Publications by authors named "Ronald Swanstrom"

149 Publications

β-D-N 4-hydroxycytidine (NHC) Inhibits SARS-CoV-2 Through Lethal Mutagenesis But Is Also Mutagenic To Mammalian Cells.

J Infect Dis 2021 May 7. Epub 2021 May 7.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, USA.

Mutagenic ribonucleosides can act as broad-based antiviral agents. They are metabolized to the active ribonucleoside triphosphate form and concentrate in the genomes of RNA viruses during viral replication. β-D-N 4-hydroxycytidine (NHC, the initial metabolite of molnupiravir) is more than 100-fold more active than ribavirin or favipiravir against SARS-CoV-2, with antiviral activity correlated to the level of mutagenesis in virion RNA. However, NHC also displays host mutational activity in an animal cell culture assay, consistent with RNA and DNA precursors sharing a common intermediate of a ribonucleoside diphosphate. These results indicate that highly active mutagenic ribonucleosides may hold risk for the host.
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http://dx.doi.org/10.1093/infdis/jiab247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8136050PMC
May 2021

Primer ID Next-Generation Sequencing for the Analysis of a Broad Spectrum Antiviral Induced Transition Mutations and Errors Rates in a Coronavirus Genome.

Bio Protoc 2021 Mar 5;11(5):e3938. Epub 2021 Mar 5.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, USA.

Next generations sequencing (NGS) has become an important tool in biomedical research. The Primer ID approach combined with the MiSeq platform overcomes the limitation of PCR errors and reveals the true sampling depth of population sequencing, making it an ideal tool to study mutagenic effects of potential broad-spectrum antivirals on RNA viruses. In this report we describe a protocol using Primer ID sequencing to study the mutations induced by antivirals in a coronavirus genome from an cell culture model and an mouse model. Viral RNA or total lung tissue RNA is tagged with Primer ID-containing cDNA primers during the initial reverse transcription step, followed by two rounds of PCR to amplify viral sequences and incorporate sequencing adaptors. Purified and pooled libraries are sequenced using the MiSeq platform. Sequencing data are processed using the template consensus sequence (TCS) web-app. The Primer ID approach provides an accurate sequencing protocol to measure mutation error rates in viral RNA genomes and host mRNA. Sequencing results suggested that β-D-N4-hydroxycytidine (NHC) greatly increased the transition substitution rate but not the transversion substitution rate in the viral RNA genomes, and cytosine (C) to uridine (U) was found as the most frequently seen mutation.
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http://dx.doi.org/10.21769/BioProtoc.3938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005877PMC
March 2021

HIV-1: To Splice or Not to Splice, That Is the Question.

Viruses 2021 01 26;13(2). Epub 2021 Jan 26.

Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.

The transcription of the HIV-1 provirus results in only one type of transcript-full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must undergo splicing but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full-length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 must circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy. Finally, splicing has been implicated in latency, but to date there is little supporting evidence for such a mechanism. In this review we apply what is known of cellular splicing to understand splicing in HIV-1, and present data from our newer and more sensitive deep sequencing assays quantifying the different HIV-1 transcript types.
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http://dx.doi.org/10.3390/v13020181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912102PMC
January 2021

The HIV-1 latent reservoir is largely sensitive to circulating T cells.

Elife 2020 10 6;9. Epub 2020 Oct 6.

Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, United States.

HIV-1-specific CD8+ T cells are an important component of HIV-1 curative strategies. Viral variants in the HIV-1 reservoir may limit the capacity of T cells to detect and clear virus-infected cells. We investigated the patterns of T cell escape variants in the replication-competent reservoir of 25 persons living with HIV-1 (PLWH) durably suppressed on antiretroviral therapy (ART). We identified all reactive T cell epitopes in the HIV-1 proteome for each participant and sequenced HIV-1 outgrowth viruses from resting CD4+ T cells. All non-synonymous mutations in reactive T cell epitopes were tested for their effect on the size of the T cell response, with a≥50% loss defined as an escape mutation. The majority (68%) of T cell epitopes harbored no detectable escape mutations. These findings suggest that circulating T cells in PLWH on ART could contribute to control of rebound and could be targeted for boosting in curative strategies.
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http://dx.doi.org/10.7554/eLife.57246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593086PMC
October 2020

Two Genetic Differences between Closely Related Zika Virus Strains Determine Pathogenic Outcome in Mice.

J Virol 2020 09 29;94(20). Epub 2020 Sep 29.

