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1.
Sheng Wu Gong Cheng Xue Bao ; 40(7): 2195-2210, 2024 Jul 25.
Article in Chinese | MEDLINE | ID: mdl-39044584

ABSTRACT

In recent years, virological, pathological, and immunological studies need to be carried out for the emerging anti-human immunodeficiency virus (HIV) therapies such as gene therapy, broadly neutralizing antibodies, and the derived chimeric antigen receptor (CAR)-T immunotherapy, which necessitates suitable, simple, and inexpensive small-animal models and methods for accurate quantification of the viral genome in the HIV-1 infected. In our research, the HIV-∆ENV-Jurkat-EGFP-mCherry cell line was engineered through the infection with a dual-labelled HIV pseudovirus. A nested quantitative PCR (nested-qPCR) method with the cellular genome as the integrated standard was established for the quantification of HIV proviral copies. We administered intravenous injections of healthy human peripheral blood mononuclear cell (PBMC) into NOD/Prkdcscid/IL2rgnull (NPG) mice. To verify engraftment kinetics, we analyzed the percentages of hCD45+, hCD3+, hCD4+, and hCD8+ cells in the peripheral blood of hu-PBMC-NPG mice. To evaluate HIV-1 infection in hu-PBMC-NPG mice, we inoculated these mice with HIV NL4-3-NanoLuc by intraperitoneal (IP) injection. We then monitored the luciferase expression by the small animal imaging system and measured the viral load in the spleen by qPCR. The infiltration of human PBMCs in mice was detected 3-5 weeks after intravenous injection, and the percentage of hCD45 in humanized mouse PBMCs were more than 25% five weeks after IP inoculation. The expression of the virus-associated luciferase protein was detected by luciferase imaging 27 days post infection. Moreover, the viral total DNA, RNA, and proviral DNA copies reached 18 000 copies/106 cells, 15 000 copies/µg RNA, and 15 000 copies/106 cells, respectively, in the mouse spleen. Taken together, we reported a convenient method for building a simple humanized mouse model of HuPBMC-NPG/severe combined immunodeficiency (SCID) by intravenous injection with hu-PBMCs without advanced surgical skills and irradiation. Furthermore, we established a convenient method for the efficient determination of proviral DNA to assess HIV replication in vivo, viral reservoir sizes, and efficacy of novel anti-HIV therapies including CAR-T immunotherapy and gene therapy.


Subject(s)
DNA, Viral , Disease Models, Animal , HIV Infections , HIV-1 , Proviruses , Animals , HIV-1/genetics , HIV-1/immunology , Mice , Humans , Proviruses/genetics , HIV Infections/immunology , HIV Infections/virology , DNA, Viral/genetics , Mice, Inbred NOD , Mice, SCID , Leukocytes, Mononuclear/immunology , Viral Load
2.
Int J Mol Sci ; 25(13)2024 Jun 22.
Article in English | MEDLINE | ID: mdl-38999966

ABSTRACT

Increased human T-cell leukemia virus type 1 (HTLV-1) proviral load (PVL) is a significant risk factor for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is controversy surrounding whether HTLV-1-specific cytotoxic T lymphocytes (CTLs) are beneficial or harmful to HAM/TSP patients. Recently, HTLV-1 Tax 301-309 has been identified as an immunodominant epitope restricted to HLA-A*2402. We investigated whether HLA-A*24 reduces HTLV-1 PVL and the risk of HAM/TSP using blood samples from 152 HAM/TSP patients and 155 asymptomatic HTLV-1 carriers. The allele frequency of HLA-A*24 was higher in HAM/TSP patients than in asymptomatic HTLV-1 carriers (72.4% vs. 58.7%, odds ratio 1.84), and HLA-A*24-positive patients showed a 42% reduction in HTLV-1 PVL compared to negative patients. Furthermore, the PVL negatively correlated with the frequency of Tax 301-309-specific CTLs. These findings are opposite to the effects of HLA-A*02, which reduces HTLV-1 PVL and the risk of HAM/TSP. Therefore, we compared the functions of CTLs specific to Tax 11-19 or Tax 301-309, which are immunodominant epitopes restricted to HLA-A*0201 or HLA-A*2402, respectively. The maximum responses of these CTLs were not different in the production of IFN-γ and MIP-1ß or in the expression of CD107a-a marker for the degranulation of cytotoxic molecules. However, Tax 301-309-specific CTLs demonstrated 50-fold higher T-cell avidity than Tax 11-19-specific CTLs, suggesting better antigen recognition at low expression levels of the antigens. These findings suggest that HLA-A*24, which induces sensitive HTLV-1-specific CTLs, increases the risk of HAM/TSP despite reducing HTLV-1 PVL.


