Retroviruses
Retrovirus replication is strongly dependent on the cellular machinery to produce progeny virus. Elucidation of the interactions between the viruses and the host cell at the molecular and structural level is important for understanding replication mechanisms and the subsequent cytopathogenesis in the infected cell, which will aid in the development of more efficient antiviral drugs. We are interested in the underlying structural basis by which retroviral proteins interact with cellular constituents during virus replication. A major component of our research program is directed towards understanding key protein-protein and protein-membrane interactions that are critical for virus assembly. The results generated from this research provide new insights into these mechanisms and may identify new attractive targets that will ultimately aid in rational drug design. Retroviruses we are studying:
HIV-1
HIV Assembly:
Human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immune deficiency syndrome (AIDS), is responsible for one of the deadliest viral pandemics in human history. Even though advances in treatment, particularly in antiretroviral therapies (ARTs) and patient care, have drastically reduced the morbidity and mortality of infected individuals worldwide, there are still ~36 million adults and 1.7 million children (<15 years) living with HIV-1 today (UNAIDS report, 2019). While ARTs have been effective in combating the AIDS epidemic, they are not capable of eradicating the virus from the body entirely, requiring patients to undergo indefinite treatments, which can lead to adverse effects including drug toxicity and drug resistant mutations in the viral genome. Because HIV-1 still represents a major risk to human health and safety around the globe, it is vital that efforts continue to focus on new targets within the virus replication cycle. Three-dimensional molecular structures often provide detailed information on biological mechanisms, which significantly aid in the development of therapeutic interventions. The development of new drugs designed to disrupt the viral assembly process could enhance the effectiveness of modern treatment options or overcome issues of drug resistance and toxicity. Research in our lab focuses on the structure and function of Gag polyprotein and its domains and the cytoplasmic domain of the gp41 subunit of Env (gp41CT), both of which are implicated in virus assembly. Detailed molecular characterization of this key step may aid in the development of new therapeutic strategies that inhibit virus assembly and Env incorporation.
Tat Neurotoxicity:
Despite otherwise successful antiretroviral therapy, HIV-associated neurocognitive disorder (HAND) is detected in ~50% of infected individuals. The presence of an HIV-1 reservoir in the brain is considered a contributor to an inflammatory milieu and to the neuronal injury in infected individuals. In the brain, HIV-1 infection is driven mostly by resident microglia cells and infiltrated macrophages. ART is less efficient in the brain tissue because of reduced drug penetration through the blood-brain barrier. Moreover, other than HIV-1 infected T cells that succumb to the cytopathic effect of the virus, microglia/macrophages are resistant to the viral cytopathic effect and can act as long-term producers. Given the long half-life of microglia and the ability of different lineages of macrophages and likely microglia to proliferate, the brain presents itself as a likely reservoir of HIV-1 infection despite ART. In the context of HAND, even if ART were to completely suppress HIV-1 replication, persistently infected microglia/macrophages can actively secrete the HIV-1 transactivator of transcription (Tat) protein, which is known to be particularly toxic for neurons. Tat is known to synergize with drugs of abuse and exacerbate disease progression and severity, further confounding the adverse effects on the central nervous system (CNS). There is mounting evidence that HIV-1 Tat exerts its neurotoxicity through interaction with human dopamine transporter (hDAT) in the CNS. Tat secretion thus presents itself as an attractive target to improve HAND. However, our knowledge of how Tat is secreted by infected cells is limited. Conversely, Tat needs to cross membranes of target cells (such as neurons) to exert its neurotoxic effect. Therefore, inhibition of uptake is a second candidate mechanism to improve HAND. Research in our lab is focused on investigating the molecular rearrangements of Tat upon binding to membranes prior to secretion and during uptake, which may offer a new platform to target the membrane-bound form of Tat, which altogether will lead to the development of new therapeutic strategies for treatment of HAND.
HTLV-1
Human T-cell leukemia virus (HTLV) is a zoonotic virus with simian T-cell leukemia virus counterparts found in monkeys. HTLV-1 and HTLV-2 are the most studied subtypes of HTLV. Even though they share ~ 70% nucleotide identity and have a similar genome structure, HTLV-1 is considered more pathogenic as it is associated with adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 transmission occurs mainly through cell-to-cell contacts rather than free virus particles. In addition, HTLV-1 infected T-cells can multiply by clonal expansion, consequently increasing the viral burden without the need for virus replication and reinfection. In order to develop effective antiretroviral treatments for HTLV-1, it is paramount to gain a more complete understanding of the molecular processes that govern HTLV-1 replication. However, many fundamental aspects of HTLV-1 replication, including particle assembly, are incompletely understood. Projects in our lab are focused on elucidating the mechanism by which the HTLV-1 Gag polyprotein is targeted to the host plasma membrane for assembly.