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Recent Zika virus (ZIKV) outbreaks and unexpected clinical manifestations of ZIKV infection have prompted an increase in ZIKV-related research. Here, we identify two strain-specific determinants of ZIKV virulence in mice. We found that strain H/PF/2013 caused 100% lethality in mice, whereas PRVABC59 caused no lethality; both strains caused 100% lethality in double-knockout (DKO) mice. Deep sequencing revealed a high-frequency variant in PRVABC59 not present in H/PF/2013: a G-to-T change at nucleotide 1965 producing a Val-to-Leu substitution at position 330 of the viral envelope (E) protein. We show that the V330 variant is lethal on both virus strain backgrounds, whereas the L330 variant is attenuating only on the PRVABC59 background. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain but not H/PF/2013. The consensus sequences of H/PF/2013 and PRVABC59 differ by 3 amino acids, but these were not responsible for the difference in virulence between the two strains. H/PF/2013 and PRVABC59 differ by an additional 31 noncoding or silent nucleotide changes. We made a panel of chimeric viruses with identical amino acid sequences but nucleotide sequences derived from H/PF/2013 or PRVABC59. We found that 6 nucleotide differences in the 3' quarter of the H/PF/2013 genome were sufficient to confer virulence in mice. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used ZIKV strains, in two widely used mouse models of ZIKV pathogenesis ( and DKO mice). Contemporary ZIKV strains are closely related and often used interchangeably in laboratory research. Here, we identify two strain-specific determinants of ZIKV virulence that are evident in only mice but not DKO mice. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain but not H/PF/2013. We further identify a second virulence determinant in the H/PF/2013 strain, which is driven by the viral nucleotide sequence but not the amino acid sequence. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used ZIKV strains, in two widely used mouse models of ZIKV pathogenesis. Our results highlight that even very closely related virus strains can produce significantly different pathogenic phenotypes in common laboratory models.
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http://dx.doi.org/10.1128/JVI.00618-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527068PMC
September 2020

Fact and Fiction about 1%: Next Generation Sequencing and the Detection of Minor Drug Resistant Variants in HIV-1 Populations with and without Unique Molecular Identifiers.

Viruses 2020 08 4;12(8). Epub 2020 Aug 4.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Next generation sequencing (NGS) platforms have the ability to generate almost limitless numbers of sequence reads starting with a PCR product. This gives the illusion that it is possible to analyze minor variants in a viral population. However, including a PCR step obscures the sampling depth of the viral population, the key parameter needed to understand the utility of the data set for finding minor variants. Also, these high throughput sequencing platforms are error prone at the level where minor variants are of interest, confounding the interpretation of detected minor variants. A simple strategy has been applied in multiple applications of NGS to solve these problems. Prior to PCR, individual molecules are "tagged" with a unique molecular identifier (UMI) that can be used to establish the actual sample size of viral genomes sequenced after PCR and sequencing. In addition, since PCR generates many copies of each sequence tagged to a specific UMI, a template consensus sequence (TCS) can be created from the many reads of each template, removing virtually all of the method error. From this perspective we examine our own use of a UMI, called Primer ID, in the detection of minor drug resistant variants in HIV-1 populations.
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http://dx.doi.org/10.3390/v12080850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472098PMC
August 2020

Structural Analysis of Potent Hybrid HIV-1 Protease Inhibitors Containing Bis-tetrahydrofuran in a Pseudosymmetric Dipeptide Isostere.

J Med Chem 2020 08 3;63(15):8296-8313. Epub 2020 Aug 3.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, United States.

The design, synthesis, and X-ray structural analysis of hybrid HIV-1 protease inhibitors (PIs) containing bis-tetrahydrofuran (bis-THF) in a pseudo-C-symmetric dipeptide isostere are described. A series of PIs were synthesized by incorporating bis-THF of darunavir on either side of the Phe-Phe isostere of lopinavir in combination with hydrophobic amino acids on the opposite P2/P2' position. Structure-activity relationship studies indicated that the bis-THF moiety can be attached at either the P2 or P2' position without significantly affecting potency. However, the group on the opposite P2/P2' position had a dramatic effect on potency depending on the size and shape of the side chain. Cocrystal structures of inhibitors with wild-type HIV-1 protease revealed that the bis-THF moiety retained similar interactions as observed in the darunavir-protease complex regardless of the position on the Phe-Phe isostere. Analyses of cocrystal structures and molecular dynamics simulations provide insights into optimizing HIV-1 PIs containing bis-THF in non-sulfonamide dipeptide isosteres.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971191PMC
August 2020

Near Real-Time Identification of Recent Human Immunodeficiency Virus Transmissions, Transmitted Drug Resistance Mutations, and Transmission Networks by Multiplexed Primer ID-Next-Generation Sequencing in North Carolina.

J Infect Dis 2021 Mar;223(5):876-884

University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Background: The identification of recent human immunodeficiency virus (HIV) 1 infections among people with new HIV diagnoses is important to both tailoring and assessing the impact of HIV-1 prevention strategies.

Methods: We developed a multiplexed Primer ID-next-generation sequencing approach to identify recent infections by measuring the intrahost viral diversity over multiple regions of the HIV-1 genome, in addition to detecting drug resistance mutations (DRMs) and phylogenetically linked clusters. We summarize the field implementation of this all-in-one platform among persons with newly diagnosed HIV-1 by the North Carolina State Laboratory of Public Health in 2018.

Results: Overall, recent infection was identified in 94 (35%) of 268 patients with new HIV diagnoses. People <30 years old, and people who inject drugs were more likely to have diagnoses of recent infection. The reverse-transcriptase region K103N was the most commonly detected DRM (prevalence, approximately 15%). We found a total of 28 clusters, and persons with recent infection were more likely to be cluster members than were those with chronic infections (P = .03).

Conclusions: We demonstrate the rapid identification of recent infection and pretreatment DRMs coupled with cluster analysis that will allow prioritization of linkage to care, treatment, and prevention interventions to those at highest risk of onward transmission.
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http://dx.doi.org/10.1093/infdis/jiaa417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938176PMC
March 2021

Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping.

Viruses 2020 06 27;12(7). Epub 2020 Jun 27.

Rush Medical College, Chicago, IL 60612, USA.

