Altered HIV-1 mRNA Splicing Due to Drug-Resistance-Associated Mutations in Exon 2/2b

The underlying molecular mechanism and their general effect on the replication capacity of HIV 1 drug-resistance-associated mutations is often poorly understood. To elucidate the effect of two such mutations located in a region with a high density of spicing regulatory elements on the HIV-1-splicing outcome, bioinformatic predictions were combined with transfection and infection experiments. Results show that the previously described R263K drug-resistance-associated integrase mutation has additionally a severe effect on the ESE2b splicing regulatory element (SRE) in exon 2b, which causes loss of SD2b recognition. This was confirmed by an R263R silent mutation with a similar predicted effect on the exon 2b SRE. In contrast, a V260I mutation and its silent counterpart with a lower effect on ESS2b did not exhibit any differences in the splicing pattern. Since HIV-1 highly relies on a balanced splicing reaction, changes in the splicing outcome can contribute to changes in viral replication and might add to the effect of escape mutations toward antiviral drugs. Thus, a classification of mutations purely addressing proteins is insufficient.

Download Full-text

Negative and Positive mRNA Splicing Elements Act Competitively To Regulate Human Immunodeficiency Virus Type 1 Vif Gene Expression

Journal of Virology ◽

10.1128/jvi.01558-07 ◽

2008 ◽

Vol 82 (8) ◽

pp. 3921-3931 ◽

Cited By ~ 37

Author(s):

C. M. Exline ◽

Z. Feng ◽

C. M. Stoltzfus

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Regulatory Elements ◽

Mrna Splicing ◽

Viral Mrnas ◽

Exon 2 ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3′ splice sites (3′ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3′ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5′ splice site (5′ss) downstream of exon 2 (5′ss D2). Here we show that the mutations within 5′ss D2 that are predicted to lower or increase the affinity of the 5′ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5′ss D2 was not necessary for the effect of 5′ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5′ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5′-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5′ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication.

Download Full-text

Elucidation of the Molecular Mechanism Driving Duplication of the HIV-1 PTAP Late Domain

Journal of Virology ◽

10.1128/jvi.01640-15 ◽

2015 ◽

Vol 90 (2) ◽

pp. 768-779 ◽

Cited By ~ 15

Author(s):

Angelica N. Martins ◽

Abdul A. Waheed ◽

Sherimay D. Ablan ◽

Wei Huang ◽

Alicia Newton ◽

...

Keyword(s):

Drug Resistance ◽

Antiretroviral Therapy ◽

Protease Inhibitor ◽

Protease Inhibitors ◽

Biochemical Analysis ◽

Replication Capacity ◽

Resistance Mutations ◽

Cellular Machinery ◽

Ptap Motif ◽

Hiv 1

ABSTRACTHIV-1 uses cellular machinery to bud from infected cells. This cellular machinery is comprised of several multiprotein complexes known as endosomal sorting complexes required for transport (ESCRTs). A conserved late domain motif, Pro-Thr-Ala-Pro (PTAP), located in the p6 region of Gag (p6Gag), plays a central role in ESCRT recruitment to the site of virus budding. Previous studies have demonstrated that PTAP duplications are selected in HIV-1-infected patients during antiretroviral therapy; however, the consequences of these duplications for HIV-1 biology and drug resistance are unclear. To address these questions, we constructed viruses carrying a patient-derived PTAP duplication with and without drug resistance mutations in the viral protease. We evaluated the effect of the PTAP duplication on viral release efficiency, viral infectivity, replication capacity, drug susceptibility, and Gag processing. In the presence of protease inhibitors, we observed that the PTAP duplication in p6Gagsignificantly increased the infectivity and replication capacity of the virus compared to those of viruses bearing only resistance mutations in protease. Our biochemical analysis showed that the PTAP duplication, in combination with mutations in protease, enhances processing between the nucleocapsid and p6 domains of Gag, resulting in more complete Gag cleavage in the presence of protease inhibitors. These results demonstrate that duplication of the PTAP motif in p6Gagconfers a selective advantage in viral replication by increasing Gag processing efficiency in the context of protease inhibitor treatment, thereby enhancing the drug resistance of the virus. These findings highlight the interconnected role of PTAP duplications and protease mutations in the development of resistance to antiretroviral therapy.IMPORTANCEResistance to current drug therapy limits treatment options in many HIV-1-infected patients. Duplications in a Pro-Thr-Ala-Pro (PTAP) motif in the p6 domain of Gag are frequently observed in viruses derived from patients on protease inhibitor (PI) therapy. However, the reason that these duplications arise and their consequences for virus replication remain to be established. In this study, we examined the effect of PTAP duplication on PI resistance in the context of wild-type protease or protease bearing PI resistance mutations. We observe that PTAP duplication markedly enhances resistance to a panel of PIs. Biochemical analysis reveals that the PTAP duplication reverses a Gag processing defect imposed by the PI resistance mutations in the context of PI treatment. The results provide a long-sought explanation for why PTAP duplications arise in PI-treated patients.

