Biology subjects

Metzner, K. J.

Publications and source records attributed to Metzner, K. J..

10 recordsLinked to original sources

bioRxiv2026

Long terminal repeats influence deletion propensity and genome instability in proviruses across major retroviruses

A hallmark of retroviral infection is integration of viral genomes into host chromosomes to form proviruses. Most proviruses are defective, particularly in people with HIV receiving antiretroviral therapy, with large internal deletions as a dominant outcome. The contribution of retroviral long terminal repeats (LTRs) in the formation of large internal deletions remains unclear. We used dual-fluorophore reporters based on HIV-1, HIV-2, and HTLV-1 LTRs, integrated at AAVS1 by CRISPR/Cas9, to test whether LTRs influence proviral integrity in the absence of reverse transcriptase or integrase. Cerulean expression was driven by the LTR and mCherry by an independent human promoter. Double-positive (Cerulean/mCherry) cells were a proxy for intact vectors, whereas single mCherry cells indicated latent and/or defective proviruses. Double-positive cells harboured intact vectors for all constructs. Non-inducible single mCherry populations were enriched for extensively deleted proviruses with HIV-1, HIV-2, or HTLV-1 LTRs, whereas the LTR-deficient reporter yielded few single mCherry cells, all with intact proviruses. The presence of LTRs was significantly associated with deleted genomes. HIV-1 and HTLV-1 showed higher observed deletion rates than HIV-2, but this was not statistically significant owing to limited sequence numbers. Single Cerulean populations across all constructs showed recurrent deletions affecting the mCherry cassette and upstream promoter, with short direct repeats at deletion junctions. These likely reflect microhomology-mediated end joining as a contributing repair mechanism during CRISPR/Cas9-mediated integration, in which LTRs may not directly contribute. Together, these data identify retroviral LTRs as contributors to the formation of large internal deletions in proviruses.

microbiology↗

bioRxiv2026

DNA Damage Response Proteins Are Involved in the Formation of Defective HIV-1 Proviruses

Latent HIV-1 proviruses remain the major barrier to curing HIV infection. Although many of these proviruses are defective, with large internal deletions and hypermutations, the mechanisms underlying their formation are still poorly understood. In this study, we applied CRISPR/Cas9 knockout screens to identify DNA damage response (DDR) proteins that contribute to the formation of defective HIV-1 proviruses carrying large internal deletions. Using an HIV-1-based dual-fluorophore vector as a model, we distinguished cells harbouring intact proviruses from those carrying large internal deletions by flow cytometry and cell sorting. We then validated top candidates using CRISPR-mediated gene activation and small interfering RNA-mediated knockdown. Across these approaches, the helicase-like transcription factor HLTF emerged as a consistent modulator of large internal deletions: increased HLTF expression raised the proportion of cells carrying defective proviruses, whereas reduced HLTF expression had the opposite effect. Additional repair factors, including RAD1, RAD18, TREX2, and ZRANB3, also influenced the balance between intact and defective proviruses, suggesting that multiple DNA repair pathways cooperate in this process. Our data indicate that several DNA damage response proteins, including HLTF, are involved in the generation of defective proviruses and may constitute a previously undescribed host defence mechanism against HIV-1.

molecular biology↗

bioRxiv2026

Substantial Deceleration of Adaptation of HIV-1 Within 1,500 Generations in an Experimental Evolution: A Genomic Perspective

Numerous experimental evolution studies have suggested that adaptation rate of microbial populations evolving in stable environments decline over time. Despite the generality of this phenomenon across different domains of life, the timing and magnitude of decline in adaptation can vary greatly based on the idiosyncrasies of the biological system. To investigate the characteristics of adaptation deceleration in a fast-evolving virus, we propagated HIV-1 in two human T-cell lines (MT-2 and MT-4) for approximately 4.8 years and tracked its genome evolution through next-generation sequencing. The curated sequencing data covering the whole-genome can be accessed and explored via LTEEviz, an interactive web application. Time-resolved sequencing data uncovered that despite constant fixation rate of 0.085 (MT-2) and 0.042 (MT-4) mutations per generation, the fixation kinetics of adaptive mutations changed considerably over time. The rate of fixation of adaptive parallel mutations decreased by 44% per 300 generations, while their conferred fitness gain decreased by 27% (MT-2) and 18% (MT-4) per every added adaptive mutation in their genetic background. The early and substantial deceleration of adaptation in our HIV-1 populations can, at least in part, be explained by diminishing gains of adaptive mutations. Furthermore, we identified genomic patterns consistent with a hard selective sweep that occurred in one population later in the experiment. Together, our results confirm that HIV-1 genomic evolution is characterized by a swift and substantial deceleration of adaptation, while also revealing that episodes of positive selection can occur beyond the initial adaptive phase.

evolutionary biology↗

bioRxiv2024

Anti-HIV-1 Effect of the Fluoroquinolone Enoxacin and Modulation of Pro-viral hsa-miR-132 Processing

