Date of Award
Fall 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Microbiology
First Advisor
Mothes, Walther
Abstract
The human immunodeficiency virus 1 (HIV-1), which is the causative agent responsible for Acquired Immunodeficiency Syndrome (AIDS), is a retrovirus that primarily infects CD4+ T lymphocytes. When left untreated, HIV replication results in progressive CD4+ T cell loss and leads to a myriad of immunological abnormalities in the host, escalating the risk of infections as well as oncological complications. To combat the progression of HIV-1, host cells have evolved to produce proteins known as restriction factors, which are crucial elements of the innate immune system, to inhibit the HIV-1 infection by disrupting various stages of viral replication. Such factors include the previously identified TRIM5α and APOBEC3G. Intriguingly, recent studies have unearthed a novel family of proteins, named SERINCs, that exhibit restriction activity against HIV-1 infection. Identified for their role as serine incorporators in phospholipids, SERINCs are multipass transmembrane proteins, which, in humans, comprise five distinct members. Among these, SERINC5 has been demonstrated to be the most potent antiviral inhibitor when incorporated into emerging virions, while SERINC3 appears to impose a more moderate inhibition of HIV-1. The antiviral activities of SERINCs, however, is counteracted by HIV-1 viral protein Nef, which redirects SERINC5 to an endosomal compartment, thereby precluding its incorporation into the viral envelope. In addition to HIV-1, SERINC 5 has been observed to restrict other enveloped viruses such as murine leukemia viruses (MLV), which expresses GlycoGag to combat SERINCs. SERINCs may exert multiple function that collectively obstruct the entry of HIV-1 into target cells. The incorporation of SERINC into HIV-1 particles seems to alter the conformation of the envelope glycoprotein (Env), as evident by the elevated exposure of sequestered epitopes. It also hinders membrane fusion by disrupting Env clustering, intermediates in the fusion pathway, lipid ordering, and expansion of the fusion pore. Although SERINCs may obstruct fusion due to their impact on the local lipid composition of the viral envelope, lipid quantitation and fractionation analyses have shown no discernible changes in the quantity and composition of cell and viral membrane lipids when SERINC5 is present. Given this background of mechanistic uncertainty, we conducted a comprehensive investigation into the fundamental mechanisms underlying SERINC-mediated anti-HIV effects, starting with a comparative analysis of SERINC's structural architecture. SERINC's structure bears a significant similarity to unregulated lipid transporters such as archaeal PfMATE and bacterial proteins MurJ. This inspired an investigation into SERINC's capacity to flip phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Utilizing a proteoliposome-based lipid-translocation quantification assay, we observed significant lipid transport capacity in hSERINC. This activity exhibited dose-dependency and did not rely on ATP consumption, suggesting that SERINC may act as a lipid transporter. Subsequently, we employed a FACS-based PS-quantification assay and confirmed that the phospholipid exposure on the HIV-1 surface is enhanced when SERINC is incorporated, signifying its impact on the lipid composition of HIV envelope. Our results also established a direct correlation between the antiviral effectiveness of various hSERINC5 mutants and the level of PS exposure on virus particles incorporating these SERINCs. Notably, we discovered that retroviral accessory proteins, Nef (from HIV-1) and GlycoGag (from MLV), counteract the PS-exposing activity of SERINC, which corroborates prior studies that demonstrated Nef and GlycoGag's ability to inhibit the antiviral effects of SERINC. Furthermore, using a constitutively active lipid scramblase, murine TMEM16F, which is unrelated to SERINC, we observed a similar correlation between the viral surface PS exposure and inhibition as we observed with the SERINC variants. In addition, a decrease in MLV infectivity was noted upon the incorporation of both hSERINC5 and mTMEM16F. This finding suggested a novel antiviral mechanism harnessed by SERINC proteins to restrict not only HIV-1 but potentially other retroviruses as well. Notably, the highly active mTMEM16F variant was impervious to the neutralizing effects of the retroviral accessory proteins Nef and GlycoGag. Given that this variant maintained its activity against MLV, it underscores the wide-ranging potential of this newly identified antiviral strategy. Finally, we delved deeper into the molecular mechanisms underlying the HIV inhibition induced by SERINC and TMEM. Utilizing single-molecule FRET technology, we discovered that both SERINC and TMEM induce an alteration in the conformation of the Env trimer upon their incorporation into nascent HIV virions. Specifically, both proteins prompted a shift in the conformational landscape of HIV Env, from predominantly state 1 to state 3. This indicates that the activities of SERINC and TMEM result in the HIV Env opening, which could explain the mechanism underlying the HIV inhibition induced by SERINC and TMEM.
Recommended Citation
Yang, Ziwei, "Antiviral HIV-1 SERINC Restriction Factors Disrupt Virus Membrane Asymmetry" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1241.
https://elischolar.library.yale.edu/gsas_dissertations/1241