Subject(s)
HLA-A24 Antigen , Human T-lymphotropic virus 1 , Paraparesis, Tropical Spastic , Proviruses , Viral Load , Humans , Human T-lymphotropic virus 1/immunology , Female , Male , Paraparesis, Tropical Spastic/immunology , Paraparesis, Tropical Spastic/virology , Proviruses/genetics , Middle Aged , HLA-A24 Antigen/immunology , HLA-A24 Antigen/genetics , T-Lymphocytes, Cytotoxic/immunology , Adult , HTLV-I Infections/immunology , HTLV-I Infections/virology , Gene Products, tax/immunology , Gene Products, tax/genetics , Aged , Gene Frequency
3.
Nat Commun ; 15(1): 5480, 2024 Jul 02.
Article in English | MEDLINE | ID: mdl-38956017

ABSTRACT

The primary obstacle to curing HIV-1 is a reservoir of CD4+ cells that contain stably integrated provirus. Previous studies characterizing the proviral landscape, which have been predominantly conducted in males in the United States and Europe living with HIV-1 subtype B, have revealed that most proviruses that persist during antiretroviral therapy (ART) are defective. In contrast, less is known about proviral landscapes in females with non-B subtypes, which represents the largest group of individuals living with HIV-1. Here, we analyze genomic DNA from resting CD4+ T-cells from 16 female and seven male Ugandans with HIV-1 receiving suppressive ART (n = 23). We perform near-full-length proviral sequencing at limiting dilution to examine the proviral genetic landscape, yielding 607 HIV-1 subtype A1, D, and recombinant proviral sequences (mean 26/person). We observe that intact genomes are relatively rare and clonal expansion occurs in both intact and defective genomes. Our modification of the primers and probes of the Intact Proviral DNA Assay (IPDA), developed for subtype B, rescues intact provirus detection in Ugandan samples for which the original IPDA fails. This work will facilitate research on HIV-1 persistence and cure strategies in Africa, where the burden of HIV-1 is heaviest.


Subject(s)
CD4-Positive T-Lymphocytes , Genome, Viral , HIV Infections , HIV-1 , Proviruses , Humans , HIV-1/genetics , HIV-1/drug effects , HIV-1/classification , Proviruses/genetics , HIV Infections/drug therapy , HIV Infections/virology , Male , Female , Genome, Viral/genetics , CD4-Positive T-Lymphocytes/virology , Adult , DNA, Viral/genetics , Uganda , Viral Load , Anti-HIV Agents/therapeutic use
4.
Proc Natl Acad Sci U S A ; 121(29): e2404349121, 2024 Jul 16.
Article in English | MEDLINE | ID: mdl-38985764

ABSTRACT

Intron-containing RNA expressed from the HIV-1 provirus activates type 1 interferon in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with short hairpin RNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte-derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the interferon-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with nontargetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant 2-CARD domain-deletion or phosphomimetic point mutations, indicates that IFIH1 (MDA5) filament formation, dephosphorylation, and association with MAVS are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 (MDA5) and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1 knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes by HIV-1. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 (MDA5), over two orders of magnitude, was revealed by formaldehyde cross-linking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is the innate immune receptor for intron-containing RNA from the HIV-1 provirus and that IFIH1 potentially contributes to chronic inflammation in people living with HIV-1, even in the presence of effective antiretroviral therapy.


Subject(s)
Dendritic Cells , HIV-1 , Immunity, Innate , Interferon-Induced Helicase, IFIH1 , Introns , Proviruses , RNA, Viral , Humans , HIV-1/genetics , HIV-1/immunology , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/metabolism , Proviruses/genetics , Dendritic Cells/immunology , Dendritic Cells/virology , Dendritic Cells/metabolism , Introns/genetics , RNA, Viral/genetics , RNA, Viral/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/immunology , HIV Infections/immunology , HIV Infections/virology , HIV Infections/genetics , Karyopherins/genetics , Karyopherins/metabolism
5.
Viruses ; 16(7)2024 Jun 25.
Article in English | MEDLINE | ID: mdl-39066179

ABSTRACT

Bovine leukemia virus (BLV) is prevalent worldwide, causing serious problems in the cattle industry. The BLV proviral load (PVL) is a useful index for estimating disease progression and transmission risk. We previously developed a quantitative real-time PCR (qPCR) assay to measure the PVL using the coordination of common motif (CoCoMo) degenerate primers. Here, we constructed a novel duplex BLV-CoCoMo qPCR assay that can amplify two genes simultaneously using a FAM-labeled MGB probe for the BLV LTR gene and a VIC-labeled MGB probe for the BoLA-DRA gene. This liquid duplex assay maintained its original sensitivity and reproducibility in field samples. Furthermore, we developed a dry duplex assay composed of PCR reagents necessary for the optimized liquid duplex assay. We observed a strong positive correlation between the PVLs measured using the dry and liquid duplex assays. Validation analyses showed that the sensitivity of the dry duplex assay was slightly lower than that of the other methods for the detection of a BLV molecular clone, but it showed similar sensitivity to the singleplex assay and slightly higher sensitivity than the liquid duplex assay for the PVL quantification of 82 field samples. Thus, our liquid and dry duplex assays are useful for measuring the BLV PVL in field samples, similar to the original singleplex assay.