Next-generation sequencing (NGS) is increasingly used for HIV-1 drug resistance genotyping. NGS methods have the potential for a more sensitive detection of low-abundance variants (LAV) compared to standard Sanger sequencing (SS) methods. A standardized threshold for reporting LAV that generates data comparable to those derived from SS is needed to allow for the comparability of data from laboratories using NGS and SS. Ten HIV-1 specimens were tested in ten laboratories using Illumina MiSeq-based methods. The consensus sequences for each specimen using LAV thresholds of 5%, 10%, 15%, and 20% were compared to each other and to the consensus of the SS sequences (protease 4-99; reverse transcriptase 38-247). The concordance among laboratories' sequences at different thresholds was evaluated by pairwise sequence comparisons. NGS sequences generated using the 20% threshold were the most similar to the SS consensus (average 99.6% identity, range 96.1-100%), compared to 15% (99.4%, 88.5-100%), 10% (99.2%, 87.4-100%), or 5% (98.5%, 86.4-100%). The average sequence identity between laboratories using thresholds of 20%, 15%, 10%, and 5% was 99.1%, 98.7%, 98.3%, and 97.3%, respectively. Using the 20% threshold, we observed an excellent agreement between NGS and SS, but significant differences at lower thresholds. Understanding how variation in NGS methods influences sequence quality is essential for NGS-based HIV-1 drug resistance genotyping.
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http://dx.doi.org/10.3390/v12070694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7411816PMC
June 2020

Determination of RNA structural diversity and its role in HIV-1 RNA splicing.

Nature 2020 06 6;582(7812):438-442. Epub 2020 May 6.

Whitehead Institute for Biomedical Research, Cambridge, MA, USA.

Human immunodeficiency virus 1 (HIV-1) is a retrovirus with a ten-kilobase single-stranded RNA genome. HIV-1 must express all of its gene products from a single primary transcript, which undergoes alternative splicing to produce diverse protein products that include structural proteins and regulatory factors. Despite the critical role of alternative splicing, the mechanisms that drive the choice of splice site are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis-regulatory elements. Here we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to investigate the structure of HIV-1 RNA in cells, and develop an algorithm that we name 'detection of RNA folding ensembles using expectation-maximization' (DREEM), which reveals the alternative conformations that are assumed by the same RNA sequence. Contrary to previous models that have analysed population averages, our results reveal heterogeneous regions of RNA structure across the entire HIV-1 genome. In addition to confirming that in vitro characterized alternative structures for the HIV-1 Rev responsive element also exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis that heterogeneity in RNA conformation regulates splice-site use and viral gene expression.
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http://dx.doi.org/10.1038/s41586-020-2253-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310298PMC
June 2020

Acetylation of Cytidine Residues Boosts HIV-1 Gene Expression by Increasing Viral RNA Stability.

Cell Host Microbe 2020 08 12;28(2):306-312.e6. Epub 2020 Jun 12.

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA. Electronic address:

Epitranscriptomic RNA modifications, including methylation of adenine and cytidine residues, are now recognized as key regulators of both cellular and viral mRNA function. Moreover, acetylation of the N position of cytidine (ac4C) was recently reported to increase the translation and stability of cellular mRNAs. Here, we show that ac4C and N-acetyltransferase 10 (NAT10), the enzyme that adds ac4C to RNAs, have been subverted by human immunodeficiency virus 1 (HIV-1) to increase viral gene expression. HIV-1 transcripts are modified with ac4C at multiple discrete sites, and silent mutagenesis of these ac4C sites led to decreased HIV-1 gene expression. Similarly, loss of ac4C from viral transcripts due to depletion of NAT10 inhibited HIV-1 replication by reducing viral RNA stability. Interestingly, the NAT10 inhibitor remodelin could inhibit HIV-1 replication at concentrations that have no effect on cell viability, thus identifying ac4C addition as a potential target for antiviral drug development.
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http://dx.doi.org/10.1016/j.chom.2020.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429276PMC
August 2020

Next-Generation Sequencing for HIV Drug Resistance Testing: Laboratory, Clinical, and Implementation Considerations.

Viruses 2020 06 5;12(6). Epub 2020 Jun 5.

Division of Infectious Diseases, Brown University Alpert Medical School, Providence, RI 02906, USA.

Higher accessibility and decreasing costs of next generation sequencing (NGS), availability of commercial kits, and development of dedicated analysis pipelines, have allowed an increasing number of laboratories to adopt this technology for HIV drug resistance (HIVDR) genotyping. Conventional HIVDR genotyping is traditionally carried out using population-based Sanger sequencing, which has a limited capacity for reliable detection of variants present at intra-host frequencies below a threshold of approximately 20%. NGS has the potential to improve sensitivity and quantitatively identify low-abundance variants, improving efficiency and lowering costs. However, some challenges exist for the standardization and quality assurance of NGS-based HIVDR genotyping. In this paper, we highlight considerations of these challenges as related to laboratory, clinical, and implementation of NGS for HIV drug resistance testing. Several sources of variation and bias occur in each step of the general NGS workflow, i.e., starting material, sample type, PCR amplification, library preparation method, instrument and sequencing chemistry-inherent errors, and data analysis options and limitations. Additionally, adoption of NGS-based HIVDR genotyping, especially for clinical care, poses pressing challenges, especially for resource-poor settings, including infrastructure and equipment requirements and cost, logistic and supply chains, instrument service availability, personnel training, validated laboratory protocols, and standardized analysis outputs. The establishment of external quality assessment programs may help to address some of these challenges and is needed to proceed with NGS-based HIVDR genotyping adoption.
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http://dx.doi.org/10.3390/v12060617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354449PMC
June 2020

Deep Sequencing Reveals Compartmentalized HIV-1 in the Semen of Men with and without Sexually Transmitted Infection-Associated Urethritis.

