extensively evaluating safety and toxicity of candidate microbicides utilizing in vitro studies, tissue explants and animal models, as well as an expanded and innovative organ culture model to study the interaction of HIV-1 and common sexually transmitted infections (STI) in assessing antiviral activity of microbicides in the presence and absence of STI. Therefore, the proposed study will provide new tools and strategies for the prevention of HIV-1 transmission and their sequelae for women. 

Origin and Dynamics of HIV-1 in Semen. We have demonstrated the presence of high levels of HIV-1 in semen of infected subjects at all stages of the disease, but in most subjects HIV-1 present in semen is different than those present in blood. In addition, HIV-1 is further sum compartmentalized between seminal cells and seminal fluid. By comparing sequences of HIV-1 in semen and male genital organ tissues, it seems that prostate is the origin of HIV-1 present in seminal fluid, Testis and rete testis are probably source of seminal cell associated HIV-1. Furthermore, studies are being conducted to determine the dynamics of HIV in semen and blood by measuring the viral decay in these two body compartments after antiretroviral therapy. In addition, studies are being conducted to elucidate the mechanism of sexual transmission of HIV-1 by comparing HIV-1 sequences in donor semen and blood in the female recipients and the effect of therapy on such transmission.

International Studies in India and China: Our laboratory is also involved in International study of HIV infection in India and China. Our previous genetic studies of HIV-1 circulating at different parts of India indicate that the subtype C predominates in India with a small proportion of infection caused by subtype A or B. In investigating the mechanism for this asymmetric distribution of HIV subtypes in India we found that subtype C is more replication fit and has higher transmission efficiency across cervical mucosa than subtype A. Recently we expanded our work in India by characterizing HIV-1 in semen in India. We are investigating whether HIV in semen is the same as that in blood and effect of therapy on viral load in semen and blood in HIV-infected subjects from India.

During the last 3 years we have established a collaborative study with Investigators at You An Hospital at Beijing, China to characterize HIV circulating among Chinese blood donors in central China for more than 10 years. Sequence analysis of HIV-1 env gene revealed that regardless of the stage of disease, all the HIV-1 patient’s belonged to Clade B subtype. However, the env sequences from AIDS patients clustered differently in the phylogenetic tree than those present in asymptomatic patients and showed a significantly higher divergence and lower diversity compared to those from asymptomatic patients. We have also analyzed nef and vpr sequences. We have isolated HIV-1 from Chinese subjects and characterized the genetic and biologic properties of these HIV isolates obtained at different stages of the disease. Virus isolates from asymptomatic virus grow slowly, are non-cytopathic and uses mostly CCR5 co-receptor. In contrast, isolates from patients with AIDS grow rapidly, highly cytopathic and uses both CCCR5 and CXCR4 co-receptors. Recently we are studying a group of Chinese subjects that developed AIDS within 2 years. Biologic and genetic approaches are being done to understand host and viral factors responsible for these rapid progressions in China.

Nonlytic CD8+ Mediated HIV Suppression: A number of studies are being conducted to investigate the mechanism of nonlytic CD8+ T cell mediated suppression of HIV-1 and SIV at the cellular and molecular level, and to determine its role in HIV-1 and SIV pathogenesis. To date, the precise cellular and molecular determinants mediating this CD8+ T cell effector function remain unsolved. Recently using a CD8+ T cell line displaying potent noncytotoxic HIV-1 suppression activity, we have identified a membrane-localized HIV-1 suppressing activity that is concomitantly secreted as 30-100nm sized endosome-derived tetraspanin-rich vesicles known as exosomes. Purified exosomes from CD8+ T cell culture supernatant noncytotoxically suppressed CCR5-tropic (R5) and CXCR4-tropic (X4) replication of HIV-1 in vitro through a protein moiety. The antiviral exosomes specifically inhibited HIV-1 transcription in both acute and chronic models of infection. Our results, for the first time, indicate the existence of an antiviral membrane-bound protein factor consistent with the hallmarks defining noncytotoxic CD8+ T cell suppression of HIV-1. Our recent data indicate that this HIV suppressive factor works by binding to target cell surface and transmitting an intracellular signal to repress transcription of HIV-1. Studies are underway to determine the mechanism of suppressive factor induced intracellular signaling pathway.

A novel cellular antiviral factor. Adenosine Deaminase acting on RNA 1(ADAR1) ADAR1 is a RNA editing enzyme which acts on complete or partially double-stranded RNA. Since HIV-1 RNA has such secondary structures, we have examined whether ADAR1 exhibits antiviral activity against HIV-1. Our results indicate that ADAR1 inhibits viral replication and infectious HIV-1 production in various cells including 293T, HeLa and Jurkat T cells, and is active against a number of X4 and R5 HIV-1 of different clades.  Analysis of the level of intracellular HIV-1 RNA shows no change in levels of intracellular Gag and Env RNA in the presence of ADAR1 in spite of a significant inhibition of intracellular and virion associated HIV-1 protein production. Furthermore, our data indicate that ADAR1 introduces A-> G mutations in Rev Response Element (RRE) binding and nuclear export signal (NES) regions of HIV-1 Rev RNA and env RNA. Studies are being done to understand the mechanism of ADAR1 induced antiviral activity.

Lab Personnel

Lori Caruso, Lab Technician

Yue Chen, Research Assistant Professor

Ming Ding, PCR Lab Technician

Deena Ratner, Lab Technician

Mary White, Lab Technician

Soni Sankapal, Student

Anwesha Sanyal, Student

Chengli Shen, Visiting Assistant Professor