Biology subjects

Rekosh, D.

Publications and source records attributed to Rekosh, D..

7 recordsLinked to original sources

bioRxiv2025

Identification and Characterization of Functional and Trans-dominant Negative HERV-K (HML-2) Rec Proteins Encoded in the Human Genome.

Human Endogenous Retroviruses K (HERV-K) of the HML-2 subgroup are the most recently integrated and biologically active retroviral elements within the human genome. The HERV-K Rec protein, a functional homolog of HIV Rev and HTLV Rex, is necessary for the nuclear export of viral mRNAs with retained introns. However, the diversity of Rec proteins encoded in the human genome and their functional capacities have remained largely unexplored. We identified full-length Rec protein sequences in the human genome and selected 23 variants for functional characterization. Using a dual-color fluorescent reporter and a complementary ELISA assay, we found that Rec proteins from only 7 genomic loci were functional. In addition, a subset of the non-functional Rec proteins exerted trans-dominant negative effects. Detailed mutational analysis of the most potent inhibitory variant, encoded by the HERV-K provirus 12q14.1, displayed only 2 amino acid changes (H2N and del34E) relative to the prototypical functional Rec protein encoded by the reconstructed consensus HERV-K (HERV-K Con). Insertion of a glutamic acid at position 34 restored the functional activity, while the substitution of histidine with asparagine at position 2 did not. Our results unveil an unexpected complexity in HERV-K (HML-2) post-transcriptional regulation, with Rec variants displaying a spectrum of activities ranging from robust nuclear export of RNA with retained introns to trans-dominant negative inhibition of Rec function. These findings expand our understanding of the regulatory landscape governing HERV-K (HML-2) expression and suggest mechanisms by which different HERV-K Rec proteins may influence host cell biology and pathology, including oncogenesis.

cell biology↗

bioRxiv2025

Rev-RRE activity modulates HIV-1 replication and latency reactivation: Implications for viral persistence and cure strategies

The HIV-1 Rev-RRE regulatory axis plays a crucial role in viral replication by facilitating the nucleo-cytoplasmic export and expression of viral mRNAs with retained introns. In this study, we investigated the impact of variation in Rev-RRE functional activity on HIV-1 replication kinetics and reactivation from latency. Using a novel HIV-1 clone with an interchangeable Rev cassette, we engineered viruses with different Rev functional activities and demonstrated that higher Rev-RRE activity confers greater viral replication capacity while maintaining a constant level of Nef expression. In addition, a low Rev activity virus rapidly acquired a compensatory mutation in the RRE that significantly increased Rev-RRE activity and replication. In a latency model, proviruses with differing Rev-RRE activity levels varied in the efficiency of viral reactivation, affecting both initial viral release and subsequent replication kinetics. These results demonstrate that activity differences in the Rev-RRE axis among different viral isolates have important implications for HIV replication dynamics and persistence. Importantly, our findings indicate that bolstering Rev/RRE activity could be explored as part of latency reversal strategies in HIV cure efforts. Author SummaryThe activity of the HIV-1 Rev-RRE axis is essential for viral replication and varies among primary viral isolates. However, the role of this for viral fitness, evolution, and persistence has not previously been investigated. Our results show that during in vitro replication, there is a selective fitness advantage for viruses with higher Rev-RRE activity and that HIV has the ability to fine tune this regulatory system with minimal sequence changes. Additionally, the maintenance of Nef expression in low Rev activity viruses suggests a potential mechanism for balancing immune evasion and replication capacity in different selection landscapes within a host. We also show that viruses with low Rev-RRE activity are more difficult to reverse from latency than viruses with higher Rev-RRE activity. Thus, differences in provirus Rev-RRE activity may be a barrier to developing effective latency reversal strategies. These findings provide new insights into the complex roles that the Rev/RRE axis plays in functionality, viral fitness, evolution, and persistence.

microbiology↗

bioRxiv2023

Rev-Rev Response Element Activity Selection Bias at the HIV Transmission Bottleneck

HIV is not efficiently transmitted between hosts, and selection of viral variants occurs during the process of sexual transmission. The factors that confer selective advantage at the transmission bottleneck remain incompletely understood. We explored whether differences in the Rev-Rev Response Element (RRE) regulatory axis of HIV affect transmission fitness, since functional variation in the Rev-RRE axis in different viral isolates has been shown to affect replication kinetics and relative expression of many HIV proteins. Single genome HIV sequences were identified from nine linked subject pairs near the time of female-to-male transmission. Using a rapid flow-cytometric assay, we found that the functional Rev-RRE activity varied significantly between isolates. Moreover, it was generally lower in recipients viruses compared to the corresponding donor viruses. In six of nine transmission events, recipient virus Rev-RRE activity clustered at the extreme low end of the range of donor virus activity. Rev-RRE pair activity was an unpredictable product of component Rev and RRE activity variation. These data indicate selection pressure on the Rev-RRE axis during female-to-male sexual transmission. Variation in the activity of the Rev-RRE axis may permit viral adaptation to different fitness landscapes and could play an important role in HIV pathogenesis.