Download Full-text

Analysis of Competing HIV-1 Splice Donor Sites Uncovers a Tight Cluster of Splicing Regulatory Elements within Exon 2/2b

Journal of Virology ◽

10.1128/jvi.00389-17 ◽

2017 ◽

Vol 91 (14) ◽

Cited By ~ 9

Author(s):

Anna-Lena Brillen ◽

Lara Walotka ◽

Frank Hillebrand ◽

Lisa Müller ◽

Marek Widera ◽

...

Keyword(s):

Protein Expression ◽

Viral Replication ◽

Particle Production ◽

Mutational Analysis ◽

Regulatory Elements ◽

Host Cells ◽

Mass Spectrometry Analysis ◽

Start Codon ◽

Exon 2 ◽

Hiv 1

ABSTRACT The HIV-1 accessory protein Vif is essential for viral replication by counteracting the host restriction factor APOBEC3G (A3G), and balanced levels of both proteins are required for efficient viral replication. Noncoding exons 2/2b contain the Vif start codon between their alternatively used splice donors 2 and 2b (D2 and D2b). For vif mRNA, intron 1 must be removed while intron 2 must be retained. Thus, splice acceptor 1 (A1) must be activated by U1 snRNP binding to either D2 or D2b, while splicing at D2 or D2b must be prevented. Here, we unravel the complex interactions between previously known and novel components of the splicing regulatory network regulating HIV-1 exon 2/2b inclusion in viral mRNAs. In particular, using RNA pulldown experiments and mass spectrometry analysis, we found members of the heterogeneous nuclear ribonucleoparticle (hnRNP) A/B family binding to a novel splicing regulatory element (SRE), the exonic splicing silencer ESS2b, and the splicing regulatory proteins Tra2/SRSF10 binding to the nearby exonic splicing enhancer ESE2b. Using a minigene reporter, we performed bioinformatics HEXplorer-guided mutational analysis to narrow down SRE motifs affecting splice site selection between D2 and D2b. Eventually, the impacts of these SREs on the viral splicing pattern and protein expression were exhaustively analyzed in viral particle production and replication experiments. Masking of these protein binding sites by use of locked nucleic acids (LNAs) impaired Vif expression and viral replication. IMPORTANCE Based on our results, we propose a model in which a dense network of SREs regulates vif mRNA and protein expression, crucial to maintain viral replication within host cells with varying A3G levels and at different stages of infection. This regulation is maintained by several serine/arginine-rich splicing factors (SRSF) and hnRNPs binding to those elements. Targeting this cluster of SREs with LNAs may lead to the development of novel effective therapeutic strategies.

Download Full-text

Impaired Replication Capacity of Acute/Early Viruses in Persons Who Become HIV Controllers

Journal of Virology ◽

10.1128/jvi.00286-10 ◽

2010 ◽

Vol 84 (15) ◽

pp. 7581-7591 ◽

Cited By ~ 93

Author(s):

Toshiyuki Miura ◽

Zabrina L. Brumme ◽

Mark A. Brockman ◽

Pamela Rosato ◽

Jennifer Sela ◽

...

Keyword(s):

