Divergent HIV-1 Restriction Phenotypes of IFITMs Expressed in Target Cells and Incorporated into Virions

Interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral factors that restrict the entry of many enveloped viruses, including HIV-1, by modifying host membrane properties and trapping fusion at the hemifusion stage. Beyond blocking entry in target cells, IFITMs also reduce the infectivity of virions produced from IFITM-expressing cells, a phenomenon termed "negative imprinting". Conserved motifs, such as the amphipathic helix and oligomerization motifs, have been reported to be essential for IFITM-mediated protection of target cells from viral infection. Yet, the impact of IFITM incorporation on progeny virion infectivity remains poorly defined. Here, we show that IFITM3 mutants defective in target cell protection activity still markedly impair HIV-1 fusion/infection upon incorporating into virions, without affecting viral maturation or Env incorporation. Immunofluorescence studies suggest mislocalization of the IFITM3 mutants as the reason for the lack of antiviral activity in target cells. Testing the antiviral activity of chimeras between antiviral and non-antiviral IFITM orthologs failed to clearly identify a domain responsible for reduction of HIV-1 infectivity, suggesting that multiple domains may be required for negative imprinting. Interestingly, co-incorporation of non-antiviral dog IFITM1 with human IFITM3 did not interfere with IFITM3's negative imprinting activity, despite forming mixed hetero-oligomers. This finding implies a dominant, oligomerization-independent antiviral phenotype of IFITM3 in virions. Our findings suggest that IFITMs may protect target cells and negatively imprint progeny virions through distinct mechanisms, underscoring the need to further characterize the molecular basis for the reduced fusion competence of IFITM-containing HIV-1 particles.