BackgroundDespite tremendous advances in antiretroviral therapy (ART) against HIV-1 infections no cure or vaccination is available. Therefore, discovering novel therapeutic strategies remains an urgent need. In that sense, miRNAs and miRNA therapeutics have moved intensively into the focus of recent HIV-1 related investigations. A strong reciprocal interdependence has been demonstrated between HIV-1 infection and changes of the intrinsic cellular miRNA milieu. This interrelationship may direct potential alterations of the host cells environment beneficial for the virus or its suppression of replication. Whether this tightly balanced and controlled battle can be exploited therapeutically, remains to be further addressed. In this context, the fluoroquinolone antibiotic Enoxacin has been demonstrated as a potent modulator of miRNA processing. Here, we test the hypothesis that this applies also to selected HIV-1 related miRNAs. MethodsWe studied the effect of Enoxacin on HIV-1 replication coupled with miRNA qRT-PCR analysis of HIV-1 related miRNAs in CEM-SS and MT-4 T-cells. The effects of miRNA mimic transfections combined with Enoxacin treatment on HIV-1 replication were assessed. Finally, we employed an in vitro DICER1 cleavage assay to study the effects of Enoxacin on a pro-HIV-1 miRNA hsa-miR-132 processing. ResultsWe established that Enoxacin, but not the structurally similar compound nalidixic acid, exhibits strong anti-HIV-1 effects in the T-cell line CEM-SS, but not MT-4. We provide experimental data that this effect of Enoxacin is partly attributed to the specific downregulation of mature hsa-miR-132-3p, but not other pro- or anti-HIV-1 miRNAs, which is likely due to affecting DICER1 processing. ConclusionsOur findings show an anti-retroviral activity of Enoxacin at least in part by downregulation of hsa-miR-132-3p, which may be relevant for future antiviral therapeutic applications by modulation of the RNA interference pathway.

molecular biology↗

bioRxiv2024

Rare twin cysteine residues in the HIV-1 envelope variable region 1 link to neutralization escape and breadth development

The identification of HIV-1 Envelope glycoprotein (Env) traits associated with development of neutralization cross-reactivity in natural infection is critical for vaccine design. Here we describe the presence of additional Cysteine (Cys) residues in V1 that are enriched among people with elite neutralization breadth. Using >65,000 V1 sequences from the CATNAP database, the AMP trials and three large longitudinal HIV infection cohorts, the SHCS, ZPHI and CAPRISA studies, we show that Env variants with extra V1 Cys are present at low levels throughout infection and fluctuate in frequency over time within participants. We demonstrate an independent association of extra V1 Cys with elite plasma neutralization, and a strong preference for two versus one extra Cys, suggesting certain Envs introduce an additional disulfide bond for stabilization. We observed high levels of neutralization resistance among Envs from 34 bNAb donors, of which 17.6% had elongated V1 regions with extra Cys. We show that extra V1 Cys moderately increase neutralization resistance in an Env from a V2- Apex bNAb-inducer. Modulation of the accessibility of bNAb epitopes on this Env by extra V1 Cys enhanced epitope shielding of several regions, but increased V2 exposure. This suggests that escape from autologous neutralizing activity drove insertion of the extra V1 Cys, creating a modified antigen that may have favored V2 bNAb induction in this donor. Overall, we identify a rare motif of twin Cys in V1 that confers increased neutralization resistance and Env stabilization, is associated with bNAb induction, and may hold potential for incorporation into future HIV bNAb immunogens.

immunology↗

bioRxiv2023

Targeting HIV-1 with CRISPR/Cas9 delivered by retargeted adenoviruses effectively suppresses viral replication

Integrated, intact, latent HIV-1 viruses in infected cells are the main obstacle to curing HIV-1 infections. Targeted inactivation of HIV-1 proviruses with CRISPR/Cas9 is a promising strategy to eradicate HIV-1. In addition, CRISPR/Cas9 is able to target replicating HIV-1 and could be used as a therapy during productive infection. Here, we combine the CRISPR/Cas9 system with a novel adenovirus (Ad) targeted delivery technology to test it as a therapeutic approach to inhibit HIV-1. First, we selected six HIV-1-specific gRNAs targeting the HIV-1 LTRs and the gag gene and tested their efficacy in inhibiting HIV-1 virion production in an HEK 293T cell co-transfection screen. The gRNA-TAR showed the most robust and potent inhibition of HIV-1 by >99% alone or in combination with the gRNA-p24, which induced a [~]1 kb deletion between both gRNA target sites in HIV-1 DNA. Delivery of this dual gRNA-TAR/p24 CRISPR/Cas9 system with CD3-CD28-IL2-retargeted Ads was highly effective, transducing 62.3{+/-}23.3% of cells and suppressing HIV-1 replication by 88.0{+/-}4.5% in primary CD4+ T cells from three independent donors. Our dual gRNA-TAR/p24-CRISPR/Cas9-Ad strategy represents a novel therapeutic approach to effectively inhibit HIV-1 in a highly HIV-1 and T cell-specific manner.

