HIV is a devastating virus that causes a life-long infection and can lead to acquired immunodeficiency syndrome (AIDS). RNA modifications play important roles in regulating a wide range of biological processes linked to human health and disease. Humans also possess multileveled defenses against invading pathogens, such as Type I interferon (IFN), which is a potent antiviral cytokine that induces expression of IFN-stimulated genes (ISGs) to induce an antiviral state. Both RNA modifications and IFN signaling are proposed to play a major role in the biology of HIV infection.Several distinct RNA modifications have been identified in human cellular mRNA such as N6-methyladenosine (m6A) and pseudouridine (Ѱ). HIV RNA contains numerous m6A modifications, which are read by the YTHDF1-3 reader proteins. There remains some controversy about its impact on viral replication. It is unknown whether HIV has Ѱ modifications. We hypothesize that HIV engages several distinct parts of RNA modification machinery to optimize its replication without changing its genome. Our work demonstrates that YTHDF1-3 incorporate into HIV particles in a nucleocapsid-dependent manner, YTHDF3 incorporation into the viral particle limits the next round of infection by blocking reverse transcription, and YTHDF3 is cleaved by HIV protease in the viral particle, a process which is blocked by HIV protease inhibitors used to treat HIV-infected patients. We also identified seven Ѱ sites in HIV RNA, found that single Ѱ mutant viruses are less infectious than wildtype virus, and identified PUS1 and PUS3 to be potential writers of Ѱ on HIV RNA.ISGs act as antiviral effectors, but also as negative regulators of IFN signaling to prevent over-inflammation. ISG15 is one such negative regulator of IFN signaling. Individuals lacking ISG15 have elevated levels of ISGs and chronic mild auto-inflammation, which is sufficient to provide increased cellular resistance to viral infections. We hypothesize that mild auto-inflammation and deregulation of IFN responses may contribute to HIV control. IFN-primed cells from ISG15-deficient patients are less susceptible to HIV infection compared to wildtype cells, complementation with ISG15 rescues this phenotype, and transcriptome analysis of the primary CD4+ T cells with and without ISG15 and with and without IFN stimulation reveal a dedicated set of ISGs is sufficient to reduce susceptibility of primary CD4+ T cells to HIV infection.Overall, these studies demonstrate that RNA modifications and IFN signaling pathways play major roles in regulating HIV infection biology.