Differential Control of HIV-1 Replication by IFN-α14 Compared to IFN-α2 Relates to Differences in the Modulation of Host Antiretroviral Restriction Factors

Type I IFN, including IFN-, induces the expression of antiviral restriction factors that can interfere with multiple steps of the HIV-1 replication cycle. Humans have 13 IFN- genes which encode 12 different IFN- subtypes. Our previous work in HIV-1 infected humanized mice showed that IFN-14 treatment more potently controlled HIV-1 than treatment with the clinically approved IFN-2 subtype. However, the mechanisms behind the more potent control of HIV-1 by IFN-14 are unknown. The IFN-14 subtype is known to more potently induce the expression of the restriction factors MX2 and ISG15 and increased APOBEC3G signature mutations in vivo compared to IFN-2. To study the importance of each of these restriction factors in mediating the potent control of HIV-1, we used a CRISPR-Cas9 lentivirus system to create stable knockouts in the MT4C5 cell line that is susceptible to HIV-1 but does not produce measurable amounts of endogenous IFN-. Knock out of ISG15, but not MX2, eliminated differences in viral suppression after IFN-14 and IFN-2 treatment. Similarly, APOBEC3G deletion eliminated differences in viral suppression and the number of infectious particles produced after IFN-14 and IFN-2 treatment. Furthermore, APOBEC3G deletion resulted in significantly fewer GG[->]AG mutations in viral DNA isolated from target cells incubated with supernatant from IFN-14 treated groups. However, APOBEC3G knock out did not result in significant increases in vDNA compared to the wild type in any experimental group. Overall, elimination of APOBEC3G and ISG15 impaired IFN-14-mediated suppression of HIV-1, highlighting them as downstream effectors of IFN-14s more potent anti-HIV-1 activity. IMPORTANCEThis study uncovers the molecular basis for the more potent antiviral activity of IFN-14 compared to the clinically used IFN-2 subtype against HIV-1. Although interferons are known to induce numerous restriction factors, the mechanisms underlying subtype-specific antiviral potency remained unclear. By using CRISPR-Cas9 knockout MT4C5 cell lines, the study identifies ISG15 and APOBEC3G as key effectors mediating IFN-14s enhanced suppression of HIV-1 replication. Loss of either ISG15 or APOBEC3G abolished the differential antiviral effect between IFN-14 and IFN-2, demonstrating their essential roles in IFN-14 driven viral restriction. These findings highlight that individual IFN- subtypes engage distinct downstream pathways and that subtype diversity encodes functional specialization rather than redundancy. Overall, this work advances our understanding of innate immune control of HIV-1 and provides a foundation for developing targeted interferon-based therapies that exploit the unique mechanisms of potent subtypes like IFN- 14.