microbiology↗

bioRxiv2023

Mycobacterium tuberculosis infection associated immune perturbations correlate with antiretroviral immunity

Infection with Mycobacterium tuberculosis (MTB) remains one of the most important opportunistic infections in people with HIV-1 (PWH). While active Tuberculosis (TB) leads to rapid progression of immunodeficiency in PWH, the interaction between MTB and HIV-1 during the asymptomatic phase of both infections remains poorly understood. In a cohort of individuals with HIV (PWH) with and without suppressed HIV-1 viral load, the transcriptomic profiles of peripheral blood mononuclear cells (PBMC) clustered in individuals infected with Mycobacterium tuberculosis (MTB) compared to carefully matched controls. Subsequent functional annotation analysis disclosed alterations in the IL-6, TNF, and KRAS pathways. Notably, MTB-associated genes demonstrated an inverse correlation with HIV-1 viremia, evident at both on individual gene level and when employed as a gene score. In sum, our data show that MTB infection in PWH is associated with a shift in the activation state of the immune system, displaying an inverse relationship with HIV-1 viral load. These results could provide an explanation for the observed increased antiretroviral control associated with MTB infection in PWH.

immunology↗

bioRxiv2020

V-pipe: a computational pipeline for assessing viral genetic diversity from high-throughput sequencing data

High-throughput sequencing technologies are used increasingly, not only in viral genomics research but also in clinical surveillance and diagnostics. These technologies facilitate the assessment of the genetic diversity in intra-host virus populations, which affects transmission, virulence, and pathogenesis of viral infections. However, there are two major challenges in analysing viral diversity. First, amplification and sequencing errors confound the identification of true biological variants, and second, the large data volumes represent computational limitations. To support viral high-throughput sequencing studies, we developed V-pipe, a bioinformatics pipeline combining various state-of-the-art statistical models and computational tools for automated end-to-end analyses of raw sequencing reads. V-pipe supports quality control, read mapping and alignment, low-frequency mutation calling, and inference of viral haplotypes. For generating high-quality read alignments, we developed a novel method, called ngshmmalign, based on profile hidden Markov models and tailored to small and highly diverse viral genomes. V-pipe also includes benchmarking functionality providing a standardized environment for comparative evaluations of different pipeline configurations. We demonstrate this capability by assessing the impact of three different read aligners (Bowtie 2, BWA MEM, ngshmmalign) and two different variant callers (LoFreq, ShoRAH) on the performance of calling single-nucleotide variants in intra-host virus populations. V-pipe supports various pipeline configurations and is implemented in a modular fashion to facilitate adaptations to the continuously changing technology landscape. V-pipe is freely available at https://github.com/cbg-ethz/V-pipe.

bioinformatics↗

bioRxiv2020

HIV-1 promoter is gradually silenced when integrated into BACH2

The persistence of the latent HIV-1 reservoir is a major obstacle to cure HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene BTB domain and CNC homology 2 (BACH2). We investigated HIV-1 promoter activity after integration into specific sites in BACH2. The HIV-1-based vector LTatCL[M] contains two fluorophores: 1.) Cerulean, which reports the activity of the HIV-1 promoter, and 2.) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of BACH2 of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of BACH2, and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean+/mCherry+) and inactive (Cerulean-/mCherry+) HIV-1 promoters were characterized. Upon targeted integration of the 5.3 kb vector LTatCL[M] into BACH2, active HIV-1 promoters were gradually silenced as reflected by decrease in Cerulean expression over a period of 162 days in culture. Silenced HIV-1 promoters could be reactivated by TNF- and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. BACH2 mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M]. Our results show that the HIV-1 promoter is silenced when integrated into BACH2 without impairing BACH2 mRNA and protein expression. This might contribute to HIV-1 persistence, enabling infected T-cells to complete differentiation into a memory phenotype, persist, and clonally expand over time.

microbiology↗

bioRxiv2017

Convergent evolution as an indicator for selection during acute HIV-1 infection

Convergent evolution describes the process of different populations acquiring similar phenotypes or genotypes. Complex organisms with large genomes only rarely and only under very strong selection converge to the same genotype. In contrast, independent virus populations with very small genomes often acquire identical mutations. Here we test the hypothesis of whether convergence in early HIV-1 infection is common enough to serve as an indicator for selection. To this end, we measure the number of convergent mutations in a well-studied dataset of full-length HIV-1 env genes sampled from HIV-1 infected individuals during early infection. We compare this data to a neutral model and find an excess of convergent mutations. Convergent mutations are not evenly distributed across the env gene, but more likely to occur in gp41, which suggests that convergent mutations provide a selective advantage and hence are positively selected. In contrast, mutations that are only found in an HIV-1 population of a single individual are significantly affected by purifying selection. Our analysis suggests that comparisons between convergent and private mutations with neutral models allow us to identify positive and negative selection in small viral genomes. Our results also show that selection significantly shapes HIV-1 populations even before the onset of the adaptive immune system.

evolutionary biology↗