Browsing by Subject "Immune evasion"
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Item Open Access Novel mechanisms of antiviral innate immune regulation by the hepatitis C virus NS3-NS4A protease(2019) Vazquez, ChristineHepatitis C virus (HCV) evasion of the host immune system is largely mediated by the actions of the HCV NS3-NS4A protease complex, which consists of the serine protease and RNA helicase NS3 and its membrane targeting co-factor NS4A. NS3-NS4A has multiple functions in the HCV life cycle, with roles in both HCV replication and regulation of innate immune signaling. To regulate innate immune signaling, NS3-NS4A inactivates multiple signaling proteins, including MAVS, an adaptor protein in the RIG-I antiviral signaling pathway, and Riplet, an E3 ubiquitin ligase that activates RIG-I. Inactivation of these host proteins results in an inhibition of downstream signaling through the transcription factor IRF3 and inhibition of the subsequent induction of IFN-β. What directs the multiple functions of NS3-NS4A throughout the HCV life cycle is largely undetermined. Here, we identify a tyrosine residue within the transmembrane domain of NS4A that uncouples the various function of NS3-NS4A.
First, to uncouple the roles of NS3-NS4A in replication and immune evasion, I focused on the NS4A transmembrane domain and generated an NS4A mutant (Y16F) in a full-length HCV infectious clone, a subgenomic replicon, and an over-expression construct. I then assessed viral replication of HCV wild-type (WT) and Y16F viruses by measuring replication of a subgenomic HCV replicon in two related liver hepatoma cell lines: Huh7, which have functional RIG-I signaling, and Huh-7.5 cells, which lack functional RIG-I signaling. The HCV Y16F virus replicated to similar levels as WT HCV in Huh-7.5 cells. However, in Huh7 cells, replication of HCV Y16F was decreased compared to the HCV WT. I used CRISPR-Cas9 gene editing to delete proteins in the RIG-I pathway, including RIG-I, MAVS, and IRF3, in Huh7 cells, infected these cells with HCV WT or Y16F viruses, and then measured virus replication. I found that Y16F viral replication was not restored to the levels of WT in Huh7-RIG-I KO cells or the Huh7-MAVS KO cells, but it was restored to the levels of WT in the Huh7-IRF3 KO cells. I also found that the HCV NS3-NS4A Y16F mutation reduced the ability of over-expressed NS3-NS4A to block IRF3 activation, as measured through nuclear translocation via immunofluorescence microscopy. Further the NS3-NS4A Y16F mutation also had a reduced ability to block the induction of interferon-stimulated genes during both HCV replication and infection. This reveals that HCV NS4A Y16 can regulate a RIG-I-independent, yet IRF3-dependent, signaling pathway that limits viral replication.
Second, to further characterize this RIG-I-independent, IRF3-dependent signaling pathway, I examined the interactions of HCV NS3-NS4A with two of its known host substrates, MAVS and Riplet. To test whether the Y16F mutation prevented NS3-NS4A cleavage of MAVS, I performed a MAVS cleavage assay during both overexpression of NS3-NS4A and MAVS and also during infection with HCV WT and Y16F viruses. NS3-NS4A Y16F was able to cleave MAVS just like WT during both conditions. Next, I found that over-expression of NS3-NS4A WT changed Riplet intracellular localization and that NS4A interacted with Riplet. However, the NS4A Y16F mutation prevented NS4A-Riplet interactions in both of these contexts. Interestingly, I found that Huh-7.5 cells express lower levels of Riplet protein and mRNA compared to Huh7 cells. When full-length Riplet was added exogenously to Huh-7.5 cells, HCV Y16F virus replication was reduced compared to WT. However, when a Riplet construct missing the RING domain, which is essential for Riplet signaling, was added exogenously to Huh-7.5, both WT and Y16F viruses now replicated similarly. Taken together, these data identify NS3-NS4A Y16 as important for regulating a previously uncharacterized Riplet-mediated signaling pathway that limits HCV infection.
Item Embargo The Role of Maternal Antibodies in Prevention of Congenital Cytomegalovirus Infection(2023) Otero, ClaireCytomegalovirus (CMV) is the most common congenital infection and a problematic opportunistic pathogen for immunocompromised patient populations. Despite the immense global burden of CMV and many years of research, the licensed interventions for prevention of CMV disease, and congenital CMV in particular, are very limited. The goal of the work presented in this dissertation is to inform vaccine design for the prevention of congenital CMV by enhancing maternal humoral immunity. We initially investigated the humoral immune responses that associate with protection from vertical CMV transmission in a rhesus macaque model of congenital CMV, evaluating Fc mediated effector responses, which had not previously been measured in the context of rhesus CMV (RhCMV) infection. This study suggested that the humoral response develops too late following primary infection to play a significant role in prevention of vertical transmission but demonstrated a role for pre-existing, potently neutralizing antibodies in prevention of vertical CMV transmission. While this study did not find an association between Fc mediated antibody effector responses, clinical observational studies have implicated these antibody functions in protection from congenital CMV. Interestingly, human CMV (HCMV) is known to encode multiple proteins capable of binding to immunoglobulin G (IgG) antibodies, which have demonstrated the ability to interfere with host Fcγ receptor (FcγR) activation and effector function. We have identified homolog viral FcγRs (vFcγRs) in RhCMV, which allows us a unique opportunity to study these proteins in vivo for the first time by infecting RhCMV-seronegative rhesus macaques with RhCMV lacking all three identified vFcγRs and validating the role vFcγRs have demonstrated in vitro in immune evasion. Lastly, we evaluated a novel HCMV vaccine strategy in which we targeted this immune evasion mechanism through active vaccination against glycoprotein B (gB) alone or in combination with one of the vFcγRs. This proof-of-concept study demonstrated that immune responses against the vFcγRs, gp34 in particular, can improve host FcγR activation and effector function, with initial focus on FcRI (CD64). These results suggest that a simple addition of one or more vFcγRs to vaccines already in development may have a significant impact on the effectiveness of Fc mediated effector responses, which could in turn reduce the risk of vertical CMV transmission.