Direct RNA sequencing confirms how RNA chemical modifications benefit HIV-1

Sequencing of full-length viral RNA reveals molecular-level effect

Numerous HIV-1 virus particles replicate from a segment of an H9 T cell, a step in HIV infection supported by the RNA chemical modifications described in the study.

A chemical modification in the HIV-1 RNA genome whose function has been a matter of scientific debate is now confirmed to be key to the virus’s ability to survive and thrive after infecting host cells, a new study has found.

This change to HIV-1 RNA, a tiny chemical modification on the adenosine building block of RNA known as m6A, is a common RNA editing process in all life forms that involves altering gene expression and protein production. The functional effect often represents a cellular solution but, in some cases, leads to disease.

By developing technological advances to observe a full length of HIV-1 RNA, researchers at The Ohio State University discovered the m6A modification occurs nearly exclusively at three specific locations on the HIV-1 RNA genome – out of the total 242 potential sites that can harbor an m6A – and these three m6As are crucial in viral replication. The finding suggested that redundancy was built into the system, and further analyses suggested that is, indeed, the case with HIV-1.

“These sites are very important for producing virus proteins and for producing viral genomic RNA,” said senior study author Sanggu Kim, associate professor of veterinary biosciences and an investigator in the Center for Retrovirus Research at The Ohio State University.

“An intriguing question is, why does HIV maintain multiple m6As? Our conclusion is that m6A is so important that HIV wants to have multiples to have redundancy. If it loses one or two, it’s OK. If it loses all three, it’s a problem.”

Though any drug development associated with this work is years away, Kim said the finding suggests targeting the site-specific m6A modifications could be the basis of designing an important new treatment for HIV infection.

The study is published today (April 11, 2024) in the journal Nature Microbiology.

HIV-1, the most common type of the human immunodeficiency virus, attacks immune cells and uses them to make copies of itself. An estimated 1.2 million people in the United States have HIV, according to the 2023 report from the Centers for Disease Control and Prevention.

The virus is a good example of why research on RNA modification has been getting a lot of attention in recent years, Kim said. Once thought of as the “middle guy” between DNA’s genes and life-sustaining proteins, RNA is now known to contain not just genetic information, but also to possess functional significance – in part because of the chemical modifications that accompany its messenger task.

“Especially because HIV is an RNA virus with a very compact RNA genome, it has to encode all of the survival information within its RNA genome – it’s using not only nucleotide sequences, but all of the chemical and structural features of RNA as codes to execute its infection of host cells,” Kim said. “We know every aspect of RNA function is very important, but we don’t really know how exactly these chemical and structural modifications of RNAs regulate virus infection.”

Though the m6A (short for N6-methyladenosine) modification was known to exist in HIV-1, previous studies had produced conflicting results about whether it helped or harmed the virus, primarily because its location was unknown and efforts to understand its effect were based on knocking out host cell genes rather than mutating the virus genome itself.

Kim and colleagues used – and refined – a technique called nanopore direct RNA sequencing to view a full length of HIV-1’s RNA genome, which is tricky to observe because RNA is a notoriously unstable and complex molecule.

Nanopore direct RNA sequencing of full-length HIV-1 RNA
points to the site-specific function of m6As

Fig. 1

a, A schematic view of multiplex RT. The polyadenylated RNAs were selectively sequenced using a RT adaptor (RTA). b, Read length distribution. Unlike the conventional methods, our multiplex RT with 111 oligos enabled a consistent and reproducible recovery of full-length HIV-1 RNA. The arrowheads denote 9.2 Kb virion RNA (left) and intracellular US, PS and CS HIV-1 RNAs (right). Rep1–3 (left) and rep1–4 (right) denote repeated experiments using independently prepared samples. c, A total of 3,985 full-length virion RNA reads, 5,411 reads of IVT RNAs (canonical control) and 1,450 IVT RNA subreads (baseline control) were analysed using Tombo-MSC, Eligos2 and Nanocompore. ‘d values’ from Tombo-MSC, ‘odd ratios’ from Eligos2 and absolute values (ABS) of ‘logit LOR score’ from Nanopore are shown. Site-specific modifications exhibit a robust and distinct signal, while the signals from non-site-specific modifications are diluted in these population-level analyses. d, To identify modification signals common in the three analyses, the top 149 peaks of Tombo-MSC (d value >0.05 based), 167 of Eligos2 data (odd ratios >2.4) and 156 peaks from Nanocompore results (logit LOR score >0.73) were cross-compared (Supplementary Fig. 7). A total of 25 signal peaks common in Tombo-MSC and Eligos2 (open circles) and seven peaks common in all three analyses (purple circles) are shown. Crosses denote DRACH sites. The 25 common sites are significantly enriched in DRACH sites. e, The magnified HIV-1 genome from 7,916 to 9,172 of HIV-1 genome (NL4-3 strain) and sequence logo plots for circulating HIV-1 are shown. Two adjacent sites (8,975 and 8,989) were located immediately upstream of the G-quadruplexes (G4s) in the U3, and the m6A at 8,079 and 8,110 are located immediately upstream of the potential G4 within the rev/env region downstream of the A7 splicing acceptor.

The team first discovered the three m6A modifications and their specific locations. From there, the researchers analyzed individual RNA molecules with distinct ensembles of m6A modifications, including those with multiple m6As and those with just one of the three m6As. They found that any ensemble of m6A modifications, regardless of the number or the position of m6As, produced similar functional changes. Removal of all three, however, caused devastating effects to viruses – a dead giveaway that these m6As are redundant.

“Until now we didn’t know which exact nucleotides are modified and how they function, and how it’s important for viruses or how it’s important for cells. Our paper addresses the keys to these important questions,” Kim said.

“Why would HIV need all three modifications if they’re functioning in the same way?” he said. “Our study is the first to show that HIV-1 utilizes this unique, important mechanism at the RNA level for its evolutionary benefit.”

Almost all existing HIV drugs block virus replication, but no medications inhibit viral RNA and protein production. There is more to learn about the RNA modification in HIV-1, but Kim said the work hints at the potential to develop therapies that could target these later steps.

SourceThe Ohio State University

Baek A, Lee GE, Golconda S, Rayhan A, Manganaris AA, Chen S, Tirumuru N, Yu H, Kim S, Kimmel C, Zablocki O, Sullivan MB, Addepalli B, Wu L, Kim S. (2024) Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m6As. Nat Microbiol [Epub ahead of print]. [article]

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