Subject(s)
Enzootic Bovine Leukosis , Leukemia Virus, Bovine , Proviruses , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity , Viral Load , Leukemia Virus, Bovine/genetics , Leukemia Virus, Bovine/isolation & purification , Animals , Cattle , Proviruses/genetics , Viral Load/methods , Enzootic Bovine Leukosis/virology , Enzootic Bovine Leukosis/diagnosis , Reproducibility of Results , Real-Time Polymerase Chain Reaction/methods , DNA Primers/genetics , Multiplex Polymerase Chain Reaction/methods
6.
Viruses ; 16(7)2024 Jul 20.
Article in English | MEDLINE | ID: mdl-39066330

ABSTRACT

Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs' eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A/B/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 °C with a color-based lateral flow readout. The detection limit of the assay for ALV-A/B/J subgroups was 50 copies with no cross reactivity with ALV-C/D/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A/B/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs.


Subject(s)
Avian Leukosis Virus , Avian Leukosis , CRISPR-Cas Systems , DNA, Viral , Proviruses , Avian Leukosis Virus/genetics , Avian Leukosis Virus/isolation & purification , Avian Leukosis Virus/classification , Animals , Proviruses/genetics , Proviruses/isolation & purification , Avian Leukosis/virology , Avian Leukosis/diagnosis , DNA, Viral/genetics , Poultry Diseases/virology , Poultry Diseases/diagnosis , Chickens/virology , Sensitivity and Specificity , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/metabolism
7.
Vopr Virusol ; 69(2): 127-133, 2024 May 06.
Article in English | MEDLINE | ID: mdl-38843019

ABSTRACT

OBJECTIVES: The HTLV-1 infection persists for life, remaining as asymptomatic viral reservoirs in most patients, ensuring the chain of transmission, but around 4% develop adult T-cell leukemia/lymphoma (ATLL). HTLV-1 is an oncogenic retrovirus that transforms CD4+ T lymphocytes and deregulates the lymphoproliferative pathways that contribute to the development of ATLL. To achieve cell transformation, most oncogenic retroviruses use proto-oncogene capture transduction, with proviral integration disrupting the expression of tumor suppressors or proto-oncogenes. THE AIM: We conducted this study on the prevalence of HTLV-1 infection in blood donors to expand the HTLV-1 database, assess the risk of transmission via blood products, as well as evaluate the risk of persistent infection or development of neoplastic diseases in HTLV-1 carriers. MATERIALS AND METHODS: This is a cross-sectional study of blood donors of all categories. For this study, 265 blood donors were recruited at the Centre National de Transfusion Sanguine in Brazzaville. After testing for HTLV-1 antibodies by ELISA, proviral DNA was extracted from all ELISA-positive samples for detection by nested PCR, followed by RT qPCR using specific primers p53 and c-myc for gene expression. RESULTS: 20/265 were positive for anti-HTLV-1 antibody, 5 donors were positive for proviral DNA. The prevalence of HTLV-1 was 1.8%. All HTLV-1-positive donors were male (1.8%), with a positive correlation (p = 0.05); the 1.1% of positive donors were regular, with the majority aged between 31 and 45 years (1.5%), and concubine donors were the most frequent (1.1%). All samples showed normal expression of the p53 and c-myc genes. CONCLUSION: The prevalence, though low, remains a serious problem. No abnormal p53 or c-myc gene expression was detected in HTLV-1-positive donors, which could mean that none of the T lymphocytes in these donors had been transformed by HTLV-1.


Subject(s)
Blood Donors , HTLV-I Infections , Human T-lymphotropic virus 1 , Proto-Oncogene Proteins c-myc , Tumor Suppressor Protein p53 , Humans , Human T-lymphotropic virus 1/genetics , Male , HTLV-I Infections/epidemiology , HTLV-I Infections/virology , HTLV-I Infections/genetics , HTLV-I Infections/blood , Adult , Female , Tumor Suppressor Protein p53/genetics , Middle Aged , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Mas , Cross-Sectional Studies , Gene Expression Profiling , Leukemia-Lymphoma, Adult T-Cell/virology , Leukemia-Lymphoma, Adult T-Cell/epidemiology , Leukemia-Lymphoma, Adult T-Cell/genetics , Leukemia-Lymphoma, Adult T-Cell/blood , Proviruses/genetics , Adolescent
8.
J Clin Invest ; 134(14)2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38833307