microbiology↗

bioRxiv2022

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 have to be exported efficiently from the host cell nucleus to the cytoplasm in order to complete the replication cycle. To overcome cellular restrictions to export incompletely spliced transcripts, HIV encodes a protein, Rev, that is constitutively expressed from a completely spliced transcript. Rev is then imported into the nucleus where it binds to an RNA structure on intron-containing viral mRNAs called the Rev Response Element (RRE). Bound Rev multimerizes and recruits cellular factors that permit the nuclear export of the resulting ribonucleoprotein complex. Primary HIV isolates display substantial variation in the functional activity of the Rev-RRE axis, which may permit viral adaptation to differing immune environments. We describe two subtype G primary isolates with disparate Rev activity. Rev activity was correlated with in vitro fitness of replication-competent viral constructs. Amino acid differences within the oligomerziation domain, but not within the arginine-rich motif or nuclear export signal, determined the different levels of Rev activity. Two specific amino acid substitutions were demonstrated to be able to alter the low-activity Rev to a high-activity phenotype. However, introducing the original amino acids from the the low activity Rev into high activity Rev in this position did not result in significant alterations in activity, highlighting the importance of the broader sequence context for functional activity. These results demonstrate that studies of Rev and RRE activity variation, which may have broader implications for HIV transmission and pathogenesis, should include sequences from primary isolates, as findings using only laboratory-adapted strains cannot be generalized.

microbiology↗

bioRxiv2019

HIV-1 Rev interacts with HERV-K RcREs present in the human genome and promotes export of unspliced HERV-K proviral RNA

BackgroundThe HERV-K (HML-2) viruses are the youngest of the human endogenous retroviruses. They are present as several almost complete proviral copies and numerous fragments in the human genome. Many HERV-K proviruses express a regulatory protein Rec, which binds to an element present in HERV-K mRNAs called the RcRE. This interaction is necessary for the nucleo-cytoplasmic export and expression of HERV-K mRNAs that retain introns and plays a role analogous to that of Rev and the RRE in HIV replication. There are over 900 HERV-K RcREs distributed throughout the human genome. Thus, it was of interest to determine if Rev could functionally interact with selected RcRE elements that map either to HERV-K proviruses or human gene regions. This interaction would have the potential to alter the expression of both HERV-K mRNAs and cellular mRNAs during HIV-1 infection. ResultsIn this study we employed a combination of RNAseq, bioinformatics and cell-based functional assays. Potential RcREs were identified through a number of bioinformatic approaches. They were then tested for their ability to promote export and translation of a reporter mRNA with a retained intron in conjunction with Rev or Rec. Some of the selected elements functioned well with either Rev, Rec or both, whereas some showed little or no function. Rev function on individual RcREs varied and was also dependent on the Rev sequence. We also performed RNAseq on total and cytoplasmic RNA isolated from SupT1 cells expressing HIV Rev, with or without Tat, or HERV-K Rec. Proviral mRNA from three HERV-K loci (4p16.1b, 22q11.23 and most significantly 3q12.3) accumulated in the cytoplasm in the presence of Rev or Tat and Rev, but not Rec. Consistent with this, the 3 RcRE from 3q12.3 functioned well with HIV-Rev in our reporter assay. In contrast, this RcRE showed little or no function with Rec. ConclusionsThe HIV Rev protein can functionally interact with many RcREs present in the human genome, depending on the RcRE sequence, as well as the Rev sequence. This leads to export of some of the HERV-K proviral mRNAs and also has the potential to change the expression of non-viral genes.

cell biology↗

bioRxiv2019

An assay to measure the activity of RNA elements and proteins that promote the expression of mRNA with retained introns

The ability to overcome cellular restrictions that exist for the export and translation of mRNAs with retained introns is a requirement for the replication of retroviruses and also for the expression of many mRNA isoforms transcribed from cellular genes. In some cases, RNA structures have been identified in the mRNA that directly interact with cellular factors to promote the export and expression of isoforms with retained introns. In other cases, a viral protein is also required to act as an adapter. In this report we describe a novel vector system that allows measurement of the ability of cis- and trans-acting factors to promote the export and translation of mRNA with retained introns. One reporter vector used in this system is derived from an HIV proviral clone engineered to express two different fluorescent proteins from spliced and unspliced transcripts. The ratio of fluorescent signals is a measurement of the efficiency of export and translation. A second vector utilizes a third fluorescent protein to measure the expression of viral export proteins that interact with some of the export elements. Both vectors can be packaged into viral particles and be used to transduce cells, allowing expression at physiological levels from the integrated vector.

molecular biology↗

bioRxiv2018

Evolution of the HIV-1 RRE during natural infection reveals key nucleotide changes that correlate with altered structure and increased activity over time

AbstractThe HIV-1 Rev Response Element (RRE) is a cis-acting RNA element characterized by multiple stem-loops. Binding and multimerization of the HIV Rev protein on the RRE promotes nucleocytoplasmic export of incompletely spliced mRNAs, an essential step in HIV replication. Most of our understanding of the Rev-RRE regulatory axis comes from studies of lab-adapted HIV clones. However, in human infection, HIV evolves rapidly and mechanistic studies of naturally occurring Rev and RRE sequences are essential to understanding this system. We previously described the functional activity of two RREs found in circulating viruses in a patient followed during the course of HIV infection. The "early" RRE was less functionally active than the "late" RRE despite differing in sequence by only four nucleotides. In this study, we describe the sequence, function, and structural evolution of circulating RREs in this patient using plasma samples collected over six years of untreated infection. RRE sequence diversity varied over the course of infection with evidence of selection pressure that led to sequence convergence as disease progressed. An increase in RRE functional activity was observed over time, and a key mutation was identified that correlates with a major conformational change in the RRE and increased functional activity. Additional mutations were found that may have contributed to increased activity as a result of greater Shannon entropy in RRE stem-loop II, which is key to primary Rev binding. ImportanceHIV-1 replication requires interaction of the viral Rev protein with a cis-acting regulatory RNA, the Rev Response Element (RRE), whose sequence changes over time during infection within a single host. In this study, we show that the RRE is subject to selection pressure and that RREs from later time points in infection tend to have higher functional activity. Differences in RRE functional activity are attributable to specific changes in RNA structure. Our results suggest that RRE evolution during infection may be important for HIV pathogenesis and that efforts to develop therapies acting on this viral pathway should take this into account.

molecular biology↗