J Virol 2020 06 1;94(12). Epub 2020 Jun 1.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Concurrent sexually transmitted infections (STI) can increase the probability of HIV-1 transmission primarily by increasing the viral load present in semen. In this study, we explored the relationship of HIV-1 in blood and seminal plasma in the presence and absence of urethritis and after treatment of the concurrent STI. Primer ID deep sequencing of the V1/V3 region of the HIV-1 gene was done for paired blood and semen samples from antiretroviral therapy (ART)-naive men living in Malawi with ( = 19) and without ( = 5) STI-associated urethritis; for a subset of samples, full-length genes were generated for sequence analysis and to test entry phenotype. Cytokine concentrations in the blood and semen were also measured, and a reduction in the levels of proinflammatory cytokines was observed following STI treatment. We observed no difference in the prevalence of diverse compartmentalized semen-derived lineages in men with or without STI-associated urethritis, and these viral populations were largely stable during STI treatment. Clonal amplification of one or a few viral sequences accounted for nearly 50% of the viral population, indicating a recent bottleneck followed by limited viral replication. We conclude that the male genital tract is a site where virus can be brought in from the blood, where localized sustained replication can occur, and where specific genotypes can be amplified, perhaps initially by cellular proliferation but further by limited viral replication. HIV-1 infection is a sexually transmitted infection that coexists with other STI. Here, we examined the impact of a concurrent STI resulting in urethritis on the HIV-1 population within the male genital tract. We found that viral populations remain largely stable even with treatment of the STI. These results show that viral populations within the male genital tract are defined by factors beyond transient inflammation associated with a concurrent STI.
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http://dx.doi.org/10.1128/JVI.00151-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307086PMC
June 2020

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice.

Sci Transl Med 2020 04 6;12(541). Epub 2020 Apr 6.

Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog β-d-N-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (β-d-N-hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.
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http://dx.doi.org/10.1126/scitranslmed.abb5883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7164393PMC
April 2020

Curing HIV: Seeking to Target and Clear Persistent Infection.

Cell 2020 04 26;181(1):189-206. Epub 2020 Mar 26.

International Center for the Advancement of Translational Science, Division of Infectious Diseases, Department of Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA; Center for AIDS Research, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA.

Human immunodeficiency virus type 1 (HIV-1) infection persists despite years of antiretroviral therapy (ART). To remove the stigma and burden of chronic infection, approaches to eradicate or cure HIV infection are desired. Attempts to augment ART with therapies that reverse viral latency, paired with immunotherapies to clear infection, have advanced into the clinic, but the field is still in its infancy. We review foundational studies and highlight new insights in HIV cure research. Together with advances in ART delivery and HIV prevention strategies, future therapies that clear HIV infection may relieve society of the affliction of the HIV pandemic.
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http://dx.doi.org/10.1016/j.cell.2020.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896558PMC
April 2020

HIV-1 Central Nervous System Compartmentalization and Cytokine Interplay in Non-Subtype B HIV-1 Infections in Nigeria and Malawi.

AIDS Res Hum Retroviruses 2020 06 17;36(6):490-500. Epub 2020 Feb 17.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

HIV-1 compartmentalization in the central nervous system (CNS) and its contribution to neurological disease have been well documented. Previous studies were conducted among people infected with subtypes B or C where CNS compartmentalization has been observed when comparing viral sequences in the blood to virus in cerebrospinal fluid (CSF). However, little is known about CNS compartmentalization in other HIV-1 subtypes. Using a deep sequencing approach with Primer ID, we conducted a cross-sectional study among Nigerian and Malawian HIV-1 cohorts with or without fungal Cryptococcus infection diagnosed as cryptococcal meningitis (CM) to determine the extent of CSF/CNS compartmentalization with CM. Paired plasma and CSF samples from 45 participants were also analyzed for cytokine/chemokine levels. Viral populations comparing virus in the blood and the CSF ranged from compartmentalized to equilibrated, including minor or partial compartmentalization or clonal amplification of a single viral sequence. The frequency of compartmentalized viral populations in the blood and CSF was similar between the CM- and CM+ participants. We confirmed the potential to see compartmentalization with subtype C infection and have also documented CNS compartmentalization of an HIV-1 subtype G infection. Cytokine profiles indicated a proinflammatory environment, especially within the CSF/CNS. However, sCD163 was suppressed in the CSF in the presence of CM, perhaps due to elevated levels of IL-4, which were also a feature of the cytokine profile, showing a distinct cytokine profile with CM.
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http://dx.doi.org/10.1089/AID.2019.0245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262640PMC
June 2020

The replication-competent HIV-1 latent reservoir is primarily established near the time of therapy initiation.

