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Research Interests

Research Interests

The primary research focus of the Sharma laboratory is to study how pathogens like retroviruses regulate multiple steps of their replication cycle by interfacing with host cell biology. Ongoing research in our lab is characterizing the retrovirus-host interactions in the non-human primate models of HIV-1 infection. We study the replication of chimeric HIV/SIV viruses (SHIVs) in the macaque model of HIV-1 infection. We are also using our expertise and molecular tools from HIV-1 Env-CD4 research to identify peptides and small-molecule inhibitors of SARS-CoV-2 entry.

Description of the research projects:

1. Determinants of SHIV replication. The host type-1 interferon (IFN) response is the first line of defense against viral infections. IFN upregulates expression of a number of IFN-stimulated genes (ISGs) and the proteins encoded by certain ISGs, referred to as restriction factors, potently block HIV-1 and SIV replication. HIV-1 infection in humans and SIV infection in macaques induces a robust IFN response in vivo. Despite the induction of the IFN response, HIV-1 and SIV replication persists in their respective natural hosts. This is because restriction factors are less active against wild-type viruses replicating in their natural host due to virally encoded evasion/escape mechanisms but act as potent barriers against cross-species transmission.

HIV-1 does not persistently infect macaques due to restriction by several macaque-specific restriction factors. To overcome these restrictions chimeric SIV/HIV-1 viruses (SHIVs), which encode the SIV antagonists of the known restriction factors and HIV-1 Envelope glycoprotein (Env) to permit viral entry, are used to infect macaques to model HIV-1 infection. Existing SHIV/macaque models typically employ SHIVs that encode HIV-1 variants isolated from chronic stages of infection that were further adapted by viral passage in cell culture (lab-adapted viruses). Moreover, these SHIVs require further adaptation in vitro in macaque cells and/or in vivo by serial macaque-passage. The adaptation of env sequences in macaques increases replication and pathogenicity of SHIVs but also leads to antigenic changes in Env that limit their utility for vaccine and therapeutic approaches. Thus, SHIVs encoding HIV-1 variants isolated directly from infected humans near the time of transmission without any adaptation (unadapted SHIVs) that maintain the antigenic properties of the transmitted variants are desired as challenge viruses for vaccine and therapeutic studies. However, most attempts at generating these SHIVs have failed as unadapted SHIVs replicate poorly in macaque cells and do not establish persistent infection.

The research in our lab is characterizing the viral and host determinants that restrict replication of unadapted SHIVs in macaque cells. Thus, our lab is tackling a fundamental weakness in the HIV-1/AIDS field that affects all downstream aspects of HIV-1 pathogenesis, vaccine, and prevention research by developing more relevant SHIV/macaque models of HIV-1 infection.

Projects in the Sharma lab are focused on answering the following questions:

  1.     What are the viral and host factors that drive selection, adaptation, and pathogenicity of SHIVs in macaque cells?
  2.     What is the molecular and mechanistic basis of adaptation of SHIVs to the macaque immune responses?
  3.     Can the molecular mechanisms of SHIV replication in macaque cells be exploited to rationally engineer SHIVs for HIV-1 vaccine and prevention research?
  4.     Can the genetic repertoire of non-human primate species be explored to identify novel innate immune factors that limit the cross-species transmission of lentiviruses?

2. SARS-CoV-2 Entry Inhibitors: Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is a novel and highly pathogenic coronavirus and is the causative agent of the coronavirus disease 2019 (COVID-19), an ongoing pandemic that has posed a serious threat to public health and global economy. The high morbidity and mortality associated with COVID-19 and the severe bottlenecks in vaccine production and distribution underscores the urgent need for developing effective antiviral therapies. Therapeutics that target essential viral proteins are effective at controlling virus spread and replication. Similar to all coronaviruses, SARS-CoV-2 virions display the characteristic club-shaped projections formed by trimers of viral Spike glycoprotein on their surface. To invade the host cell, the receptor-binding domain (RBD) of viral Spike protein binds to the host cell’s angiotensin-converting enzyme 2 (ACE2) receptor, followed by cleavage events that allow the viral Spike protein to fuse with the host cell membrane. Thus, the essential role of Spike protein in ACE2 receptor binding and viral fusion makes it a prime target for therapeutic interventions.

We rationally designed a panel of ACE2-derived peptides based on the RBD-ACE2 binding interfaces of SARS-CoV-2 and SARS-CoV. Using SARS-CoV-2 and SARS-CoV Spike-pseudotyped viruses, we found that a subset of peptides inhibited Spike-mediated infection with IC50 values in the low millimolar range. We identified two peptides that bound Spike RBD in affinity precipitation assays and inhibited infection with genuine SARS-CoV-2. Moreover, these peptides inhibited the replication of a common cold causing coronavirus, HCoV-NL63, which also uses ACE2 as its entry receptor. Results from the infection experiments and structural modeling of the peptides with Spike RBD identified a six amino acid (Glu37-Gln42) ACE2 motif that is important for SARS-CoV-2 inhibition. Importantly, our work provided proof-of-principle and demonstrated feasibility of inhibiting SARS-CoV-2 infection with peptide-based inhibitors.

We are building on our proof-of-principle study to develop therapeutic leads with inhibitory profiles in the nanomolar range. Using structure modeling and molecular dynamic simulation approaches, we have characterized the binding mode of our ACE2-derived peptides with the SARS-CoV-2 Spike RBD and have identified the dominant contacts between them. Importantly, these approaches have resulted in discovery of a novel “druggable” target on SARS-CoV-2 Spike protein. We are exploring the druggability of this cavity to block the critical interactions between ACE2 and Spike RBD, thereby inhibiting SARS-CoV-2 infection.