ABSTRACT

Despite effective antiretroviral therapy (ART), persons living with HIV harbor reservoirs of persistently infected CD4+ cells, which constitute a barrier to cure. Initiation of ART during acute infection reduces the size of the HIV reservoir, and we hypothesized that in addition, it would favor integration of proviruses in HIV-specific CD4+ T cells, while initiation of ART during chronic HIV infection would favor relatively more proviruses in herpesvirus-specific cells. We further hypothesized that proviruses in acute ART initiators would be integrated into antiviral genes, whereas integration sites (ISs) in chronic ART initiators would favor genes associated with cell proliferation and exhaustion. We found that the HIV DNA distribution across HIV-specific versus herpesvirus-specific CD4+ T cells was as hypothesized. HIV ISs in acute ART initiators were significantly enriched in gene sets controlling lipid metabolism and HIF-1α-mediated hypoxia, both metabolic pathways active in early HIV infection. Persistence of these infected cells during prolonged ART suggests a survival advantage. ISs in chronic ART initiators were enriched in a gene set controlling EZH2 histone methylation, and methylation has been associated with diminished long terminal repeat transcription. These differences that we found in antigen specificities and IS distributions within HIV-infected cells might be leveraged in designing cure strategies tailored to the timing of ART initiation.


Subject(s)
CD4-Positive T-Lymphocytes , HIV Infections , HIV-1 , Proviruses , Virus Integration , Humans , HIV Infections/drug therapy , HIV Infections/genetics , HIV Infections/virology , HIV Infections/immunology , Proviruses/genetics , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , HIV-1/genetics , Male , Female , Adult , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , DNA, Viral/genetics , Anti-Retroviral Agents/administration & dosage , Anti-Retroviral Agents/therapeutic use
9.
Viruses ; 16(6)2024 May 31.
Article in English | MEDLINE | ID: mdl-38932184

ABSTRACT

Endogenous retroviruses (ERVs) are related to long terminal repeat (LTR) retrotransposons, comprising gene sequences of exogenous retroviruses integrated into the host genome and inherited according to Mendelian law. They are considered to have contributed greatly to the evolution of host genome structure and function. We previously characterized HERV-K HML-9 in the human genome. However, the biological function of this type of element in the genome of the chimpanzee, which is the closest living relative of humans, largely remains elusive. Therefore, the current study aims to characterize HML-9 in the chimpanzee genome and to compare the results with those in the human genome. Firstly, we report the distribution and genetic structural characterization of the 26 proviral elements and 38 solo LTR elements of HML-9 in the chimpanzee genome. The results showed that the distribution of these elements displayed a non-random integration pattern, and only six elements maintained a relatively complete structure. Then, we analyze their phylogeny and reveal that the identified elements all cluster together with HML-9 references and with those identified in the human genome. The HML-9 integration time was estimated based on the 2-LTR approach, and the results showed that HML-9 elements were integrated into the chimpanzee genome between 14 and 36 million years ago and into the human genome between 18 and 49 mya. In addition, conserved motifs, cis-regulatory regions, and enriched PBS sequence features in the chimpanzee genome were predicted based on bioinformatics. The results show that pathways significantly enriched for ERV LTR-regulated genes found in the chimpanzee genome are closely associated with disease development, including neurological and neurodevelopmental psychiatric disorders. In summary, the identification, characterization, and genomics of HML-9 presented here not only contribute to our understanding of the role of ERVs in primate evolution but also to our understanding of their biofunctional significance.


Subject(s)
Endogenous Retroviruses , Evolution, Molecular , Genome , Pan troglodytes , Phylogeny , Terminal Repeat Sequences , Animals , Endogenous Retroviruses/genetics , Humans , Genome, Human , Proviruses/genetics , Virus Integration , Retroelements
10.
Viruses ; 16(5)2024 05 09.
Article in English | MEDLINE | ID: mdl-38793632

ABSTRACT

People with HIV exhibit persistent inflammation that correlates with HIV-associated comorbidities including accelerated aging, increased risk of cardiovascular disease, and neuroinflammation. Mechanisms that perpetuate chronic inflammation in people with HIV undergoing antiretroviral treatments are poorly understood. One hypothesis is that the persistent low-level expression of HIV proviruses, including RNAs generated from defective proviral genomes, drives the immune dysfunction that is responsible for chronic HIV pathogenesis. We explore factors during HIV infection that contribute to the generation of a pool of defective proviruses as well as how HIV-1 mRNA and proteins alter immune function in people living with HIV.


