HIV protease cleaves the antiviral m6A reader protein YTHDF3 in the viral particle

Author summary The human transcriptome contains a large number of post-transcriptional modifications such as N6-methyladenosine (m(6)A). Several recent studies indicate that the HIV RNA contains numerous m(6)A modifications but their impact on viral replication (e.g., antiviral or proviral) remains controversial. Here we report that the m(6)A reader protein YTHDF3 is incorporated into HIV particles in a nucleocapsid-dependent manner and reduces viral infectivity in the next cycle of infection. Importantly, we show that HIV protease cleaves the virion-incorporated full-length YTHDF3 protein, a process which can be blocked by FDA-approved HIV protease inhibitors. Mass-spectrometry analyses confirmed the proteolytic processing of YTHDF3 in the virion and identified at least two distinct cleavage sites. These results point to virus incorporated YTHDF3 acting as a regulator of HIV biology if left unchecked by the HIV protease. N-6-methyladenosine (m(6)A) is the most abundant HIV RNA modification but the interplay between the m(6)A reader protein YTHDF3 and HIV replication is not well understood. We found that knockout of YTHDF3 in human CD4+ T-cells increases infection supporting the role of YTHDF3 as a restriction factor. Overexpression of the YTHDF3 protein in the producer cells reduces the infectivity of the newly produced viruses. YTHDF3 proteins are incorporated into HIV particles in a nucleocapsid-dependent manner permitting the m(6)A reader protein to limit infection in the new target cell at the step of reverse transcription. Importantly, HIV protease cleaves the virion-incorporated full-length YTHDF3 protein, a process which is blocked by HIV protease inhibitors used to treat HIV infected patients. Mass-spectrometry confirmed the proteolytic processing of YTHDF3 in the virion. Thus, HIV protease cleaves the virion-encapsidated host m(6)A effector protein in addition to the viral polyproteins to ensure optimal infectivity of the mature virion.