Proviral Sequences Research Articles

Complete Sequences of the Human T-Cell Leukemia Virus Type 1 Proviral Genomes from Newly Established Adult T-Cell Leukemia Cell Lines in Oita Prefecture, Japan.

ABSTRACTWe report two complete proviral genome sequences of human T-cell leukemia virus type 1 (HTLV-1) isolated from the peripheral blood specimens of acute type adult T-cell leukemia (ATL) patients in Oita Prefecture, Japan.

Genetic characterization of the HIV-1 reservoir after Vacc-4x and romidepsin therapy in HIV-1-infected individuals.

Objective:Therapeutic HIV-1 immunization followed by latency reversal has been suggested as a strategy to eradicate HIV-1. Here we investigate the phylogenetic composition of the HIV-1 regions targeted by the therapeutic HIV-1 peptide vaccine Vacc-4x in participants in a clinical trial.Design:Seventeen participants on suppressive antiretroviral therapy were vaccinated with six doses of Vacc-4x followed by three doses of romidepsin. Seven study participants were selected for sequencing analysis. All participants underwent an analytical treatment interruption.Methods:Single-genome/proviral sequencing of the p24-RT region was performed to genetically characterize proviral DNA, cell-associated RNA and outgrowth viruses during therapy as well as plasma HIV-1 RNA during an analytical treatment interruption.Results:There were no changes in cell-associated HIV-1 RNA (P = 0.83) and DNA (P = 0.09) diversity over the course of the study and no difference between cell-associated HIV-1 RNA and DNA diversity (P = 0.32). Only one participant showed signs of potential vaccine-related selection in the rebounding plasma virus. In five of seven participants, we identified human leukocyte antigen-specific cytotoxic T lymphocytes (CTL) epitopes containing nonsilent mutations in 100% of the sequences.Conclusion:We detected no evidence of selective immune pressure reflected in proviral diversity or by occurrence of specific mutation in the vaccine-targeted epitopes. Preexisting CTL epitope mutations may affect the potency of this therapeutic vaccine. This highlights the challenges of developing effective HIV-1 therapeutic vaccines.

Safety of intra-articular transplantation of lentivirally transduced mesenchymal stromal cells for haemophilic arthropathy in a non-human primate

Joint bleeding and resultant arthropathy are major determinants of quality of life in haemophilia patients. We previously developed a mesenchymal stromal cell (MSC)-based treatment approach for haemophilic arthropathy in a mouse model of haemophilia A. Here, we evaluated the long-term safety of intra-articular injection of lentivirally transduced autologous MSCs in non-human primates. Autologous bone-marrow-derived MSCs transduced with a lentiviral vector expressing coagulation factor VIII (FVIII) were injected into the left knee joint of cynomolgus monkeys. We first conducted codon optimization to increase FVIII production in the cells. Lentiviral transduction of autologous MSCs resulted in a significant increase of FVIII in the culture supernatant before transplantation. We did not find any tumour generation around the knee structure at 11-16 months after injection by magnetic resonance imaging. The proviral sequence of the simian immunodeficiency virus lentiviral vector was not detected in the heart, lungs, spleen, liver, testis, or bone marrow by real-time quantitative PCR. We confirmed the long-term safety of intra-articular injection of transduced MSCs in a non-human primate. The procedure may be an attractive therapeutic approach for joint diseases in haemophilia patients.

D-104 Clonal proliferation of CD4 T cells encoding intact HIV-1

HIV-1 causes a chronic, incurable disease due to CD4 T cells that contain replication-competent provirus but exhibit little or no active viral gene expression, and effectively resist combination antiretroviral therapy (cART). Such latently-infected CD4 T cells possess a remarkable long-term stability and typically persist life-long, for reasons that are not fully understood. We have used massive single-genome, near full-length next-generation sequencing of HIV-1 DNA derived from unfractionated PBMC, ex vivo-isolated CD4 T cells, and phenotypically complex memory CD4 T cells from peripheral blood and lymphoid tissues to characterize the dynamics and underlying mechanisms supporting viral persistence. These studies demonstrated multiple sets of independent, near full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4 T cells harboring intact HIV-1. Interestingly, we found that Th1 CD4 T cells, typically responsible for antiviral immune defense, seem to harbor the majority of such clonally-expanded intact proviruses in cells from peripheral blood. In addition, we noted that cells harboring clonally-expanded proviral sequences frequently expressed cell surface markers known to protect CD4 T cells during the vulnerable phase of clonal proliferation, suggesting that HIV-1-infected CD4 T cells rely on physiological mechanisms for maintaining viral reservoir stability through clonal expansion. A closer longitudinal analysis of intact proviruses in distinct CD4 T cell subsets in future studies will be highly informative for developing targeted interventions for viral reservoir manipulation in clinical settings.

