Scientific Officers: R. Mukhopadhyaya, Ph.D. (Head)

Research Fellows: M.C. Tamhane, M.C. Patel.

Viruses have been implicated in ~15% of malignancies, prominent ones being HPV in cervical cancer and human herpes virus-8 (HHV-8) in Kaposi’s sarcoma, and the relative contribution of other viruses in cancer formation is still under investigation. The Virology laboratory is evaluating the role of HHV-6 and HPV in lymphoma and oesophageal cancers, respectively. Having gained considerable knowledge and expertise in HIV biology, a thrust area now is understanding the biology of HIV-2, which is less studied as the virus has spread only in parts of Africa and in India. Having an Indian isolate of HIV-2 has provided an impetus to develop an indigenous retroviral vector for gene transfer as well as to focus on developing cell lines and biological reagents as research components.

Biology of HHV-6 and its activation in lymphoproliferative disorders

HHV-6 is ubiquitous in distribution, being found in a latent form in the cellular genome of 80-90% of the population. While circulating monocytes and epithelial cells of the bronchus and salivary glands are believed to be reservoirs, the exact sites of viral persistence and latency in vivo are still uncertain. In this study, the sub-cellular distribution of HHV-6 in peripheral blood mononuclear cells (PBMC) of immuno-competent healthy individuals is being evaluated. HHV-6 has been detected in non-adherent as well as adherent cells, and in unfractionated PBMC. In 3 samples, HHV-6 has been detected only in one of the enriched fractions but not in total PBMC, indicating the presence of very low amounts of the virus detectable only after enrichment. Thus, even if the total PBMC is negative by PCR, an individual may harbour HHV-6 in a few cells. Negativity of 3 cases in all the fractions indicates either total absence of HHV-6 or copy numbers that are below the detection limit of the particular PCR. Thus, no generalised pattern has emerged of any particular cell type/s in the PBMC compartment harbouring detectable level of latent HHV-6; both lymphoid and monocytic cells may harbour the virus. The study has also indicated an absolute prevalence of HHV-6 sub type B, and not A, in the Indian population. In future studies, a B cell line with very high copy integration of HHV-6 - developed earlier in this laboratory, will be used as a model to study the replication of HHV-6.

HPV and oesophageal carcinoma

The involvement of HPV has been strongly indicated in oesophageal squamous cell carcinoma. Preliminary screening of 85 endoscopically-excised oesophageal cancer biopsies has revealed the presence of HPV in 67% samples; with a majority having the HPV-16 serotype. An ELISA is being developed to evaluate the anti-HPV antibody status of these and future cases. A prokaryotic clone of HPV-16 L1 region has been made for expressing the HPV coat protein in the yeast system.

Biology of HIV-2 infection

The effect of HIV-1 and HIV-2 infection on NFkB activation is being studied using CRIT2 as a system to show constitutive activation of NFkB and the T cell line Jurkat as a system to show inducible activation of NFkB. Chronically infected CRIT2 cells show apparent down-regulation of NFkB on infection with HIV-1, while HIV-2 does not alter the activation status. Jurkat cells do not show activation of NFkB upon infection with either HIV-1 or HIV-2. To evaluate the NFkB status of cells in a dual infection scenario, CEM/tat cells infected with HIV-1 or HIV-2 have been used to look for NFkB activation by EMSA. CEM/tat cells do not show activation of NFkB on infection with HIV-1 or HIV-2, implying that chronic infection of cells by HIV-2 does not result in activation of NFkB in Jurkat cells or cause any change in NFkB levels in the constitutively expressing cell line, CRIT2. NFkB activation in HIV infection may thus be contextual and a function of various host cell and viral factors, whose interplay may result in different observations in different systems. While investigating the activation profile of NFkB in HIV-2 infection, it is found that 2 indigenously developed cell lines, CRIT2 (a CD4+ T cell line) and PJH6 (an EBV transformed B cell line with very high copy integrated HHV-6), show unusually high constitutive level of NFkB activation. In the light of the recently demonstrated importance of NFkB activation in tumorigenesis, various agents will be used to study NFkB inhibition in these cell lines.

Indigenous HIV-based vector development for gene transfer

It is proposed to develop indigenous HIV-1 / HIV-2 based viral vectors using the Indian isolates propagated in this laboratory. For HIV-1, a 9.2 kb fragment generated by long PCR has been cloned in pCR-TOPO-XL vector, while for HIV-2, the sub-genomic 3.6 kb amplicon of full envelope and 3’ LTR has been cloned in the same vector. This has been attained by amplifying 2 overlapping fragments, cloning them separately and using them in a fusion PCR to obtain the original 3.6 kb fragment encompassing the full envelope and 3’ LTR. A 7.6 kb gag-pol-env (part) fragment has also been cloned in the same vector. These will be used to make the packaging constructs. An HIV-2 LTR-GFP-LTR construct has also been prepared, which is found to act as a reporter on co-transfection with a HIV-2 tat construct in HeLa and 293 cells. Efforts are now on to evaluate, by transduction, if a complete functional vector system based on HIV-2 is ready.