Coronavirus
The COVID-19 pandemic has run rampant and caused widespread harm across the globe. There have been, thankfully, many breakthroughs in vaccine production and the technology that enabled them. However, it is possible that COVID-19 may operate similarly to the Influenza virus in that mutations may lead to new variants, requiring new vaccine treatments yearly. As a result, scientists are in a hurried state to identify other vaccine candidates and treatment strategies. SARS-CoV-2 is comprised of four primary structural proteins that enable this virus to function properly including the Spike (S), Envelope (E), Matrix (M), and Nucleocapsid (N) proteins. SARS-Cov2 E is a membrane protein that is thought to act form ion channel activity that transverses the ER Golgi Intermediate Complex (ERGIC). The structure and function of the SARS-CoV-2 E protein are not well understood. Our lab is interested in studying the structure and function of the full SARS-CoV-2 E protein and how it interacts with other cellular factors during replication and the viral life-cycle.
Cancer
Fas-Mediated Apoptosis and Mechanisms of Inhibition:
The extrinsic apoptotic pathway is initiated by surface death receptors such as Fas. The long-term goal is to develop new strategies to stimulate Fas/DR5-mediated apoptosis as a potential therapy for many types of cancers. Binding of Fas ligand (FasL) to Fas ectodomain or TRAIL to DR5 is the initial step leading to formation of the death-inducing signaling complex (DISC), subsequent activation of caspases, and ultimately cell death. The DISC is a critical component of the Fas/DR5–mediated apoptotic pathway. FasL binding to Fas or TRAIL to DR5 triggers recruitment of FADD, allowing direct interactions between the death domains (DD) of Fas/DR5 and FADD and release of death effector domain (DED) of FADD to interact with procaspases-8/10 (cas-8/10) via its two tandem DEDs (DED1/2), finalizing an active DISC. c-FLIP, a key anti-apoptotic protein overexpressed in multiple types of tumor cells, is effectively recruited into the DISC. The anti-apoptotic effect of c-FLIP lies in its ability to block the recruitment of cas-8/10 into the DISC by interacting with FADD DED, which is recognized as the gateway to an unfortunate cell survival. Calmodulin (CaM), a calcium-binding protein is also recruited into the DISC and plays a novel regulatory role by interacting with Fas/DR5 DD. In addition to its interaction with Fas/DR5 DD, CaM also binds to c-FLIP to regulate its anti-apoptotic effect and to maintain survival signals under basal conditions. In summary, a significant body of research indicates that the exact same core group of proteins that execute cell death (Fas/DR5, FADD, cas-8/10, c-FLIP, and CaM) also function to promote proliferative and survival signals. However, how these components interact or compete with each other is not known. The overall objectiveis to examine at the structural and cellular level how proteins interact or compete with each other to execute cell death or promote cell survival.
Akt translocation and activation:
One of the most frequent cell survival pathways involves activation of Akt, a serine/threonine kinase that is critical in regulating apoptosis and oncogenesis. Hyperactivation of Akt is a common tumorigenic event in many types of cancers including breast cancer. Akt interferes and inhibits programmed cell death by phosphorylating and thereby inactivating a number of proteins involved in apoptosis. Binding of Akt to the plasma membrane (PM) via direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) is considered a hallmark in the activation pathway and is required for Akt phosphorylation and subsequent initiation of downstream events including inhibition of apoptotic pathways. Additionally, it has been demonstrated that epidermal growth factor (EGF)-induced membrane translocation and activation of Akt in breast cancer cells is modulated by calmodulin (CaM). Interaction of CaM with Akt is mediated by the PH domain. Perturbation of this targeting mechanism led to apoptotic cell death in tumorigenic mammary carcinoma cells. We employ NMR, biochemical, and biophysical techniques to understand how Akt binds to PI(3,4,5)P3 and CaM. Elucidation of the structural details of how Akt binds to the PM and CaM may offer a better understanding of the molecular basis for Akt activation, which may help in the development of new therapeutic strategies against breast and other types of cancer.
