This study presents a single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs, revealing the functional roles of site-specific m⁶A modifications. Using nanopore direct RNA sequencing (DRS), the researchers analyzed the chemical modifications on HIV-1 RNAs at the single RNA level and nucleotide resolution. The results show that HIV-1 has a relatively simple modification landscape, with three predominant m⁶A modifications near the 3' end of the RNA genome. These m⁶A modifications are more densely installed in spliced viral messenger RNAs than in genomic RNAs and play a crucial role in maintaining normal levels of HIV-1 RNA splicing and translation. The study also shows that HIV-1 generates diverse RNA subspecies with distinct m⁶A ensembles, and maintaining multiple of these m⁶A modifications on its RNAs provides additional stability and resilience to HIV-1 replication, suggesting an unexplored viral RNA-level evolutionary strategy. The study highlights the importance of m⁶A modifications in HIV-1 replication, as their removal or mutation significantly reduces the levels of US RNAs, which are essential for producing structural proteins and genomic RNA. The researchers also found that the three m⁶A sites are highly conserved among HIV-1 subtypes and are associated with major m⁶A peaks in previous short-read sequencing studies. The study further shows that the three m⁶A sites are involved in regulating RNA splicing and translation, with the triple mutation of all three sites leading to an over-splicing phenotype and reduced viral fitness. The results suggest that m⁶A modifications play a critical role in the regulation of HIV-1 replication and that their site-specific roles are important for the virus's survival and replication. The study also demonstrates the potential of DRS technology for the study of HIV-1 RNA biology, as it allows for a comprehensive analysis of individual reads of HIV-1 RNAs in virions and in virus-producing cells. The findings provide new insights into the functional roles of m⁶A modifications in HIV-1 replication and highlight the importance of these modifications in the virus's life cycle.This study presents a single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs, revealing the functional roles of site-specific m⁶A modifications. Using nanopore direct RNA sequencing (DRS), the researchers analyzed the chemical modifications on HIV-1 RNAs at the single RNA level and nucleotide resolution. The results show that HIV-1 has a relatively simple modification landscape, with three predominant m⁶A modifications near the 3' end of the RNA genome. These m⁶A modifications are more densely installed in spliced viral messenger RNAs than in genomic RNAs and play a crucial role in maintaining normal levels of HIV-1 RNA splicing and translation. The study also shows that HIV-1 generates diverse RNA subspecies with distinct m⁶A ensembles, and maintaining multiple of these m⁶A modifications on its RNAs provides additional stability and resilience to HIV-1 replication, suggesting an unexplored viral RNA-level evolutionary strategy. The study highlights the importance of m⁶A modifications in HIV-1 replication, as their removal or mutation significantly reduces the levels of US RNAs, which are essential for producing structural proteins and genomic RNA. The researchers also found that the three m⁶A sites are highly conserved among HIV-1 subtypes and are associated with major m⁶A peaks in previous short-read sequencing studies. The study further shows that the three m⁶A sites are involved in regulating RNA splicing and translation, with the triple mutation of all three sites leading to an over-splicing phenotype and reduced viral fitness. The results suggest that m⁶A modifications play a critical role in the regulation of HIV-1 replication and that their site-specific roles are important for the virus's survival and replication. The study also demonstrates the potential of DRS technology for the study of HIV-1 RNA biology, as it allows for a comprehensive analysis of individual reads of HIV-1 RNAs in virions and in virus-producing cells. The findings provide new insights into the functional roles of m⁶A modifications in HIV-1 replication and highlight the importance of these modifications in the virus's life cycle.