Subject(s)
HIV Infections , HIV-1 , Inflammation , Transcription, Genetic , Humans , HIV Infections/virology , HIV Infections/complications , HIV Infections/immunology , HIV-1/genetics , Proviruses/genetics , Protein Biosynthesis , RNA, Viral/genetics
11.
J Antimicrob Chemother ; 79(7): 1673-1676, 2024 07 01.
Article in English | MEDLINE | ID: mdl-38804140

ABSTRACT

OBJECTIVES: Resistance associated mutations (RAMs) are archived in the HIV reservoir and can re-emerge with an inappropriate ART use limiting treatment options. However, recent studies, using ultra-deep sequencing (UDS), showed a decrease of quasispecies harbouring RAMs, suggesting that recycling some antiretrovirals could be considered. The aim of this study was to characterize, in HIV treated PLWHIV, the M184V mutation decrease kinetics in proviral DNA and associated factors of M184V mutation clearance over time. METHODS: UDS was performed on HIV-DNA from blood cells at different time points to quantify the percentage of M184V positive quasispecies. The sequence reads were analysed with a minimum coverage set at 50 and an ambiguity filter at 5% or 2%. RESULTS: At 2.5 years after the first time point, the M184V lost was observed in 50% of PLWHIV. Moreover, univariate analyses highlight that a higher nadir CD4 count and a lower zenith HIV1 RNA viral load were correlated with a faster clearance of the mutation. In multivariate analysis, a higher zenith was negatively associated with the M184V clearance at the 5% threshold. Interestingly, lamivudine/emtricitabine presence in the ART therapy regiment during the 5 years was not associated with the persistence of the M184V. CONCLUSIONS: Our study provides new information concerning the clearance speed of M184V mutation over time in PLWHIV with fully suppressed viremia, opens the discussion about the duration needed to consider a lamivudine/emtricitabine recycling and reinforces the association of the nadir and zenith values with the M184V mutation clearance.


Subject(s)
Anti-HIV Agents , Drug Resistance, Viral , HIV Infections , HIV-1 , Mutation , Viral Load , Humans , HIV Infections/drug therapy , HIV Infections/virology , Drug Resistance, Viral/genetics , HIV-1/genetics , HIV-1/drug effects , CD4 Lymphocyte Count , Male , Anti-HIV Agents/therapeutic use , Anti-HIV Agents/pharmacology , Female , Adult , Middle Aged , Proviruses/genetics , High-Throughput Nucleotide Sequencing , DNA, Viral/genetics , DNA, Viral/blood , HIV Reverse Transcriptase/genetics , Antiretroviral Therapy, Highly Active
12.
BMC Vet Res ; 20(1): 195, 2024 May 13.
Article in English | MEDLINE | ID: mdl-38741095

ABSTRACT

Small ruminant lentiviruses (SRLVs) are widespread and infect goats and sheep. Several reports also suggest that SRLVs can infect wild ruminants. The presence of specific antibodies against SRLVs has been identified in wild ruminants from Poland, but no studies have been conducted to detect proviral DNA of SRLVs in these animals. Therefore, the purpose of this study was to examine samples from Polish wild ruminants to determine whether these animals can serve as reservoirs of SRLVs under natural conditions. A total of 314 samples were tested from red deer (n = 255), roe deer (n = 52) and fallow deer (n = 7) using nested real-time PCR. DNA from positive real-time PCR samples was subsequently used to amplify a CA fragment (625 bp) of the gag gene, a 1.2 kb fragment of the pol gene and an LTR-gag fragment. Three samples (0.95%) were positive according to nested real-time PCR using primers and probe specific for CAEV (SRLV group B). All the samples were negative for the primers and probe specific for MVV (SRLV A group). Only SRLV LTR-gag sequences were obtained from two red deer. Phylogenetic analysis revealed that these sequences were more closely related to CAEV than to MVV. Our results revealed that deer can carry SRLV proviral sequences and therefore may play a role in the epidemiology of SRLVs. To our knowledge, this is the first study describing SRLV sequences from red deer.


Subject(s)
DNA, Viral , Deer , Lentivirus Infections , Proviruses , Animals , Deer/virology , Poland/epidemiology , Proviruses/genetics , Lentivirus Infections/veterinary , Lentivirus Infections/virology , Lentivirus Infections/epidemiology , DNA, Viral/genetics , Lentivirus/isolation & purification , Lentivirus/genetics , Lentivirus/classification , Phylogeny , Real-Time Polymerase Chain Reaction/veterinary
13.
Methods Mol Biol ; 2807: 15-30, 2024.
Article in English | MEDLINE | ID: mdl-38743218