Pinpointing recurrent proviral integration sites in new models for latent HIV-1 infection

HIV infection is characterized by accumulation of proviral sequences within the human host genome. Integration of viral-derived DNA occurs at preferential loci, suggesting a site-specific crosstalk between viral sequences and human genes. We here describe a genome engineering workflow to generate models for HIV-1 infection that for the first time recapitulate proviral integration at selected genomic loci and provide unique tools to study effects of HIV proviral integration site choice. Using this workflow, we have derived two BACH2–HIV-1 reporter models that mimic largely latent integration in the clinically relevant BACH2 gene locus, which has been associated with recurrent integration and HIV-reservoir maintenance in chronically infected patients.

Recent progress in understanding HIV reservoirs.

Reservoirs of HIV-1-infected cells persist long-term despite highly effective antiretroviral suppression therapy and represent the main barrier against a cure for HIV-1. This review summarizes recent advances in understanding the complexity and diversity of viral reservoir cells. Latently infected memory CD4 T cells are the predominant cell compartment responsible for viral persistence, but some studies suggest that myeloid cells, and possibly hematopoietic progenitors, can also serve as long-term viral reservoirs. Specific phenotypic markers, including T-cell activation and exhaustion molecules, may denote CD4 T cells enriched for replication-competent proviruses. Clonal proliferation of infected CD4 T cells in vivo represents an important mechanism responsible for the remarkable long-term stability of the viral reservoir. Multiple new assays, including near full-genome proviral sequencing and simplified versions of viral outgrowth assays, are being developed to analyze and quantify persisting reservoirs of HIV-1-infected cells. Recent technological advances allow to profile the molecular structure and composition of viral reservoir cells in great detail. Continuous progress in understanding phenotypic and functional properties of viral reservoir cells provides clues for novel clinical interventions to destabilize viral persistence during antiretroviral therapy.

Prevalence of Mouse Mammary Tumor Virus (MMTV)-like sequences in human breast cancer tissues and adjacent normal breast tissues in Saudi Arabia

BackgroundBreast cancer is considered the most common cancer in women worldwide and is the leading cause of cancer mortality. Sequences similar to Mouse Mammary Tumor Virus (MMTV) were detected in human breast cancer in several studies from different geographical areas. However, the role played by this virus in breast cancer tumorigenesis is not completely understood. These MMTV-like sequences were found to be associated with breast cancer of more malignant types. The aim of this study is to determine the prevalence of MMTV-like envelope gene (env) positivity in breast cancer and non-cancerous breast tissue from Saudi Arabia.MethodsDetection of MMTV-like env proviral sequences was done using newly designed primers for conventional polymerase chain reaction (PCR). One hundred nighty four samples were collected from 103 females with breast cancer in addition to 51 control breast tissue obtained from individuals without cancer. We additionally investigated the association of proviral positivity with age of the patients, grade of breast cancer and presence of lymph node metastasis. The results were confirmed by sequencing.ResultsThe prevalence of MMTV-like env proviral positivity was 8.7% (9/103). MMTV env proviral sequences were detected in 5.9% (6/101) of breast cancer tissues and 9.7% (9/93) of non-cancerous adjacent tissues obtained from the same patients. None of the 51 control sample showed positive result for the MMTV env gene. No significant association was found between detection of the virus and the age of the patient, grade of the cancer or presence of metastasis.ConclusionWe document the presence of low frequency of MMTV env provirus sequence among breast cancer patients from Saudi Arabia. Further studies are needed to explore the role of the MMTV in breast cancer.

Blood CXCR3+ CD4 T Cells Are Enriched in Inducible Replication Competent HIV in Aviremic Antiretroviral Therapy-Treated Individuals

We recently demonstrated that lymph nodes (LNs) PD-1+/T follicular helper (Tfh) cells from antiretroviral therapy (ART)-treated HIV-infected individuals were enriched in cells containing replication competent virus. However, the distribution of cells containing inducible replication competent virus has been only partially elucidated in blood memory CD4 T-cell populations including the Tfh cell counterpart circulating in blood (cTfh). In this context, we have investigated the distribution of (1) total HIV-infected cells and (2) cells containing replication competent and infectious virus within various blood and LN memory CD4 T-cell populations of conventional antiretroviral therapy (cART)-treated HIV-infected individuals. In the present study, we show that blood CXCR3-expressing memory CD4 T cells are enriched in cells containing inducible replication competent virus and contributed the most to the total pool of cells containing replication competent and infectious virus in blood. Interestingly, subsequent proviral sequence analysis did not indicate virus compartmentalization between blood and LN CD4 T-cell populations, suggesting dynamic interchanges between the two compartments. We then investigated whether the composition of blood HIV reservoir may reflect the polarization of LN CD4 T cells at the time of reservoir seeding and showed that LN PD-1+ CD4 T cells of viremic untreated HIV-infected individuals expressed significantly higher levels of CXCR3 as compared to CCR4 and/or CCR6, suggesting that blood CXCR3-expressing CD4 T cells may originate from LN PD-1+ CD4 T cells. Taken together, these results indicate that blood CXCR3-expressing CD4 T cells represent the major blood compartment containing inducible replication competent virus in treated aviremic HIV-infected individuals.

