Direct Analysis of HIV mRNA m6A Methylation by Nanopore Sequencing.

The post-transcriptional processing and chemical modification of HIV RNA are understudied aspects of HIV virology, primarily due to the limited ability to accurately map and quantify RNA modifications. Modification-specific antibodies or modification-sensitive endonucleases coupled with short-read RNA sequencing technologies have allowed for low-resolution or limited mapping of important regulatory modifications of HIV RNA such as N6-methyladenosine (m6A). However, a high-resolution map of where these sites occur on HIV transcripts is needed for detailed mechanistic understanding. This has recently become possible with new sequencing technologies. Here, we describe the direct RNA sequencing of HIV transcripts using an Oxford Nanopore Technologies sequencer and the use of this technique to map m6A at near single nucleotide resolution. This technology also provides the ability to identify splice variants with long RNA reads and thus, can provide high-resolution RNA modification maps that distinguish between overlapping splice variants. The protocols outlined here for m6A also provide a powerful paradigm for studying any other RNA modifications that can be detected on the nanopore platform.

HIV-1 Rev-RRE functional activity in primary isolates is highly dependent on minimal context-dependent changes in Rev.

During HIV infection, intron-containing viral mRNAs are exported from the cell nucleus to the cytoplasm to complete the replication cycle. Cellular restrictions on the export of incompletely spliced transcripts are overcome by a viral protein, Rev, and an RNA structure found in all unspliced and incompletely spliced viral mRNAs, the Rev Response Element (RRE). Primary HIV isolates display substantial variation in the sequence and functional activity of Rev proteins. We analyzed Rev from two primary isolates with disparate activity that resulted in differences in in vitro fitness of replication-competent viral constructs. The results showed that amino acid differences within the oligomerization domain, but not the arginine-rich motif or the nuclear export signal, determined the level of Rev activity. Two specific amino acid substitutions were sufficient to alter the low-activity Rev to a high-activity phenotype. Other mutations in Rev sequences had unpredictable effects on activity that differed between the two Rev backbones. The sensitivity of Rev function level to small sequence changes likely permits modulation of Rev-RRE activity during HIV infection, which may play a role in pathogenesis. The functional consequences of Rev mutations differed between primary isolates, highlighting the challenge of generalizing studies of Rev conducted using laboratory HIV strains.

Assisters Succeed in Insurance Navigation for People Living with HIV and People at Increased Risk of HIV in a Complex Coverage Landscape.

Insurance enrollment is complex for people living with HIV (PLWH) and people at increased risk for HIV, in part, owing to needing to ensure access to adequate provider networks and appropriate formularies. Insurance for PLWH facilitates access to HIV care/treatment and, ultimately, viral suppression, which has the individual benefit of longevity and the public health benefit of decreased HIV transmission. For people at increased risk for HIV, access to insurance facilitates improved access to HIV biomedical prevention, which has the individual benefit of elimination of transmission risk and the public health benefit of decreased HIV transmission. The objective of this study was to explore perceptions of priorities related to plan navigation, barriers and facilitators for enrolling and maintaining insurance coverage, and questions related to regional, state, and federal policies impacting plans provided both on and off the Affordable Care Act (ACA) marketplace. We interviewed a national sample of assisters (n = 40), who specialize in insurance plan selection for these populations. We found that assisters tailor their approaches to HIV-specific and person-specific concerns by navigating challenges related to affordability, formularies, and provider networks. In a complex coverage landscape during a time of uncertainty about the long-term future of the ACA, assisters have mastered the ability to simplify the insurance selection process for a vulnerable population. Assisters have excelled at incorporating insurance literacy education and encouraging client engagement in the process. Assisters play an essential role in the current complicated and fragmented United States' health care delivery system for PLWH and people at increased risk for HIV and could be incorporated into the Ending the HIV Epidemic initiative.