ABSTRACT

Live-cell imaging has become a powerful tool for dissecting the behavior of viral complexes during HIV-1 infection with high temporal and spatial resolution. Very few HIV-1 particles in a viral population are infectious and successfully complete replication (~1/50). Single-particle live-cell imaging enables the study of these rare infectious viral particles, which cannot be accomplished in biochemical assays that measure the average property of the entire viral population, most of which are not infectious. The timing and location of many events in the early stage of the HIV-1 life cycle, including nuclear import, uncoating, and integration, have only recently been elucidated. Live-cell imaging also provides a valuable approach to study interactions of viral and host factors in distinct cellular compartments and at specific stages of viral replication. Successful live-cell imaging experiments require careful consideration of the fluorescent labeling method used and avoid or minimize its potential impact on normal viral replication and produce misleading results. Ideally, it is beneficial to utilize multiple virus labeling strategies and compare the results to ensure that the virion labeling did not adversely influence the viral replication step that is under investigation. Another potential benefit of using different labeling strategies is that they can provide information about the state of the viral complexes. Here, we describe our methods that utilize multiple fluorescent protein labeling approaches to visualize and quantify important events in the HIV-1 life cycle, including docking HIV-1 particles with the nuclear envelope (NE) and their nuclear import, uncoating, and proviral transcription.


Subject(s)
Active Transport, Cell Nucleus , HIV-1 , Transcription, Genetic , Virus Replication , HIV-1/physiology , HIV-1/genetics , Humans , Virus Uncoating , Proviruses/genetics , Proviruses/physiology , Cell Nucleus/metabolism , Cell Nucleus/virology , HIV Infections/virology , HIV Infections/metabolism , Virion/metabolism , Virion/genetics
14.
Methods Mol Biol ; 2807: 45-59, 2024.
Article in English | MEDLINE | ID: mdl-38743220

ABSTRACT

Latent HIV-1 reservoirs are a major obstacle to the eradication of HIV-1. Several cure strategies have been proposed to eliminate latent reservoirs. One of the key strategies involves the reactivation of latent HIV-1 from cells using latency-reversing agents. However, currently it is unclear whether any of the latency-reversing agents are able to completely reactivate HIV-1 provirus transcription in all latent cells. An understanding of the reactivation of HIV-1 provirus at single-cell single-molecule level is necessary to fully comprehend the reactivation of HIV-1 in the reservoirs. Furthermore, since reactivable viruses in the pool of latent reservoirs are rare, combining single-cell imaging techniques with the ability to visualize a large number of reactivated single cells that express both viral RNA and proteins in a pool of uninfected and non-reactivated cells will provide unprecedented information about cell-to-cell variability in reactivation. Here, we describe the single-cell single-molecule RNA-FISH (smRNA-FISH) method to visualize HIV-1 gag RNA combined with the immunofluorescence (IF) method to detect Gag protein to characterize the reactivated cells. This method allows the visualization of subcellular localization of RNA and proteins before and after reactivation and facilitates absolute quantitation of the number of transcripts per cell using FISH-quant. In addition, we describe a high-speed and high-resolution scanning (HSHRS) fluorescence microscopy imaging method to visualize rare and reactivated cells in a pool of non-reactivated cells with high efficiency.


Subject(s)
Fluorescent Antibody Technique , HIV-1 , In Situ Hybridization, Fluorescence , RNA, Viral , Single Molecule Imaging , Single-Cell Analysis , Virus Activation , Virus Latency , HIV-1/physiology , HIV-1/genetics , Humans , In Situ Hybridization, Fluorescence/methods , RNA, Viral/genetics , Single-Cell Analysis/methods , Single Molecule Imaging/methods , Fluorescent Antibody Technique/methods , HIV Infections/virology , Proviruses/genetics
15.
J Vet Med Sci ; 86(6): 653-655, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38631888

ABSTRACT

The present study analyzed B-cell clonality and bovine leukemia virus (BLV) provirus integration sites in cattle with enzootic bovine leukosis (EBL) having BLV proviral copy numbers less or greater than the number of bovine nucleated cells. EBL cattle with BLV copy numbers less than the number of bovine nucleated cells showed monoclonal and biclonal proliferation of B-cells with one BLV provirus integration site. On the other hand, EBL cattle with BLV copy numbers greater than the number of bovine nucleated cells showed monoclonal proliferation of B-cells with two BLV provirus integration sites. These results suggest that superinfection of BLV can occur in EBL cattle.


Subject(s)
B-Lymphocytes , DNA, Viral , Enzootic Bovine Leukosis , Leukemia Virus, Bovine , Proviruses , Animals , Leukemia Virus, Bovine/genetics , Enzootic Bovine Leukosis/virology , Cattle , Proviruses/genetics , DNA, Viral/genetics , B-Lymphocytes/virology , Virus Integration , Cell Proliferation
16.
Viruses ; 16(4)2024 03 27.
Article in English | MEDLINE | ID: mdl-38675857

