The ability of HIV to hide in a latent state, lying dormant within host cells, is one of the biggest challenges in combating the virus. Researchers have long sought to understand how the virus manipulates the host’s genetic machinery to maintain its grip, especially during latency. A recent study sheds light on this mystery by focusing on the process of alternative splicing (AS) and introduces a powerful new tool, MrHAMER2, for exploring HIV’s behavior during latency.

What is Alternative Splicing?

Alternative splicing is a natural process where cells can produce multiple versions of proteins from a single gene. This is like cutting and reassembling pieces of genetic “blueprints” to create different final products. Viruses, including HIV, have evolved to exploit this process to control their gene expression.

In the case of HIV, the virus integrates its genetic material into the host genome and uses alternative splicing to decide which viral proteins to produce. While scientists have gained some understanding of these processes during active replication, much less is known about what happens during latency—a period when the virus is inactive but still present in the body.

The Challenge of Studying HIV in Latency

Studying HIV during latency is tough because the amount of viral RNA present is extremely low, and it is hidden within specific subsets of host cells. Traditional sequencing methods often miss these subtle signals, making it difficult to fully decode the virus’s behavior.

MrHAMER2

To overcome these challenges, researchers at Seattle Children’s Research Institute developed MrHAMER2, a high-accuracy long-read RNA sequencing technology. This tool uses advanced molecular tagging techniques (called Unique Molecular Identifier or UMI tagging) to accurately identify and quantify full-length viral RNA molecules. It boasts incredible precision, with an error rate as low as 0.032%, making it ideal for studying rare and complex RNA molecules like those from latent HIV.

MrHAMER2 assay and bioinformatic components

(A) The assay component first involves reverse transcription of target RNAs with Oligo-d(T) priming. UMIs containing flanking synthetic priming sites are added to both 5’ and 3’ ends of the first-strand cDNA via gene-specific priming using respective 5’/3’ UTR sequences shared in all target isoforms. Dual UMI-tagged molecules are amplified via emulsion PCR, and libraries are prepared from the resulting amplicons using the Native Barcoding kits from ONT followed by Nanopore sequencing using R.10.4.1 chemistry. (B) The bioinformatic component involves extraction of the patterned UMIs from each read, followed by clustering of reads into bins based on dual UMI identity. A minimum cluster size filter is then applied so that a minimum number of balanced (i.e. sense and antisense) raw reads are available per dual UMI cluster to ensure sufficient error-correction. Dual UMI clusters passing the size filter are then assembled and polished into high-accuracy consensus assemblies originating from single cDNA molecules. A post-hoc chimera filtering module is then used to identify partial UMI matches present in other clusters and remove the less enriched recombinant clusters, resulting in high-accuracy single molecule sequences free of PCR sampling or chimeric artefacts.

Key Findings

Using MrHAMER2, the team explored the spliced transcriptome of HIV in a laboratory model of latency. They uncovered significant changes in the virus’s RNA, including variations with retained introns—sections of RNA that are typically removed during splicing. These retained introns can alter the ability of the virus to produce proteins, offering new insights into how HIV maintains latency and potentially reactivates.

Implications for HIV Research

Understanding how HIV manipulates alternative splicing during latency provides valuable clues for developing therapies. By targeting these unique RNA isoforms or the splicing process itself, researchers may uncover new ways to prevent the virus from reactivating or even eliminate it entirely.

A Step Toward the Future

The introduction of MrHAMER2 is a game-changer for RNA research, particularly for studying diseases like HIV where small changes in gene expression have big consequences. This breakthrough not only expands our understanding of HIV latency but also paves the way for more effective treatments in the fight against the virus.

The road to curing HIV remains challenging, but tools like MrHAMER2 are helping scientists piece together the intricate puzzle of the virus’s biology, bringing us closer to innovative and lasting solutions.

Availability – https://github.com/gallardo-seq/MrHAMER2