Functional Mapping of Regions Involved in the Negative Imprinting of Virion Particle Infectivity and in Target Cell Protection by Interferon-Induced Transmembrane Protein 3 against HIV-1

Abstract

The interferon-induced transmembrane proteins (IFITMs) are a family of highly related antiviral factors that affect numerous viruses at two steps: in target cells by sequestering incoming viruses in endosomes and in producing cells by leading to the production of virions that package IFITMs and exhibit decreased infectivity. While most studies have focused on the former, little is known about the regulation of the negative imprinting of virion particle infectivity by IFITMs and about its relationship with target cell protection. Using a panel of IFITM3 mutants against HIV-1, we have explored these issues as well as others related to the biology of IFITM3, in particular virion packaging, stability, the relation to CD63/multivesicular bodies (MVBs), the modulation of cholesterol levels, and the relationship between negative imprinting of virions and target cell protection. The results that we have obtained exclude a role for cholesterol and indicate that CD63 accumulation does not directly relate to an antiviral behavior. We have defined regions that modulate the two antiviral properties of IFITM3 as well as novel domains that modulate protein stability and that, in so doing, influence the extent of its packaging into virions. The results that we have obtained, however, indicate that, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for negative imprinting. Finally, while most mutations concomitantly affect target cell protection and negative imprinting, a region in the C-terminal domain (CTD) exhibits a differential behavior, potentially highlighting the regulatory role that this domain may play in the two antiviral activities of IFITM3. IMPORTANCE IFITM proteins have been associated with the sequestration of incoming virions in endosomes (target cell protection) and with the production of virion particles that incorporate IFITMs and exhibit decreased infectivity (negative imprinting of virion infectivity). How the latter is regulated and whether these two antiviral properties are related remain unknown. By examining the behavior of a large panel of IFITM3 mutants against HIV-1, we determined that IFITM3 mutants are essentially packaged into virions proportionally to their intracellular levels of expression. However, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for the antiviral effects. Most mutations were found to concomitantly affect both antiviral properties of IFITM3, but one CTD mutant exhibited a divergent behavior, possibly highlighting a novel regulatory role for this domain. These findings

Macaque interferon-induced transmembrane proteins limit replication of SHIV strains in an Envelope-dependent manner

“…The cell and virion lysate were immunoblotted and infectivity of cell-free virions were measured on TZM-bl reporter cell line. Consistent with other studies [37, 40, 43], we did not observe any differential effects of Ptm IFITM expression on Env processing in the producer cells and Env incorporation into viral particles (S4A and S4B Fig). In addition, Ptm IFITM expression did not affect infectivity of SHIV virions produced during 48 hours in these experiments (S4C Fig).…”

IFITM Genes, Variants, and Their Roles in the Control and Pathogenesis of Viral Infections

“…In addition to the protective function of IFITM proteins to reduce viral infection of host cells, IFITM proteins, particularly IFITM2 and 3, can lead to the production of virions that package IFITMs and display reduced entry into target cells (Compton et al, 2014; Tartour et al, 2014, 2017). One study found that IFITM proteins interact with HIV envelope protein in viral producer cells to disrupt envelope protein processing and virion incorporation, which impairs virion infectivity (Yu et al, 2015); however, another found that IFITM3 expression in producing cells did not affect the amount of envelope protein incorporation into progeny HIV-1 virions (Appourchaux et al, 2018). Both studies reported that the level of IFITM protein incorporation into progeny virions does not correlate with the extent of infectivity reduction.…”

“…More strikingly, deletion of C-terminal 12-aa or 18-aa converted IFITM1 to a potent enhancer of MERS-CoV and HCoV-OC43 entry (Zhao et al, 2014, 2018). A recent mutagenesis study indicated that amino acid residues 127–132 in the CTD domain of IFITM3 differentially modulates its activities in target cell protection and negative imprinting of progeny HIV-1 infectivity (Appourchaux et al, 2018). These studies clearly indicate that the CTD of IFITMs contains multiple structure motifs that regulate the subcellular localization and antiviral functions.…”

Human IFITM3 restricts Chikungunya virus and Mayaro virus infection and is susceptible to virus-mediated counteraction

“…Remarkably, IFITM proteins display a second layer of antiviral activity, which consists in diminishing the infectivity and fusogenicity of enveloped virus particles produced in IFITM protein-expressing cells (Compton et al, 2014, Tartour, Appourchaux et al, 2014. This process is often, but not always, accompanied by incorporation of IFITM proteins into the membrane of secreted virions (Appourchaux, Delpeuch et al, 2019). Future studies are required to characterize susceptibility of CHIKV particles to IFITM-mediated negative imprinting and whether the mode of viral counteraction identified in this study is related to this site of action rather than to entry inhibition.…”