Patel, Trushar

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    Molecular characterization of the RNA-protein complex directing -2/-1 programmed ribosomal frameshifting during arterivirus replilcase expression
    (American Society for Biochemistry and Molecular Biology, 2020) Patel, Ankoor; Treffers, Emmely E.; Meier, Markus; Patel, Trushar R.; Stetefeld, Jörg; Snijder, Eric J.; Mark, Brian L.
    Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs −1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical −1 and −2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate −1 and −2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1β and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1β:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1β within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1β and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1β and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.
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    Host transcription factors in hepatitis B virus RNA synthesis
    (MDPI, 2020) Turton, Kristi L.; Meier-Stephenson, Vanessa; Badmalia, Maulik D.; Coffin, Carla S.; Patel, Trushar R.
    The hepatitis B virus (HBV) chronically infects over 250 million people worldwide and is one of the leading causes of liver cancer and hepatocellular carcinoma. HBV persistence is due in part to the highly stable HBV minichromosome or HBV covalently closed circular DNA (cccDNA) that resides in the nucleus. As HBV replication requires the help of host transcription factors to replicate, focusing on host protein–HBV genome interactions may reveal insights into new drug targets against cccDNA. The structural details on such complexes, however, remain poorly defined. In this review, the current literature regarding host transcription factors’ interactions with HBV cccDNA is discussed.
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    Biodefense implications of new-world hantaviruses
    (Frontiers Media, 2020) D'Souza, Michael H.; Patel, Trushar R.
    Hantaviruses, part of the Bunyaviridae family, are a genus of negative-sense, single-stranded RNA viruses that cause two major diseases: New-World Hantavirus Cardiopulmonary Syndrome and Old-World Hemorrhagic Fever with Renal Syndrome. Hantaviruses generally are found worldwide with each disease corresponding to their respective hemispheres. New-World Hantaviruses spread by specific rodent-host reservoirs and are categorized as emerging viruses that pose a threat to global health and security due to their high mortality rate and ease of transmission. Incidentally, reports of Hantavirus categorization as a bioweapon are often contradicted as both US National Institute of Allergy and Infectious Diseases and the Centers for Disease Control and Prevention refer to them as Category A and C bioagents respectively, each retaining qualitative levels of importance and severity. Concerns of Hantavirus being engineered into a novel bioagent has been thwarted by Hantaviruses being difficult to culture, isolate, and purify limiting its ability to be weaponized. However, the natural properties of Hantaviruses pose a threat that can be exploited by conventional and unconventional forces. This review seeks to clarify the categorization of Hantaviruses as a bioweapon, whilst defining the practicality of employing New-World Hantaviruses and their effect on armies, infrastructure, and civilian targets.
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    Molecular mechanisms of viral hepatitis induced hepatocellular carcinoma
    (Baishideng Publishing, 2020) D'Souza, Simmone; Lau, Keith C. K.; Coffin, Carla S.; Patel, Trushar R.
    Chronic infection with viral hepatitis affects half a billion individuals worldwide and can lead to cirrhosis, cancer, and liver failure. Liver cancer is the third leading cause of cancer-associated mortality, of which hepatocellular carcinoma (HCC) represents 90% of all primary liver cancers. Solid tumors like HCC are complex and have heterogeneous tumor genomic profiles contributing to complexity in diagnosis and management. Chronic infection with hepatitis B virus (HBV), hepatitis delta virus (HDV), and hepatitis C virus (HCV) are the greatest etiological risk factors for HCC. Due to the significant role of chronic viral infection in HCC development, it is important to investigate direct (viral associated) and indirect (immune-associated) mechanisms involved in the pathogenesis of HCC. Common mechanisms used by HBV, HCV, and HDV that drive hepatocarcinogenesis include persistent liver inflammation with an impaired antiviral immune response, immune and viral protein-mediated oxidative stress, and deregulation of cellular signaling pathways by viral proteins. DNA integration to promote genome instability is a feature of HBV infection, and metabolic reprogramming leading to steatosis is driven by HCV infection. The current review aims to provide a brief overview of HBV, HCV and HDV molecular biology, and highlight specific viral-associated oncogenic mechanisms and common molecular pathways deregulated in HCC, and current as well as emerging treatments for HCC.
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    Human DDX3X unwinds Japanese encephalitis and Zika viral 5' terminal regions
    (MDPI, 2021) Nelson, Corey R.; Mrozowich, Tyler; Gemmill, Darren L.; Park, Sean M.; Patel, Trushar R.
    Flavivirus genus includes many deadly viruses such as the Japanese encephalitis virus (JEV) and Zika virus (ZIKV). The 5′ terminal regions (TR) of flaviviruses interact with human proteins and such interactions are critical for viral replication. One of the human proteins identified to interact with the 5′ TR of JEV is the DEAD-box helicase, DDX3X. In this study, we in vitro transcribed the 5′ TR of JEV and demonstrated its direct interaction with recombinant DDX3X (Kd of 1.66 ± 0.21 µM) using microscale thermophoresis (MST). Due to the proposed structural similarities of 5′ and 3′ TRs of flaviviruses, we investigated if the ZIKV 5′ TR could also interact with human DDX3X. Our MST studies suggested that DDX3X recognizes ZIKV 5′ TR with a Kd of 7.05 ± 0.75 µM. Next, we performed helicase assays that suggested that the binding of DDX3X leads to the unwinding of JEV and ZIKV 5′ TRs. Overall, our data indicate, for the first time, that DDX3X can directly bind and unwind in vitro transcribed flaviviral TRs. In summary, our work indicates that DDX3X could be further explored as a therapeutic target to inhibit Flaviviral replication