Transcription start site heterogeneity controls MDA5 sensing of unspliced HIV-1 RNAs

Heterogenous transcription start site (TSS) usage dictates the 5' leader structure and function of unspliced HIV-1 RNAs (usRNA). We and others have previously reported that expression and Rev/CRM1-mediated nuclear export of HIV-1 usRNA in macrophages activates MDA5, MAVS, and innate immune signaling cascades. In this study, we reveal that MDA5 sensing of viral usRNA is largely determined by TSS and cytoplasmic RNA function in macrophages. We show that HIV-1 usRNAs (^cap1G) that are preferentially destined for dimerization and viral genome packaging are specifically targeted by MDA5, while efficiently translated (^cap3G) usRNAs are immunologically silent. Using mutant viruses which generate usRNA with altered 5' leader structure, or inclusion of a retroviral constitutive transport element which drives mRNA-like NXF1-dependent nuclear export of viral usRNA, we show that transcription initiation site and nuclear export pathway choice are major determinants of both HIV-1 usRNA immunogenicity and cytoplasmic function. In total, we identify innate immune response modulation as a consequence of the well-conserved heterogenous TSS usage among ancestral and extant HIV-1 isolates in humans, and shed light on how MDA5 discriminates between self and non-self RNAs.