Sci Transl Med 2019 10;11(513)

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Although antiretroviral therapy (ART) is highly effective at suppressing HIV-1 replication, the virus persists as a latent reservoir in resting CD4 T cells during therapy. This reservoir forms even when ART is initiated early after infection, but the dynamics of its formation are largely unknown. The viral reservoirs of individuals who initiate ART during chronic infection are generally larger and genetically more diverse than those of individuals who initiate therapy during acute infection, consistent with the hypothesis that the reservoir is formed continuously throughout untreated infection. To determine when viruses enter the latent reservoir, we compared sequences of replication-competent viruses from resting peripheral CD4 T cells from nine HIV-positive women on therapy to viral sequences circulating in blood collected longitudinally before therapy. We found that, on average, 71% of the unique viruses induced from the post-therapy latent reservoir were most genetically similar to viruses replicating just before ART initiation. This proportion is far greater than would be expected if the reservoir formed continuously and was always long lived. We conclude that ART alters the host environment in a way that allows the formation or stabilization of most of the long-lived latent HIV-1 reservoir, which points to new strategies targeted at limiting the formation of the reservoir around the time of therapy initiation.
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http://dx.doi.org/10.1126/scitranslmed.aaw5589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7233356PMC
October 2019

Female genital tract shedding of HIV-1 is rare in women with suppressed HIV-1 in plasma.

AIDS 2020 01;34(1):39-46

UNC Center for AIDS Research Department of Microbiology and Immunology Department of Medicine Department of Biochemistry and Biophysics, School of Medicine Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Departments of Pediatrics and Microbiology-Immunology Department of Medicine, Albert Einstein College of Medicine, Bronx Department of Obstetrics and Gynecology, Maimonides Medical Center, Brooklyn, New York Division of Infectious Diseases and Travel Medicine, Georgetown University, Washington, DC Department of Obstetrics, Gynecology & Reproductive Sciences, University of California, San Francisco, California The Core Center, Cook County Health and Hospital System, Rush University Medical College, Chicago, Illinois Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, Georgia Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA.

Objective: Determine the frequency of genital HIV-1 shedding in a large cohort of women on long-term suppressive antiretroviral therapy (ART) and its association with mucosal inflammation.

Design: We measured levels of HIV-1 RNA and inflammation biomarkers in cervicovaginal lavage (CVL) from HIV-seropositive women enrolled in the Women's Interagency HIV Study (WIHS).

Methods: HIV-1 was quantified (Abbott RealTime HIV-1 assay) from CVL samples of 332 WIHS participants with and without clinical evidence of genital inflammation at the time of CVL collection; participants had suppressed plasma viral load (PVL; limit of quantitation less than 20-4000 copies/ml depending on year of collection) for a median of 7.1 years [interquartile range (IQR) 3.4-9.8, Group 1] or for a median of 1.0 years (IQR = 0.5-1.0, Group 2). Twenty-two biomarkers of inflammation were measured in CVL to compare with clinical markers.

Results: HIV-1 was detected in 47% of 38 pre-ART CVL samples (median 668 copies/ml) and detection in CVL was associated with higher pre-ART PVL. HIV-1 was detected in only 1 of 38 CVL samples from these women on suppressive antiretroviral therapy for 1 year. No HIV-1 RNA was detected in 294 CVL samples from a cross-sectional set of women with suppressed PVL for a median of 7 years. Clinical inflammation markers were correlated with inflammatory biomarkers in CVL specimens, although genital inflammation was not associated with measurable genital HIV-1 shedding in these WIHS participants on ART.

Conclusion: ART that suppresses HIV-1 in the plasma of women also prevents genital tract HIV-1 shedding, even in the presence of genital tract inflammation.
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http://dx.doi.org/10.1097/QAD.0000000000002373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910230PMC
January 2020

Epitranscriptomic Addition of mC to HIV-1 Transcripts Regulates Viral Gene Expression.

Cell Host Microbe 2019 08;26(2):217-227.e6

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA. Electronic address:

How the covalent modification of mRNA ribonucleotides, termed epitranscriptomic modifications, alters mRNA function remains unclear. One issue has been the difficulty of quantifying these modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears more epitranscriptomic modifications than the average cellular mRNA, with 5-methylcytosine (mC) and 2'O-methyl modifications being particularly prevalent. The methyltransferase NSUN2 serves as the primary writer for mC on HIV-1 RNAs. NSUN2 inactivation inhibits not only mC addition to HIV-1 transcripts but also viral replication. This inhibition results from reduced HIV-1 protein, but not mRNA, expression, which in turn correlates with reduced ribosome binding to viral mRNAs. In addition, loss of mC dysregulates the alternative splicing of viral RNAs. These data identify mC as a post-transcriptional regulator of both splicing and function of HIV-1 mRNA, thereby affecting directly viral gene expression.
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http://dx.doi.org/10.1016/j.chom.2019.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714563PMC
August 2019

Genome-Wide Analysis of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) Binding to HIV-1 RNA Reveals a Key Role for hnRNP H1 in Alternative Viral mRNA Splicing.

J Virol 2019 11 15;93(21). Epub 2019 Oct 15.

