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Vincent Gerard Francis

Bio: Vincent Gerard Francis is an academic researcher from Indian Institute of Technology Madras. The author has contributed to research in topics: Phospholipid scramblase & Cell biology. The author has an hindex of 7, co-authored 9 publications receiving 126 citations. Previous affiliations of Vincent Gerard Francis include Montreal Neurological Institute and Hospital.

Papers
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Posted ContentDOI
13 Nov 2020-bioRxiv
TL;DR: It is demonstrated that following engagement with the plasma membrane, SARS-CoV-2 undergoes rapid clathrin-mediated endocytosis, which suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system, and importantly clATHrin-heavy chain knockdown, which blocks clathin-mediatedendocytotic, reduces viral infectivity.
Abstract: With more than 51 million cases and 1.3 million deaths, and with the resulting social upheaval, the COVID-19 pandemic presents one of the greatest challenges ever to human society. It is thus vital to fully understand the biology of SARS-CoV-2, the causative agent of COVID-19. SARS-CoV-2 uses its spike glycoprotein to interact with the cell surface as a first step in the infection process. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, we now demonstrate that following engagement with the plasma membrane, SARS-CoV-2 undergoes rapid clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system, and importantly clathrin-heavy chain knockdown, which blocks clathrin-mediated endocytosis, reduces viral infectivity. This discovery reveals important new information about the basic biology of SARS-CoV-2 infectivity.

25 citations

Journal ArticleDOI
TL;DR: The CTH domain is deleted and it is concluded that CTH is required for membrane insertion and Ca2+ coordination and also plays an important role in the functional conformation of hPLSCR1.
Abstract: Human phospholipid scramblase 1 (hPLSCR1) belongs to the ATP-independent class of phospholipid translocators which possess a single EF-hand-like Ca2+-binding motif and also a C-terminal helix (CTH). The CTH domain of hPLSCR1 was believed to be a putative single transmembrane helix at the C-terminus. Recent homology modeling studies by Bateman et al. predicted that the hydrophobic nature of this helix is due to its packing in the core of the protein domain and proposed that this is not a true transmembrane helix [Bateman A, Finn RD, Sims PJ, Wiedmer T, Biegert A & Johannes S. Bioinformatics 2008, 25, 159]. To determine the exact function of the CTH of hPLSCR1, we deleted the CTH domain and determined: (a) whether CTH plays any role beyond membrane anchorage, (b) the functional consequences of CTH deletion, and (c) any conformational changes associated with CTH in a lipid environment. In vitro reconstitution studies confirm that the predicted CTH is required for membrane insertion and scrambling activity. CTH deletion caused a 50% decrease in binding affinity of Ca2+ for ∆CTH-hPLSCR1 (Ka = 115 μm) compared with hPLSCR1 (Ka = 249 μm). Far UV-CD studies revealed that the CTH peptide adopts α-helicity only in the presence of SDS micelles and negatively charged vesicles, indicating that electrostatic interactions are required for insertion of the peptide. CTH peptide-quenching studies confirm that the predicted CTH inserts into the membrane and its ability to interact with the membrane depends on the presence of charge interactions. TOXCAT assay revealed that CTH of hPLSCR1 does not oligomerize in the membrane. We conclude that CTH is required for membrane insertion and Ca2+ coordination and also plays an important role in the functional conformation of hPLSCR1.

24 citations

Journal Article
TL;DR: The authors showed that SARS-CoV-2 uses clathrin-mediated endocytosis to gain access into cells and suggests that this process is a key aspect of virus infectivity, which suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system.

24 citations

Journal ArticleDOI
TL;DR: A method to purify recombinant membrane protein with higher yield than previously described methods involving renaturation techniques is described and revealed that the secondary structure of protein is predominantly an α-helix, and under nondenaturing conditions, the protein exists as a monomer.
Abstract: Human phospholipid scramblase (hPLSCR1) is a transmembrane protein involved in rapid bidirectional scrambling of phospholipids across the plasma membrane in response to elevated intracellular calcium (Ca2+) levels. Overexpression of recombinant hPLSCR1 in Escherichia coli BL21 (DE3) leads to its deposition in inclusion bodies (IBs). N-lauroyl sarcosine was used to solubilize IBs and to recover functionally active hPLSCR1 from them. Protein was purified to homogeneity by nickel-nitrilotriacetic acid (Ni2+–NTA) affinity chromatography and was >98% pure. Functional activity of the purified protein was validated by in vitro reconstitution studies, ~18% of 7-nitrobenz-2-oxa-1, 3-diazol-4-yl-phosphatidylcholine (NBD-PC) phospholipids was translocated across the lipid bilayer in the presence of Ca2+ ions. Far ultraviolet circular dichroism (UV-CD) studies reveal that the secondary structure of protein is predominantly an α-helix, and under nondenaturing conditions, the protein exists as a monomer. Here we describe a method to purify recombinant membrane protein with higher yield than previously described methods involving renaturation techniques.