ABSTRACT

The persistence of the latent viral reservoir is the main hurdle to curing HIV-1 infection. SIV infection of non-human primates (NHPs), namely Indian-origin rhesus macaques, is the most relevant and widely used animal model to evaluate therapies that seek to eradicate HIV-1. The utility of a model ultimately rests on how accurately it can recapitulate human disease, and while reservoirs in the NHP model behave quantitatively very similar to those of long-term suppressed persons with HIV-1 (PWH) in the most salient aspects, recent studies have uncovered key nuances at the clonotypic level that differentiate the two in qualitative terms. In this review, we will highlight differences relating to proviral intactness, clonotypic structure, and decay rate during ART between HIV-1 and SIV reservoirs and discuss the relevance of these distinctions in the interpretation of HIV-1 cure strategies. While these, to some degree, may reflect a unique biology of the virus or host, distinctions among the proviral landscape in SIV are likely to be shaped significantly by the condensed timeframe of NHP studies. ART is generally initiated earlier in the disease course, and animals are virologically suppressed for shorter periods before receiving interventions. Because these are experimental variables dictated by the investigator, we offer guidance on study design for cure-related studies performed in the NHP model. Finally, we highlight the case of GS-9620 (Vesatolimod), an antiviral TLR7 agonist tested in multiple independent pre-clinical studies in which virological outcomes may have been influenced by study-related variables.


Subject(s)
HIV Infections , HIV-1 , Simian Acquired Immunodeficiency Syndrome , Simian Immunodeficiency Virus , Virus Latency , Animals , Humans , Disease Models, Animal , Disease Reservoirs/virology , HIV Infections/virology , HIV Infections/drug therapy , HIV-1/genetics , HIV-1/drug effects , HIV-1/physiology , Macaca mulatta , Proviruses/genetics , Proviruses/physiology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Acquired Immunodeficiency Syndrome/drug therapy , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/physiology , Viral Load , Virus Latency/drug effects
17.
Microbiol Spectr ; 12(6): e0432323, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38687078

ABSTRACT

An investigation into retrovirus was conducted in six species of bats (Myotis aurascens, Myotis petax, Myotis macrodactylus, Miniopterus fuliginosus, Rhinolophus ferrumequinum, and Pipistrellus abramus) inhabiting South Korea. Exogenous retroviruses (XRVs) were detected in the tissue samples of R. ferrumequinum individuals by PCR assay. Proviruses were identified in all tissue samples through viral quantification using a digital PCR assay per organ (lung, intestine, heart, brain, wing, kidney, and liver), with viral loads varying greatly between each organ. In phylogenetic analysis based on the whole genome, the Korean bat retroviruses and the R. ferrumequinum retrovirus (RfRV) strain formed a new clade distinct from the Gammaretrovirus clade. The phylogenetic results determined these viruses to be RfRV-like viruses. In the Simplot comparison, Korean RfRV-like viruses exhibited relatively strong fluctuated patterns in the latter part of the envelope gene area compared to other gene areas. Several point mutations within this region (6,878-7,774 bp) of these viruses were observed compared to the RfRV sequence. One Korean RfRV-like virus (named Y4b strain) was successfully recovered in the Raw 264.7 cell line, and virus particles replicated in the cells were confirmed by transmission electron microscopy. RfRVs (or RfRV-like viruses) have been spreading since their first discovery in 2012, and the Korean RfRV-like viruses were assumed to be XRVs that evolved from RfRV.IMPORTANCER. ferrumequinum retrovirus (RfRV)-like viruses were identified in greater horseshoe bats in South Korea. These RfRV-like viruses were considered exogenous retroviruses (XRVs) that emerged from RfRV. Varying amounts of provirus detected in different organs suggest ongoing viral activity, replication, and de novo integration in certain organs. Additionally, the successful recovery of the virus in the Raw 264.7 cell line provides strong evidence supporting their status as XRVs. These viruses have now been identified in South Korea and, more recently, in Kenya since RfRV was discovered in China in 2012, indicating that RfRVs (or RfRV-like viruses) have spread worldwide.


Subject(s)
Chiroptera , Phylogeny , Animals , Chiroptera/virology , Republic of Korea , Mice , Proviruses/genetics , Proviruses/isolation & purification , Retroviridae Infections/virology , Retroviridae Infections/veterinary , Retroviridae/isolation & purification , Retroviridae/classification , Retroviridae/genetics , Genome, Viral , Viral Load
18.
Proc Natl Acad Sci U S A ; 121(18): e2202003121, 2024 Apr 30.
Article in English | MEDLINE | ID: mdl-38669184