Laboratory of Retrovirology, The Rockefeller University, New York, New York, USA

Alternative splicing of HIV-1 mRNAs increases viral coding potential and controls the levels and timing of gene expression. HIV-1 splicing is regulated in part by heterogeneous nuclear ribonucleoproteins (hnRNPs) and their viral target sequences, which typically repress splicing when studied outside their native viral context. Here, we determined the location and extent of hnRNP binding to HIV-1 mRNAs and their impact on splicing in a native viral context. Notably, hnRNP A1, hnRNP A2, and hnRNP B1 bound to many dispersed sites across viral mRNAs. Conversely, hnRNP H1 bound to a few discrete purine-rich sequences, a finding that was mirrored hnRNP H1 depletion and mutation of a prominent viral RNA hnRNP H1 binding site decreased the use of splice acceptor A1, causing a deficit in Vif expression and replicative fitness. This quantitative framework for determining the regulatory inputs governing alternative HIV-1 splicing revealed an unexpected splicing enhancer role for hnRNP H1 through binding to its target element. Alternative splicing of HIV-1 mRNAs is an essential yet quite poorly understood step of virus replication that enhances the coding potential of the viral genome and allows the temporal regulation of viral gene expression. Although HIV-1 constitutes an important model system for general studies of the regulation of alternative splicing, the inputs that determine the efficiency with which splice sites are utilized remain poorly defined. Our studies provide an experimental framework to study an essential step of HIV-1 replication more comprehensively and in much greater detail than was previously possible and reveal novel -acting elements regulating HIV-1 splicing.
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http://dx.doi.org/10.1128/JVI.01048-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803249PMC
November 2019

HIV-1 Protease Inhibitors Incorporating Stereochemically Defined P2' Ligands To Optimize Hydrogen Bonding in the Substrate Envelope.

J Med Chem 2019 09 21;62(17):8062-8079. Epub 2019 Aug 21.

Department of Biochemistry and Molecular Pharmacology , University of Massachusetts Medical School , Worcester , Massachusetts 01605 , United States.

A structure-guided design strategy was used to improve the resistance profile of HIV-1 protease inhibitors by optimizing hydrogen bonding and van der Waals interactions with the protease while staying within the substrate envelope. Stereoisomers of 4-(1-hydroxyethyl)benzene and 4-(1,2-dihydroxyethyl)benzene moieties were explored as P2' ligands providing pairs of diastereoisomers epimeric at P2', which exhibited distinct potency profiles depending on the configuration of the hydroxyl group and size of the P1' group. While compounds with the 4-(1-hydroxyethyl)benzene P2' moiety maintained excellent antiviral potency against a panel of multidrug-resistant HIV-1 strains, analogues with the polar 4-(1,2-dihydroxyethyl)benzene moiety were less potent, and only the ()-epimer incorporating a larger 2-ethylbutyl P1' group showed improved potency. Crystal structures of protease-inhibitor complexes revealed strong hydrogen bonding interactions of both ()- and ()-stereoisomers of the hydroxyethyl group with Asp30'. Notably, the ()-dihydroxyethyl group was involved in a unique pattern of direct hydrogen bonding interactions with the backbone amides of Asp29' and Asp30'. The SAR data and analysis of crystal structures provide insights for optimizing these promising HIV-1 protease inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941148PMC
September 2019

Picomolar to Micromolar: Elucidating the Role of Distal Mutations in HIV-1 Protease in Conferring Drug Resistance.

ACS Chem Biol 2019 11 13;14(11):2441-2452. Epub 2019 Aug 13.

Department of Biochemistry and Molecular Pharmacology , University of Massachusetts Medical School , Worcester , Massachusetts 01605 , United States.

Drug resistance continues to be a growing global problem. The efficacy of small molecule inhibitors is threatened by pools of genetic diversity in all systems, including antibacterials, antifungals, cancer therapeutics, and antivirals. Resistant variants often include combinations of active site mutations and distal "secondary" mutations, which are thought to compensate for losses in enzymatic activity. HIV-1 protease is the ideal model system to investigate these combinations and underlying molecular mechanisms of resistance. Darunavir (DRV) binds wild-type (WT) HIV-1 protease with a potency of <5 pM, but we have identified a protease variant that loses potency to DRV 150 000-fold, with 11 mutations in and outside the active site. To elucidate the roles of these mutations in DRV resistance, we used a multidisciplinary approach, combining enzymatic assays, crystallography, and molecular dynamics simulations. Analysis of protease variants with 1, 2, 4, 8, 9, 10, and 11 mutations showed that the primary active site mutations caused ∼50-fold loss in potency (2 mutations), while distal mutations outside the active site further decreased DRV potency from 13 nM (8 mutations) to 0.76 μM (11 mutations). Crystal structures and simulations revealed that distal mutations induce subtle changes that are dynamically propagated through the protease. Our results reveal that changes remote from the active site directly and dramatically impact the potency of the inhibitor. Moreover, we find interdependent effects of mutations in conferring high levels of resistance. These mechanisms of resistance are likely applicable to many other quickly evolving drug targets, and the insights may have implications for the design of more robust inhibitors.
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http://dx.doi.org/10.1021/acschembio.9b00370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941144PMC
November 2019

Rationally designed carbohydrate-occluded epitopes elicit HIV-1 Env-specific antibodies.

Nat Commun 2019 02 27;10(1):948. Epub 2019 Feb 27.

Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.

An array of carbohydrates masks the HIV-1 surface protein Env, contributing to the evasion of humoral immunity. In most HIV-1 isolates 'glycan holes' occur due to natural sequence variation, potentially revealing the underlying protein surface to the immune system. Here we computationally design epitopes that mimic such surface features (carbohydrate-occluded neutralization epitopes or CONE) of Env through 'epitope transplantation', in which the target region is presented on a carrier protein scaffold with preserved structural properties. Scaffolds displaying the four CONEs are examined for structure and immunogenicity. Crystal structures of two designed proteins reflect the computational models and accurately mimic the native conformations of CONEs. The sera from rabbits immunized with several CONE immunogens display Env binding activity. Our method determines essential structural elements for targets of protective antibodies. The ability to design immunogens with high mimicry to viral proteins also makes possible the exploration of new templates for vaccine development.
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http://dx.doi.org/10.1038/s41467-019-08876-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393580PMC
February 2019

Constrained Mutational Sampling of Amino Acids in HIV-1 Protease Evolution.

