Background
While the significance of chemical modifications on RNA has been well established, their specific roles in HIV-1 replication remain unclear and, at times, controversial. Previous studies have provided only low-resolution, type-specific RNA modification site mapping and have relied on indirect analyses of phenotypic effects through the overexpression or knockdown (knockout) of host effectors. These approaches fail to account for the site-specific and context-dependent functions of viral RNA modifications.
Methods
We conducted full-length, nucleotide-resolution mapping and characterization of chemical modifications on HIV-1 RNA using novel Nanopore direct RNA sequencing (DRS) and oligonucleotide LC-MS/MS methods, which we recently developed (Baek et al., Nature Microbiology, 2024; Baek et al., Methods & Protocols, 2024). Site-directed mutagenesis was employed to characterize the functional properties of HIV-1-specific chemical modifications.
Results
Our data revealed a surprisingly simple HIV-1 modification landscape, with three dominant site-specific N6-methyladenosine (m6A) modifications near the 3’ end, out of the 242 known m6A motifs (DRACH) on the HIV-1 RNA genome. Each of these three m6A marks up to 89% of HIV-1 RNAs, a level of stoichiometry exceptionally higher than that seen in typical cellular mRNAs (typically less than 20%). Knocking out one of these three m6As did not affect HIV-1 RNA production, isoform expression, viral protein expression, virion production, or infectivity. However, knocking out all three m6As (triple mutation) resulted in a significant reduction in full-length unspliced HIV-1 RNA and Gag expression, while increasing the fraction of completely spliced RNAs. Despite this apparent “over-splicing” phenotype, our single-RNA-level analysis revealed that the more m6As deposited in individual HIV-1 RNAs, the more splicing events occurred in those RNAs. These counterintuitive results can be explained by the additional role of these m6As in regulating the expression of HIV-1’s Rev protein, which is crucial for controlling unspliced RNA and Gag expression.
Conclusions
The potential HIV-1-specific and context-dependent roles of m6As have been overlooked in previous studies. Our data shed new light on the functional and evolutionary significance of m6As in HIV-1 biology. The technological innovations presented here will also serve as valuable tools for future exploration of the complex RNA biology of HIV-1.
Table or Figure