Drug Resistance ◽

Viral Replication ◽

Acute Infection ◽

Viral Fitness ◽

Replication Capacity ◽

Transmitted Drug Resistance ◽

Resistance Mutations ◽

Drug Resistance Mutations ◽

Hiv Controllers ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) controllers maintain viremia at <2,000 RNA copies/ml without antiretroviral therapy. Viruses from controllers with chronic infection were shown to exhibit impaired replication capacities, in part associated with escape mutations from cytotoxic-T-lymphocyte (CTL) responses. In contrast, little is known about viruses during acute/early infection in individuals who subsequently become HIV controllers. Here, we examine the viral replication capacities, HLA types, and virus sequences from 18 HIV-1 controllers identified during primary infection. g ag-protease chimeric viruses constructed using the earliest postinfection samples displayed significantly lower replication capacities than isolates from persons who failed to control viremia (P = 0.0003). Protective HLA class I alleles were not enriched in these early HIV controllers, but viral sequencing revealed a significantly higher prevalence of drug resistance mutations associated with impaired viral fitness in controllers than in noncontrollers (6/15 [40.0%] versus 10/80 [12.5%], P = 0.018). Moreover, of two HLA-B57-positive (B57+) controllers identified, both harbored, at the earliest time point tested, signature escape mutations within Gag that likewise impair viral replication capacity. Only five controllers did not express “protective” alleles or harbor viruses with drug resistance mutations; intriguingly, two of them displayed typical B57 signature mutations (T242N), suggesting the acquisition of attenuated viruses from B57+ donors. These data indicate that acute/early stage viruses from persons who become controllers have evidence of reduced replication capacity during the initial stages of infection which is likely associated with transmitted or acquired CTL escape mutations or transmitted drug resistance mutations. These data suggest that viral dynamics during acute infection have a major impact on HIV disease outcome.

Download Full-text

Splicing Regulatory Elements within tat Exon 2 of Human Immunodeficiency Virus Type 1 (HIV-1) Are Characteristic of Group M but Not Group O HIV-1 Strains

Journal of Virology ◽

10.1128/jvi.73.12.9764-9772.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 9764-9772 ◽

Cited By ~ 15

Author(s):

Patricia S. Bilodeau ◽

Jeffrey K. Domsic ◽

C. Martin Stoltzfus

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Splice Site ◽

Regulatory Elements ◽

Splice Sites ◽

Exon 2 ◽

Immunodeficiency Virus ◽

Splicing Regulatory Elements ◽

Hiv 1

ABSTRACT In the NL4-3 strain of human immunodeficiency virus type 1 (HIV-1), regulatory elements responsible for the relative efficiencies of alternative splicing at the tat, rev, and theenv/nef 3′ splice sites (A3 through A5) are contained within the region of tat exon 2 and its flanking sequences. Two elements affecting splicing of tat, rev, and env/nef mRNAs have been localized to this region. First, an exon splicing silencer (ESS2) in NL4-3, located approximately 70 nucleotides downstream from the 3′ splice site used to generatetat mRNA, acts specifically to inhibit splicing at this splice site. Second, the A4b 3′ splice site, which is the most downstream of the three rev 3′ splice sites, also serves as an element inhibiting splicing at the env/nef 3′ splice site A5. These elements are conserved in some but not all HIV-1 strains, and the effects of these sequence changes on splicing have been investigated in cell transfection and in vitro splicing assays. SF2, another clade B virus and member of the major (group M) viruses, has several sequence changes within ESS2 and uses a differentrev 3′ splice site. However, splicing is inhibited by the two elements similarly to NL4-3. As with the NL4-3 strain, the SF2 A4b AG dinucleotide overlaps an A5 branchpoint, and thus the inhibitory effect may result from competition of the same site for two different splicing factors. The sequence changes in ANT70C, a member of the highly divergent outlier (group O) viruses, are more extensive, and ESS2 activity in tat exon 2 is not present. Group O viruses also lack the rev 3′ splice site A4b, which is conserved in all group M viruses. Mutagenesis of the most downstream rev3′ splice site of ANT70C does not increase splicing at A5, and all of the branchpoints are upstream of the two rev 3′ splice sites. Thus, splicing regulatory elements in tat exon 2 which are characteristic of most group M HIV-1 strains are not present in group O HIV-1 strains.

Download Full-text

Simian-Tropic HIV as a Model To Study Drug Resistance against Integrase Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04829-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 1942-1949 ◽

Cited By ~ 6

Author(s):

Melissa Wares ◽

Said Hassounah ◽

Thibault Mesplède ◽

Paul A. Sandstrom ◽

Mark A. Wainberg

Keyword(s):