Mol Biol Evol 2019 04;36(4):798-810

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA.

The evolution of HIV-1 protein sequences should be governed by a combination of factors including nucleotide mutational probabilities, the genetic code, and fitness. The impact of these factors on protein sequence evolution is interdependent, making it challenging to infer the individual contribution of each factor from phylogenetic analyses alone. We investigated the protein sequence evolution of HIV-1 by determining an experimental fitness landscape of all individual amino acid changes in protease. We compared our experimental results to the frequency of protease variants in a publicly available data set of 32,163 sequenced isolates from drug-naïve individuals. The most common amino acids in sequenced isolates supported robust experimental fitness, indicating that the experimental fitness landscape captured key features of selection acting on protease during viral infections of hosts. Amino acid changes requiring multiple mutations from the likely ancestor were slightly less likely to support robust experimental fitness than single mutations, consistent with the genetic code favoring chemically conservative amino acid changes. Amino acids that were common in sequenced isolates were predominantly accessible by single mutations from the likely protease ancestor. Multiple mutations commonly observed in isolates were accessible by mutational walks with highly fit single mutation intermediates. Our results indicate that the prevalence of multiple-base mutations in HIV-1 protease is strongly influenced by mutational sampling.
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http://dx.doi.org/10.1093/molbev/msz022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804413PMC
April 2019

Predicting Efavirenz Concentrations in the Brain Tissue of HIV-Infected Individuals and Exploring their Relationship to Neurocognitive Impairment.

Clin Transl Sci 2019 05 27;12(3):302-311. Epub 2019 Feb 27.

Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Sparse data exist on the penetration of antiretrovirals into brain tissue. In this work, we present a framework to use efavirenz (EFV) pharmacokinetic (PK) data in plasma, cerebrospinal fluid (CSF), and brain tissue of eight rhesus macaques to predict brain tissue concentrations in HIV-infected individuals. We then perform exposure-response analysis with the model-predicted EFV area under the concentration-time curve (AUC) and neurocognitive scores collected from a group of 24 HIV-infected participants. Adult rhesus macaques were dosed daily with 200 mg EFV (as part of a four-drug regimen) for 10 days. Plasma was collected at 8 time points over 10 days and at necropsy, whereas CSF and brain tissue were collected at necropsy. In the clinical study, data were obtained from one paired plasma and CSF sample of participants prescribed EFV, and neuropsychological test evaluations were administered across 15 domains. PK modeling was performed using ADAPT version 5.0 Biomedical Simulation Resource, Los Angeles, CA) with the iterative two-stage estimation method. An eight-compartment model best described EFV distribution across the plasma, CSF, and brain tissue of rhesus macaques and humans. Model-predicted median brain tissue concentrations in humans were 31 and 8,000 ng/mL, respectively. Model-predicted brain tissue AUC was highly correlated with plasma AUC (γ = 0.99, P < 0.001) but not CSF AUC (γ = 0.34, P = 0.1) and did not show any relationship with neurocognitive scores (γ < 0.05, P > 0.05). This analysis provides an approach to estimate PK the brain tissue in order to perform PK/pharmacodynamic analyses at the target site.
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http://dx.doi.org/10.1111/cts.12620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510381PMC
May 2019

Human Immunodeficiency Virus Type 1 RNA Detected in the Central Nervous System (CNS) After Years of Suppressive Antiretroviral Therapy Can Originate from a Replicating CNS Reservoir or Clonally Expanded Cells.

Clin Infect Dis 2019 09;69(8):1345-1352

University of North Carolina Center for AIDS Research, University of North Carolina at Chapel Hill, San Francisco.

Background: Human immunodeficiency virus type 1 (HIV-1) populations are detected in cerebrospinal fluid (CSF) of some people on suppressive antiretroviral therapy (ART). Detailed analysis of these populations may reveal whether they are produced by central nervous system (CNS) reservoirs.

Methods: We performed a study of 101 asymptomatic participants on stable ART. HIV-1 RNA concentrations were cross-sectionally measured in CSF and plasma. In participants with CSF HIV-1 RNA concentrations sufficient for analysis, viral populations were genetically and phenotypically characterized over multiple time points.

Results: For 6% of participants (6 of 101), the concentration of HIV-1 RNA in their CSF was ≥0.5 log copies/mL above that of plasma (ie, CSF escape). We generated viral envelope sequences from CSF of 3 participants. One had a persistent CSF escape population that was macrophage-tropic, partially drug resistant, genetically diverse, and closely related to a minor macrophage-tropic lineage present in the blood prior to viral suppression and enriched for after ART. Two participants (1 suppressed and 1 not) had transient CSF escape populations that were R5 T cell-tropic with little genetic diversity.

