Research Interests

Over the years, we have focused on studying the molecular steps involved in the replication of retroviruses, including genomic RNA (gRNA) packaging, dimerization, and export using a number of different retroviruses, including human, simian, and feline immunodeficiency viruses (HIV, SIV, & FIV), Mason-Pfizer monkey virus (MPMV), and mouse mammary tumor virus (MMTV).  The overall goal of these efforts has been: 1) enhancement of our understanding of retrovirus replication & regulation of gene expression, 2) improvements in the design and development of retroviral/lentiviral vectors and packaging cells lines for safe and efficient human gene therapy, 3) testing of novel and classical vaccines and antivirals against HIV/AIDS, and finally, 4) development of innovative methods to detect, quantify, & identify viruses and other biological materials based on electrical parameters.  Most recently, with the ensuing COVID-19 pandemic, we have started applying the expertise learned from retroviruses to determine how we can expedite the study of SARS-CoV-2 replication, the etiological agent of COVID-19, with the hope of identifying therapeutic agents for this devastating pandemic. A summary of the research undertaken over the years is provided below:

1. Structural basis of retroviral gRNA export, dimerization, and packaging: The overall goal of these research efforts has been to gain a better understanding of gRNA export, dimerization, and packaging mechanisms and their interplay during retroviral replication.  Genomic RNA packaging is a hallmark of retroviral life cycle where two copies of the gRNA, most likely in their dimeric form, are encapsidated into the virus particles.  The specificity towards gRNA packaging is conferred by the recognition of specific cis-acting sequences, the packaging signal (?), present at the 5’end of the viral genome (that assumes a higher order structure), which interacts with the nucleocapsid (NC) domain of the Gag protein.  It has also been proposed that dimerization leads to conformational changes, exposing single-stranded NC binding motifs (generally purine-rich sequence), facilitating Gag binding during recruitment of the gRNA for packaging. Using a combination of in vivo genetic complementation assays, in vitro biochemical probing/mapping-SHAPE, and structural prediction/phylogenetic approaches, we have mapped the structural determinants of gRNA packaging and dimerization in SIV, FIV, MMTV, and MPMV as well as cross/co-packaging among retroviruses.  We are taking this work to the next level by identifying Gag binding sites on the gRNA which has already accomplished for MMTV and MPMV, while work is in progress for FIV and SIV.  This has been accomplished by expressing full-length Gag proteins from these retroviruses and testing their RNA binding potential combined with footprinting assays based on SHAPE.  Expression of full-length retroviral Gag is a feat in itself that so far has only been achieved for HIV-1 due to the toxic nature of these proteins.  Our ongoing research efforts are directed towards delineating Gag-gRNA interactions that distinguish gRNA substrates for dimerization and subsequent RNA packaging from those for Gag/Pol translation.

2. Molecular mechanism(s) of SARS-CoV-2 genomic RNA (gRNA) packaging : SARS-CoV-2 causes the current COVID-19 pandemic against which there are no approved drugs and the recently produced vaccines are facing challenges of waning immunity and reduced effectiveness against the emerging variants.  Understanding the replication process of SARS-CoV-2 can help us develop novel therapeutics and better vaccines against COVID-19.  How SARS-CoV-2 “recognizes” and “incorporates” its RNA genome (gRNA) into the newly assembling/forming viral particles is crucial for understanding and thereby controlling virus transmission.  Viral particle formation involves recognition and interaction of sequences constituting the “packaging signal” (PS) on the gRNA with the nucleocapsid (N) protein of the virus, resulting in gRNA packaging/incorporation into the virus particles.  However, not much is known about how SARS-CoV-2) packages its gRNA, a crucial aspect to continue its life cycle.  We are working towards defining the initial recognition of gRNA by N protein employing a combination of in vitro biochemical, structural, and in vivo genetic approaches.  Briefly we are working towards: 1) expressing/purifying N protein and its different domains, 2) mapping sequences responsible for gRNA packaging and their binding to N protein, 3) characterizing N protein binding sites in the context of the secondary structure of the packaging signal, and 4) establishing the biological correlation between N protein binding site(s) and gRNA packaging.  These approaches should precisely map SARS-CoV-2 packaging signal, define and validate its higher order structure, and identify specific nucleotides that interact with N protein during gRNA packaging.  Furthermore, these studies should pave way for high resolution structural analysis of the N protein-RNA interaction(s), which can be used for rational drug design as therapeutic interventions against SARS-CoV-2.

3. Development of retroviral vectors for human gene therapy: One practical application of studying retroviral replication has been the development of retroviral vectors for human gene therapy. Thus, we have been successful in creating split-genome viral replication assays not only for HIV-1 and SIV, but also FIV, MMTV, and MPMV.  These assays have allowed us to not only study retrovirus replication in a highly sensitive and specific manner, but also allow transduction of human cells with marker genes successfully without the need for biosafety level 3 (BSL-3) facilities.  Lessons learned from these studies are now being used to develop similar assays for SARS-CoV-2 to allow study of its replication in a safe, sensitive, and quantitative manner under BSL-2 conditions.

4. Vaccine/antiviral strategies against HIV/AIDS Over the years our laboratory has been involved towards facilitating the development of vaccine and novel antiviral strategies against HIV/AIDS:  These strategies have used either the more novel DNA-based approach, in collaboration with Prof. H. L. Robinson (Emory University), the guru of DNA vaccines, or the more classic passive immunization approach, in collaboration with Prof. R. M. Ruprecht, (Harvard University).  In both cases, the simian-human (SHIV)-monkey model system was used for these studies.  The passive immunization approach was used in neonatal monkeys (macaques) using human monoclonal antibodies to study protection against mother-to-child transmission and oral challenge, while the DNA vaccines were tested in combination with protein boost to study the development of protective neutralizing antibody response against challenge with live virus.  These studies have led to seminal contributions towards the development of vaccines as potential therapeutics against HIV/AIDS.  In addition, our laboratory has also been involved in the development of novel antiviral agents from the virus itself by exploiting its replicative biology.

5. Development of techniques for the detection, quantification, and identification of viruses and other biological materials based on electrical parameters: Most of the existing techniques for viral screening and quantification suffer from limitations due to the need for extensive sample preparation and labeling, which is fairly costly, requiring a great deal of time.  Therefore, in collaboration with Dr. Mahmoud Al Ahmad (Department of Electrical Engineering, UAE University), our laboratory has been working towards developing novel label-free virus screening and quantification techniques based on electrical parameters.  These studies have demonstrated that viruses can in fact be detected, quantified, and identified within minutes in tissue culture medium without the need for any protein labeling or amplification strategies and have resulted in several pivotal publications in this area.  More recently, we have expanded the scope of these studies by characterizing other biological molecules such as nucleotides (A, C, T, G) and DNA, virus-expressing cells, cancer cells, as well as blood cells in urine based on their electrical properties.  We anticipate that our electrical approach is just a starting point towards establishing the foundation for the electrical-based identification and quantification of unlimited number of biological materials, and nano-sized particles, including SARS-CoV-2.