ABSTRACT

Using an immunofluorescence assay based on CRISPR-dCas9-gRNA complexes that selectively bind to the HIV LTR (HIV Cas-FISH), we traced changes in HIV DNA localization in primary effector T cells from early infection until the cells become quiescent as they transition to memory cells. Unintegrated HIV DNA colocalized with CPSF6 and HIV capsid (CA, p24) was found in the cytoplasm and nuclear periphery at days 1 and 3 post infection. From days 3 to 7, most HIV DNA was distributed primarily in the nuclear intermediate euchromatic compartment and was transcribed. By day 21, the cells had entered quiescence, and HIV DNA accumulated in the perinucleolar compartment (PNC). The localization of proviruses to the PNC was blocked by integrase inhibitor Raltegravir, suggesting it was due to chromosomal rearrangements. During the reactivation of latently infected cells through the T cell receptor (TCR), nascent viral mRNA transcripts associated with HIV DNA in the PNC were detected. The viral trans-activator Tat and its regulatory partners, P-TEFb and 7SK snRNA, assembled in large interchromatin granule clusters near the provirus within 2 h of TCR activation. As T cell activation progressed, the HIV DNA shifted away from the PNC. HIV DNA in latently infected memory T cells from patients also accumulated in the PNC and showed identical patterns of nuclear rearrangements after cellular reactivation. Thus, in contrast to transformed cells where proviruses are found primarily at the nuclear periphery, in primary memory T cells, the nuclear architecture undergoes rearrangements that shape the transcriptional silencing and reactivation of proviral HIV.


Subject(s)
Cell Nucleus , HIV Infections , HIV-1 , Proviruses , Virus Activation , Virus Latency , Humans , Proviruses/genetics , Cell Nucleus/metabolism , Cell Nucleus/virology , HIV-1/genetics , HIV-1/physiology , HIV-1/metabolism , HIV Infections/virology , HIV Infections/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , HIV Long Terminal Repeat/genetics
19.
Front Cell Infect Microbiol ; 14: 1379962, 2024.
Article in English | MEDLINE | ID: mdl-38655281

ABSTRACT

The notion that viruses played a crucial role in the evolution of life is not a new concept. However, more recent insights suggest that this perception might be even more expansive, highlighting the ongoing impact of viruses on host evolution. Endogenous retroviruses (ERVs) are considered genomic remnants of ancient viral infections acquired throughout vertebrate evolution. Their exogenous counterparts once infected the host's germline cells, eventually leading to the permanent endogenization of their respective proviruses. The success of ERV colonization is evident so that it constitutes 8% of the human genome. Emerging genomic studies indicate that endogenous retroviruses are not merely remnants of past infections but rather play a corollary role, despite not fully understood, in host genetic regulation. This review presents some evidence supporting the crucial role of endogenous retroviruses in regulating host genetics. We explore the involvement of human ERVs (HERVs) in key physiological processes, from their precise and orchestrated activities during cellular differentiation and pluripotency to their contributions to aging and cellular senescence. Additionally, we discuss the costs associated with hosting a substantial amount of preserved viral genetic material.


Subject(s)
Endogenous Retroviruses , Endogenous Retroviruses/genetics , Endogenous Retroviruses/physiology , Humans , Animals , Cell Differentiation , Host-Pathogen Interactions/genetics , Host Microbial Interactions/genetics , Retroviridae Infections/virology , Cellular Senescence/genetics , Proviruses/genetics , Proviruses/physiology , Evolution, Molecular
20.
Curr Opin HIV AIDS ; 19(3): 124-132, 2024 05 01.
Article in English | MEDLINE | ID: mdl-38502547

ABSTRACT

PURPOSE OF REVIEW: Despite suppressive antiretroviral therapy (ART), HIV-1 reservoirs persist and reignite viral replication if therapy is interrupted. Persistence of the viral reservoir in people with HIV-1 (PWH) is the main obstacle to an HIV-1 cure. The reservoirs are not transcriptionally silent, and viral transcripts can be detected in most ART-treated individuals. Here, we review the recent progress in the characterization of persistent HIV-1 transcription during ART. RECENT FINDINGS: Evidence from several studies indicates that, although cell-associated unspliced (US) HIV-1 RNA is abundantly expressed in ART-treated PWH, intact full-length US transcripts are rare and most US RNA is derived from defective proviruses. The transcription- and translation-competent defective proviruses, previously considered irrelevant, are increasingly being linked to residual HIV-1 pathogenesis under suppressive ART. Recent data suggest a continuous crosstalk between the residual HIV-1 activity under ART and the immune system. Persistent HIV-1 transcription on ART, despite being mostly derived from defective proviruses, predicts viral rebound upon therapy interruption, suggesting its role as an indicator of the strength of the host antiviral immune response that is shaping the viral rebound. SUMMARY: In light of the recent findings, the significance of persistent HIV-1 transcription during ART for the long-term health of PWH and the cure research should be reassessed.


Subject(s)
HIV Infections , HIV-1 , Humans , HIV Infections/drug therapy , HIV-1/genetics , Anti-Retroviral Agents/therapeutic use , Virus Replication , Proviruses/genetics , RNA, Viral/genetics , CD4-Positive T-Lymphocytes , Viral Load
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