22 citations

Journal ArticleDOI
TL;DR: It is concluded that scramblases exhibit Ca2+-dependent scrambling activity by aggregation of protein, and the results suggest that PRD is crucial for the function of the protein.

19 citations


Cited by
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Journal ArticleDOI
TL;DR: In this article, structural and cellular foundations for understanding the multistep SARS-CoV-2 entry process, including S protein synthesis, S protein structure, conformational transitions necessary for association of the spike (S) protein with ACE2, engagement of the receptor-binding domain of the S protein with ACS, proteolytic activation of S protein, endocytosis and membrane fusion are provided.
Abstract: The unprecedented public health and economic impact of the COVID-19 pandemic caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been met with an equally unprecedented scientific response. Much of this response has focused, appropriately, on the mechanisms of SARS-CoV-2 entry into host cells, and in particular the binding of the spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2), and subsequent membrane fusion. This Review provides the structural and cellular foundations for understanding the multistep SARS-CoV-2 entry process, including S protein synthesis, S protein structure, conformational transitions necessary for association of the S protein with ACE2, engagement of the receptor-binding domain of the S protein with ACE2, proteolytic activation of the S protein, endocytosis and membrane fusion. We define the roles of furin-like proteases, transmembrane protease, serine 2 (TMPRSS2) and cathepsin L in these processes, and delineate the features of ACE2 orthologues in reservoir animal species and S protein adaptations that facilitate efficient human transmission. We also examine the utility of vaccines, antibodies and other potential therapeutics targeting SARS-CoV-2 entry mechanisms. Finally, we present key outstanding questions associated with this critical process.

988 citations

Journal ArticleDOI
TL;DR: The SARS-CoV-2 infection is likely from a zoonotic source and people with the infection may shed it while suggestive or asymptomatic, and clinical manifestations change generally.
Abstract: The SARS-CoV-2 infection is likely from a zoonotic source. Transmissibility between people happens principally through beads and surface contacts. People with the infection may shed it while suggestive or asymptomatic. Despite the fact that brooding period is as long as 12 days, middle term of viral shedding can be as long as 20 days. Clinical manifestations change generally and might be mellow (80%), moderate (15%), or serious (5%).

183 citations

Journal ArticleDOI
TL;DR: The characterization of PL scramblase (PLSCR) and XKR family members and TMEM16 family members which contribute to phosphatidylserine exposure in response to apoptotic stimuli are described and their potential roles in pathogenic conditions are reported.

99 citations

Journal ArticleDOI
TL;DR: In this paper, the authors discuss the recent advances in understanding the molecular events during SARS-CoV-2 entry which will contribute to developing vaccines and therapeutics, and discuss some auxiliary receptors and cofactors are also involved that expand the host/tissue tropism.

90 citations

Journal ArticleDOI
TL;DR: It is found that the stable overexpression of PLSCR1 suppressed the nuclear import of NP, hindered the virus life cycle, and significantly inhibited the replication of various influenza subtypes.
Abstract: Transcription and replication of the influenza A virus (IAV) genome occur in the nucleus of infected cells and are carried out by the viral ribonucleoprotein complex (vRNP). As a major component of the vRNP complex, the viral nucleoprotein (NP) mediates the nuclear import of the vRNP complex via its nuclear localization signals (NLSs). Clearly, an effective way for the host to antagonize IAV infection would be by targeting vRNP nuclear import. Here, we identified phospholipid scramblase 1 (PLSCR1) as a binding partner of NP by using a yeast two-hybrid (Y2H) screen. The interaction between NP and PLSCR1 in mammalian cells was demonstrated by using co-immunoprecipitation and pull-down assays. We found that the stable overexpression of PLSCR1 suppressed the nuclear import of NP, hindered the virus life cycle, and significantly inhibited the replication of various influenza subtypes. In contrast, siRNA knockdown or CRISPR/Cas9 knockout of PLSCR1 increased virus propagation. Further analysis indicated that the inhibitory effect of PLSCR1 on the nuclear import of NP was not caused by affecting the phosphorylation status of NP or by stimulating the interferon (IFN) pathways. Instead, PLSCR1 was found to form a trimeric complex with NP and members of the importin α family, which inhibited the incorporation of importin β, a key mediator of the classical nuclear import pathway, into the complex, thus impairing the nuclear import of NP and suppressing virus replication. Our results demonstrate that PLSCR1 negatively regulates virus replication by interacting with NP in the cytoplasm and preventing its nuclear import.

60 citations