Abstract
N6-methyladenosine (m6A) is the most abundant HIV RNA modification but the interplay between the m6A 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 m6A 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 m6A effector protein in addition to the viral polyproteins to ensure optimal infectivity of the mature virion.
Highlights
We report that the m6A reader protein YTHDF3 is incorporated into HIV particles in a nucleocapsid-dependent manner and reduces viral infectivity in the cycle of infection
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
YTHDF3-deficient T cells are more susceptible to HIV infection
Summary
Post-transcriptional modifications of the transcriptome, such as N6-methyladenosine (m6A), are globally referred to as epitranscriptome [1,2,3,4]. m6A is a dynamic and reversible RNA modification involved in mRNA splicing, stability, localization, and translation [5,6,7,8]. m6A modifications have been identified within the RNA genomes as well as transcripts of several viruses, including Influenza A virus, adenovirus, Rous sarcoma virus, hepatitis C virus, Zika virus, Dengue virus, West Nile virus, Yellow fever virus, and HIV-1 (HIV) [9,10,11,12,13,14,15,16]. M6A modifications that result in temporally-controlled burst of protein synthesis and mRNA decay, require the interaction with the YTHDF1-3 reader proteins [17]. YTHDF2 promotes decay of m6A-modified mRNA by directing transcripts to cytoplasmic processing bodies [5]. YTHDF3 is capable of promoting both translation and decay of m6A-modified target transcripts [17]. HIV genomic RNA (gRNA) contains multiple m6A modifications [9, 11, 12], which positively affect virus replication by enhancing nuclear export of m6A-modified viral RNA and increasing viral gene and protein expression [9, 11]. The three m6A reader proteins, YTHDF1-3, play both a negative and positive role in HIV replication. While it is well established that YTHDF1-3 bind to HIV RNA transcript in infected cells [9, 12, 19], there are conflicting reports on whether
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