Drug Resistance ◽

Nonhuman Primate ◽

Hiv Treatment ◽

Replication Capacity ◽

Primate Model ◽

Primary Resistance ◽

Immunodeficiency Virus ◽

The Impact ◽

Hiv 1

ABSTRACTDrug resistance represents a key aspect of human immunodeficiency virus (HIV) treatment failure. It is important to develop nonhuman primate models for studying issues of drug resistance and the persistence and transmission of drug-resistant viruses. However, relatively little work has been conducted using either simian immunodeficiency virus (SIV) or SIV/HIV recombinant viruses for studying resistance against integrase strand transfer inhibitors (INSTIs). Here, we used a T-cell-tropic SIV/HIV recombinant virus in which the capsid andvifregions of HIV-1 were replaced with their SIV counterparts (simian-tropic HIV-1 [stHIV-1](SCA,SVIF)) to study the impact of a number of drug resistance substitutions in the integrase coding region at positions E92Q, G118R, E138K, Y143R, S153Y, N155H, and R263K on drug resistance, viral infectivity, and viral replication capacity. Our results show that each of these substitutions exerted effects that were similar to their effects in HIV-1. Substitutions associated with primary resistance against dolutegravir were more detrimental to stHIV-1(SCA,SVIF)infectiousness than were resistance substitutions associated with raltegravir and elvitegravir, consistent with data that have been reported for HIV-1. These findings support the role of stHIV-1(SCA,SVIF)as a useful model with which to evaluate the role of INSTI resistance substitutions on viral persistence, transmissibility, and pathogenesis in a nonhuman primate model.

Download Full-text

Identification of a Novel Posttranscriptional Regulatory Element by Using a rev- and RRE-Mutated Human Immunodeficiency Virus Type 1 DNA Proviral Clone as a Molecular Trap

Journal of Virology ◽

10.1128/jvi.75.10.4558-4569.2001 ◽

2001 ◽

Vol 75 (10) ◽

pp. 4558-4569 ◽

Cited By ~ 29

Author(s):

Filomena Nappi ◽

Ralf Schneider ◽

Andrei Zolotukhin ◽

Sergey Smulevitch ◽

Daniel Michalowski ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Regulatory Element ◽

Mrna Export ◽

Regulatory Elements ◽

Control Element ◽

Rna Transport ◽

Human Lymphoid Cell ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus (HIV) and all other lentiviruses utilize the essential viral protein Rev, which binds to RRE RNA, to export their unspliced and partially spliced mRNAs from the nucleus. We used a rev- and RRE-defective HIV type 1 (HIV-1) molecular clone in complementation experiments to establish a method for the rapid isolation of posttranscriptional regulatory elements from the mammalian genome by selecting for rescue of virus replication. Viruses rescued by this method contained a novel element with homology to rodent intracisternal A-particle (IAP) retroelements. A functional element was contained within a 247-nucleotide fragment named RNA transport element (RTE), which was able to promote replication of the Rev- and RRE-defective HIV-1 in both human lymphoid cell lines and primary lymphocytes, demonstrating its potent posttranscriptional function. RTE was functional in many cell types, indicating that the cellular factors that recognize RTE are widely expressed and evolutionarily conserved. RTE also promoted RNA export fromXenopus oocyte nuclei. RTE-mediated RNA transport was CRM1 independent, and RTE did not show high affinity for binding to mRNA export factor TAP/NXF1. Since CRM1 and TAP/NXF1 are critical export receptors associated with the two recognized mRNA export pathways, these results suggest that RTE functions via a distinct export mechanism. Taken together, our results identify a novel posttranscriptional control element that uses a conserved cellular export mechanism.

Download Full-text

In Vivo Validation of a Bioinformatics Based Tool to Identify Reduced Replication Capacity in HIV-1

The Open Medical Informatics Journal ◽

10.2174/1874431101004010225 ◽

2010 ◽

Vol 4 (1) ◽

pp. 225-232

Author(s):

Christina M.R Kitchen ◽

Paul Krogstad ◽

Scott G Kitchen

Keyword(s):

Drug Resistance ◽

In Silico ◽

Viral Fitness ◽

Biological Assessment ◽

Relative Fitness ◽

Replication Capacity ◽

Resistance Mutations ◽

Hiv 1

Although antiretroviral drug resistance is common in treated HIV infected individuals, it is not a consistent indicator of HIV morbidity and mortality. To the contrary, HIV resistance-associated mutations may lead to changes in viral fitness that are beneficial to infected individuals. Using a bioinformatics-based model to assess the effects of numerous drug resistance mutations, we determined that the D30N mutation in HIV-1 protease had the largest decrease in replication capacity among known protease resistance mutations. To test thisin silicoresult in anin vivoenvironment, we constructed several drug-resistant mutant HIV-1 strains and compared their relative fitness utilizing the SCID-hu mouse model. We found HIV-1 containing the D30N mutation had a significant defectin vivo, showing impaired replication kinetics and a decreased ability to deplete CD4+ thymocytes, compared to the wild-type or virus without the D30N mutation. In comparison, virus containing the M184V mutation in reverse transcriptase, which shows decreased replication capacityin vitro, did not have an effect on viral fitnessin vivo. Thus, in this study we have verified anin silicobioinformatics result with a biological assessment to identify a unique mutation in HIV-1 that has a significant fitness defectin vivo.

Download Full-text