Conclusions: Extensive analysis of viral populations in 1 participant revealed that CSF escape was from a persistently replicating population, likely in macrophages/microglia, present in the CNS over 3 years of ART. CSF escape in 2 other participants was likely produced by trafficking and transient expansion of infected T cells in the CNS. Our results show that CNS reservoirs can persist during ART and that CSF escape is not exclusively produced by replicating CNS reservoirs.
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http://dx.doi.org/10.1093/cid/ciy1066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938202PMC
September 2019

HIV-1 Protease Uses Bi-Specific S2/S2' Subsites to Optimize Cleavage of Two Classes of Target Sites.

J Mol Biol 2018 12 7;430(24):5182-5195. Epub 2018 Nov 7.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

Retroviral proteases (PRs) have a unique specificity that allows cleavage of sites with or without a P1' proline. A P1' proline is required at the MA/CA cleavage site due to its role in a post-cleavage conformational change in the capsid protein. However, the HIV-1 PR prefers to have large hydrophobic amino acids flanking the scissile bond, suggesting that PR recognizes two different classes of substrate sequences. We analyzed the cleavage rate of over 150 combinations of six different HIV-1 cleavage sites to explore rate determinants of cleavage. We found that cleavage rates are strongly influenced by the two amino acids flanking the amino acids at the scissile bond (P2-P1/P1'-P2'), with two complementary sets of rules. When P1' is proline, the P2 side chain interacts with a polar region in the S2 subsite of the PR, while the P2' amino acid interacts with a hydrophobic region of the S2' subsite. When P1' is not proline, the orientations of the P2 and P2' side chains with respect to the scissile bond are reversed; P2 residues interact with a hydrophobic face of the S2 subsite, while the P2' amino acid usually engages hydrophilic amino acids in the S2' subsite. These results reveal that the HIV-1 PR has evolved bi-functional S2 and S2' subsites to accommodate the steric effects imposed by a P1' proline on the orientation of P2 and P2' substrate side chains. These results also suggest a new strategy for inhibitor design to engage the multiple specificities in these subsites.
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http://dx.doi.org/10.1016/j.jmb.2018.10.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292680PMC
December 2018

Endogenous retroviral signatures predict immunotherapy response in clear cell renal cell carcinoma.

J Clin Invest 2018 11 2;128(11):4804-4820. Epub 2018 Oct 2.

Department of Microbiology and Immunology, UNC School of Medicine, Chapel Hill, North Carolina, USA.

Human endogenous retroviruses (hERVs) are remnants of exogenous retroviruses that have integrated into the genome throughout evolution. We developed a computational workflow, hervQuant, which identified more than 3,000 transcriptionally active hERVs within The Cancer Genome Atlas (TCGA) pan-cancer RNA-Seq database. hERV expression was associated with clinical prognosis in several tumor types, most significantly clear cell renal cell carcinoma (ccRCC). We explored two mechanisms by which hERV expression may influence the tumor immune microenvironment in ccRCC: (i) RIG-I-like signaling and (ii) retroviral antigen activation of adaptive immunity. We demonstrated the ability of hERV signatures associated with these immune mechanisms to predict patient survival in ccRCC, independent of clinical staging and molecular subtyping. We identified potential tumor-specific hERV epitopes with evidence of translational activity through the use of a ccRCC ribosome profiling (Ribo-Seq) dataset, validated their ability to bind HLA in vitro, and identified the presence of MHC tetramer-positive T cells against predicted epitopes. hERV sequences identified through this screening approach were significantly more highly expressed in ccRCC tumors responsive to treatment with programmed death receptor 1 (PD-1) inhibition. hervQuant provides insights into the role of hERVs within the tumor immune microenvironment, as well as evidence that hERV expression could serve as a biomarker for patient prognosis and response to immunotherapy.
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http://dx.doi.org/10.1172/JCI121476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205406PMC
November 2018

Evolutionary pathways to NS5A inhibitor resistance in genotype 1 hepatitis C virus.

Antiviral Res 2018 10 3;158:45-51. Epub 2018 Aug 3.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Division of Infectious Diseases, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address:

Direct-acting antivirals (DAAs) targeting NS5A are broadly effective against hepatitis C virus (HCV) infections, but sustained virological response rates are generally lower in patients infected with genotype (gt)-1a than gt-1b viruses. The explanation for this remains uncertain. Here, we adopted a highly accurate, ultra-deep primer ID sequencing approach to intensively study serial changes in the NS5A-coding region of HCV in gt-1a- and gt-1b-infected subjects receiving a short course of monotherapy with the NS5A inhibitor, elbasvir. Low or undetectable levels of viremia precluded on-treatment analysis in gt-1b-infected subjects, but variants with the resistance-associated substitution (RAS) Y93H in NS5A dominated rebounding virus populations following cessation of treatment. These variants persisted until the end of the study, two months later. In contrast, while Y93H emerged in multiple lineages and became dominant in subjects with gt-1a virus, these haplotypes rapidly decreased in frequency off therapy. Substitutions at Q30 and L31 emerged in distinctly independent lineages at later time points, ultimately coming to dominate the virus population off therapy. Consistent with this, cell culture studies with gt-1a and gt-1b reporter viruses and replicons demonstrated that Y93H confers a much greater loss of replicative fitness in gt-1a than gt-1b virus, and that L31M/V both compensates for the loss of fitness associated with Q30R (but not Y93H) and also boosts drug resistance. These observations show how differences in the impact of RASs on drug resistance and replicative fitness influence the evolution of gt-1a and gt-1b viruses during monotherapy with an antiviral targeting NS5A.
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http://dx.doi.org/10.1016/j.antiviral.2018.07.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6146077PMC
October 2018