BEGIN:VCALENDAR VERSION:2.0 X-WR-CALNAME:EventsCalendar PRODID:-//hacksw/handcal//NONSGML v1.0//EN CALSCALE:GREGORIAN BEGIN:VTIMEZONE TZID:America/New_York LAST-MODIFIED:20240422T053451Z TZURL:https://www.tzurl.org/zoneinfo-outlook/America/New_York X-LIC-LOCATION:America/New_York BEGIN:DAYLIGHT TZNAME:EDT TZOFFSETFROM:-0500 TZOFFSETTO:-0400 DTSTART:19700308T020000 RRULE:FREQ=YEARLY;BYMONTH=3;BYDAY=2SU END:DAYLIGHT BEGIN:STANDARD TZNAME:EST TZOFFSETFROM:-0400 TZOFFSETTO:-0500 DTSTART:19701101T020000 RRULE:FREQ=YEARLY;BYMONTH=11;BYDAY=1SU END:STANDARD END:VTIMEZONE BEGIN:VEVENT CATEGORIES:College of Arts and Sciences,Lectures and Seminars,Thesis/Disser tations DESCRIPTION:Abstract HIV-1 Nef is a multifunctional accessory protein with a molecular mass of 27 kDa that plays a central role in lentiviral pathoge nesis by manipulating the host immune response to enhance viral replicatio n. One of Nef’s key functions is the downregulation of cell surface rece ptors, particularly CD4 and major histocompatibility complex class I (MHCI ). In addition to receptor downregulation, Nef modulates various host cell ular processes, including signal transduction and vesicular transport path ways. These interactions not only contribute to immune evasion but also cr eate a cellular environment favorable for viral replication. Nef promotes the internalization of CD4 from the cell surface through the clathrin/AP-2 endocytosis pathway. After internalization, Nef directs CD4 into the mult ivesicular body (MVB) pathway. The MVB pathway, regulated by the ESCRT com plex, sorts CD4 into vesicles that fuse with lysosomes for degradation. Ne f directly interacts with Alix (apoptosis-linked gene 2-interacting protei n X), a key component of the ESCRT machinery, to transport CD4 into the mu ltivesicular body (MVB) pathway, which ultimately leads to its degradation . In this dissertation project, the interaction between Alix and Nef was s tudied using biochemical assays. Chapter 2 of the dissertation focuses on these assays, providing new insights into how Nef interacts with Alix and contributing to a deeper understanding of Nef's role in viral pathogenesis . PTPN23 is a putative protein tyrosine phosphatase and a paralog of Alix. Like Alix, PTPN23 supports ESCRT function and is a key regulator in endos omal sorting and vesicular trafficking. Preliminary findings from our coll aborator, Dr. John Guatelli’s lab, suggests that HIV-1 Nef may exploit P TPN23 in a manner similar to its interaction with Alix to misroute 3 host cell surface receptors toward lysosomal degradation. In Chapter 3 of this dissertation, we explored the potential binding interaction between Nef an d PTPN23 using biochemical assays. Our results showed that the Nef-PTPN23 interaction is promoted by acidic pH, consistent with the fact that PTPN23 operates in late endosomal compartments. Chapter 4 of the dissertation de scribes experimental procedures for protein expression, purification, and assay methodologies. My additional contributions to the study of HIV-1 Nef interactions, including the cloning of MBP-FLAG-CD4(394–433) and Sumo-C D4 10aa-β2(1–25)-FLAG constructs for FRET and FP assay analysis, are al so discussed in Chapter 4. Zoom link: https://umassd.zoom.us/j/93254193551 ?pwd=117zcTaxg5qks3yxDLAE4Y2eJwT4jz.1 Meeting ID: 932 5419 3551 Passcode: 942981 Ìý\nEvent page: /events/cms/phd-dissertation- defense-by-priya-sridharan-investigating-the-molecular-bases-of-hiv-1-nef- mediated-hijacking-of-alix-and-ptpn23.php\nEvent link: https://umassd.zoom .us/j/93254193551?pwd=117zcTaxg5qks3yxDLAE4Y2eJwT4jz.1 X-ALT-DESC;FMTTYPE=text/html:

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Abstract

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HIV-1 Nef is a multifunctional accessory protein with a molecular mass of 27 kDa that plays a central role in lentiviral pathogenesis by manipula ting the host immune response to enhance viral replication. One of Nef’s key functions is the downregulation of cell surface receptors\, particula rly CD4 and major histocompatibility complex class I (MHCI). In addition t o receptor downregulation\, Nef modulates various host cellular processes\ , including signal transduction and vesicular transport pathways. These in teractions not only contribute to immune evasion but also create a cellula r environment favorable for viral replication. Nef promotes the internaliz ation of CD4 from the cell surface through the clathrin/AP-2 endocytosis p athway. After internalization\, Nef directs CD4 into the multivesicular bo dy (MVB) pathway. The MVB pathway\, regulated by the ESCRT complex\, sorts CD4 into vesicles that fuse with lysosomes for degradation.

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Nef d irectly interacts with Alix (apoptosis-linked gene 2-interacting protein X )\, a key component of the ESCRT machinery\, to transport CD4 into the mul tivesicular body (MVB) pathway\, which ultimately leads to its degradation . In this dissertation project\, the interaction between Alix and Nef was studied using biochemical assays. Chapter 2 of the dissertation focuses on these assays\, providing new insights into how Nef interacts with Alix an d contributing to a deeper understanding of Nef's role in viral pathogenes is.

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PTPN23 is a putative protein tyrosine phosphatase and a paralo g of Alix. Like Alix\, PTPN23 supports ESCRT function and is a key regulat or in endosomal sorting and vesicular trafficking. Preliminary findings fr om our collaborator\, Dr. John Guatelli’s lab\, suggests that HIV-1 Nef may exploit PTPN23 in a manner similar to its interaction with Alix to mis route 3 host cell surface receptors toward lysosomal degradation. In Chapt er 3 of this dissertation\, we explored the potential binding interaction between Nef and PTPN23 using biochemical assays. Our results showed that t he Nef-PTPN23 interaction is promoted by acidic pH\, consistent with the f act that PTPN23 operates in late endosomal compartments.

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Chapter 4 of the dissertation describes experimental procedures for protein express ion\, purification\, and assay methodologies. My additional contributions to the study of HIV-1 Nef interactions\, including the cloning of MBP-FLAG -CD4(394–433) and Sumo-CD4 10aa-β2(1–25)-FLAG constructs for FRET and FP assay analysis\, are also discussed in Chapter 4.

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Zoom link: < a href="https://umassd.zoom.us/j/93254193551?pwd=117zcTaxg5qks3yxDLAE4Y2eJ wT4jz.1">https://umassd.zoom.us/j/93254193551?pwd=117zcTaxg5qks3yxDLAE4Y2e JwT4jz.1
Meeting ID: 932 5419 3551
Passcode: 942981

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Event page: /events/cms/phd-dissertation-defense-by-priya-sridharan-investigating-the- molecular-bases-of-hiv-1-nef-mediated-hijacking-of-alix-and-ptpn23.php
Event link:

DTSTAMP:20250605T193944 DTSTART;TZID=America/New_York:20250611T110000 DTEND;TZID=America/New_York:20250611T130000 LOCATION:Zoom Meeting SUMMARY;LANGUAGE=en-us:PhD Dissertation Defense by Priya Sridharan "Investi gating the Molecular bases of HIV-1 Nef mediated hijacking of Alix and PTP N23" UID:b8465237463b31450be16f72ad644ee9@www.umassd.edu END:VEVENT END:VCALENDAR