AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human immunodeficiency virus 1 (HIV-1) are enveloped viruses that mediate viral entry via class I fusion glycoproteins. Both of these viruses demonstrate significant sequence variability in their entry glycoproteins, a result of adaptation to escape both adaptive and innate immunity intra-host. Interferon-Induced TransMembrane (IFITM) proteins are antiviral proteins that inhibit viral entry and previous reports demonstrate varying IFITM sensitivity in coronaviruses. HIV-1 has also been reported to be varyingly susceptible to IFITM-mediated inhibition in an isolate-dependent manner.
This thesis evaluates the IFITM sensitivity of the ancestral Wuhan SARS-CoV-2 virus and SARS-CoV-2 variants of concern (VOCs) that emerged between 2020 and 2022. We found that the Wuhan SARS-CoV-2 was IFITM2-sensitive in the A549-ACE2 system, however this can be overcome by overexpression of TMPRSS2. Deletion of the polybasic cleavage site of spike renders Wuhan SARS-CoV-2 pseudotyped lentiviral vectors even more IFITM2 sensitive, which cannot be rescued by TMPRSS2 overexpression, demonstrating that the polybasic cleavage site of SARS-CoV-2 spike can modulate IFITM2 sensitivity. The IFITM sensitivity of D614G, alpha, beta, gamma, kappa, delta, and omicron (BA.1 and BA.2) variants was also investigated. Strikingly, the alpha variant was resistant to IFITMs in TMPRSS2-negative cells which contributes a significant component of the resistance of the alpha virus to IFNb. Additionally, we show here that the P681H mutation in alpha is a major determinant of both IFITM and IFNb resistance.
We also investigated the IFITM sensitivity of 24 CCR5-tropic HIV-1 Envelopes isolated from a single anti-retroviral therapy negative patient over nine years of infection in order to define the determinants of IFITM sensitivity and resistance of Env. Transmitted/founder (T/F) viruses, and viruses which emerge following ART interruption, have previously been reported to be more resistant to both IFITMs and type I IFN. Here, it is shown that sensitivity to both IFITMs and IFNa fluctuate over the course of infection and isolates present at the same timepoint exhibit differing sensitivity to IFITMs and IFNa. By utilising a pair of Envs that are the most related but differentially sensitive to IFITM3, the determinant of IFITM sensitivity/resistance of both Envs was traced to the V1/V2 loop.
Overall, this piece of work highlights the complexity of the relationship between viral entry and IFITM sensitivity. The data presented here demonstrate that although different regions of spike and Env can alter IFITM sensitivity, there are also commonalties in the relationships between two unrelated viruses and IFITM-mediated restriction. The priming cleavage of both HIV-1 Env and SARS-CoV-2 spike can impact IFITM sensitivity. Additionally, the cytoplasmic tail of spike and Env appears to play a role in restriction by IFITM2, and also in enhancement of entry by IFITM3. Altogether, IFITM sensitivity of unrelated viral entry proteins can be governed by both similar and distinct mechanisms.
|Date of Award||1 Mar 2023|
|Supervisor||Stuart Neil (Supervisor) & Katherine Doores (Supervisor)|