Effect of analytical treatment interruption and reinitiation of antiretroviral therapy on HIV reservoirs and immunologic parameters in infected individuals.

Therapeutic strategies aimed at achieving antiretroviral therapy (ART)-free HIV remission in infected individuals are under active investigation. Considering the vast majority of HIV-infected individuals experience plasma viral rebound upon cessation of therapy, clinical trials evaluating the efficacy of curative strategies would likely require inclusion of ART interruption. However, it is unclear what impact short-term analytical treatment interruption (ATI) and subsequent reinitiation of ART have on immunologic and virologic parameters of HIV-infected individuals. Here, we show a significant increase of HIV burden in the CD4+ T cells of infected individuals during ATI that was correlated with the level of plasma viral rebound. However, the size of the HIV reservoirs as well as immune parameters, including markers of exhaustion and activation, returned to pre-ATI levels 6–12 months after the study participants resumed ART. Of note, the proportions of near full-length, genome-intact and structurally defective HIV proviral DNA sequences were similar prior to ATI and following reinitiation of ART. In addition, there was no evidence of emergence of antiretroviral drug resistance mutations within intact HIV proviral DNA sequences following reinitiation of ART. These data demonstrate that short-term ATI does not necessarily lead to expansion of the persistent HIV reservoir nor irreparable damages to the immune system in the peripheral blood, warranting the inclusion of ATI in future clinical trials evaluating curative strategies.

Characterization of HIV-1 Near Full-Length Proviral Genome Quasispecies from Patients with Undetectable Viral Load Undergoing First-Line HAART Therapy.

Increased access to highly active antiretroviral therapy (HAART) by human immunodeficiency virus postive (HIV+) individuals has become a reality worldwide. In Brazil, HAART currently reaches over half of HIV-infected subjects. In the context of a remarkable HIV-1 genetic variability, highly related variants, called quasispecies, are generated. HIV quasispecies generated during infection can influence virus persistence and pathogenicity, representing a challenge to treatment. However, the clinical relevance of minority quasispecies is still uncertain. In this study, we have determined the archived proviral sequences, viral subtype and drug resistance mutations from a cohort of HIV+ patients with undetectable viral load undergoing HAART as first-line therapy using next-generation sequencing for near full-length virus genome (NFLG) assembly. HIV-1 consensus sequences representing NFLG were obtained for eleven patients, while for another twelve varying genome coverage rates were obtained. Phylogenetic analysis showed the predominance of subtype B (83%; 19/23). Considering the minority variants, 18 patients carried archived virus harboring at least one mutation conferring antiretroviral resistance; for six patients, the mutations correlated with the current ARVs used. These data highlight the importance of monitoring HIV minority drug resistant variants and their clinical impact, to guide future regimen switches and improve HIV treatment success.

Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential

Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in "enhancerless" self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required.IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy.

Interest of proviral HIV-1 DNA genotypic resistance testing in virologically suppressed patients candidate for maintenance therapy

Switch of antiretroviral therapy in virologically suppressed HIV-infected patients is frequent, to prevent toxicities, for simplification or convenience reasons. Pretherapeutic genotypic resistance testing on RNA can be lacking in some patients, which could enhance the risk of virologic failure, if resistance-associated mutations of the new regimen are not taken into account. Proviral DNA resistance testing in 69 virologically suppressed patients on antiretroviral treatment with no history of virological failure were pair-wised compared with pre-ART plasma RNA resistance testing. The median time between plasma (RNA testing) and whole blood (proviral DNA testing) was 47 months (IQR 29–63). A stop codon was evidenced in 23% (16/69) of proviral DNA sequences; these strains were considered as defective, non-replicative, and not taken into consideration. Within the non defective strains, concordance rate between plasma RNA and non-defective proviral DNA was high both on protease (194/220 concordant resistance-associated mutations=88%) and reverse transcriptase (28/37 concordant resistance-associated mutations=76%) genes. This study supports that proviral DNA testing might be an informative tool before switching antiretrovirals in virologically suppressed patients with no history of virological failure, but the interpretation should be restricted to non-defective viruses.

Novel endogenous retrovirus-derived transcript expressed in the bovine placenta is regulated by WNT signaling.

Endogenous retroviruses (ERVs) are involved in placentation; perhaps, the most well-known ERVs are the syncytins, actively transcribed env genes involved in cell–cell fusion and possible morphological variations. However, ERVs other than syncytins that play an important role in placental development have not been well characterized. To identify ERV genes expressed during the onset of placentation in the bovine species, we characterized the expression profiles of bovine conceptus transcripts during the peri-attachment period using RNA-seq analysis, and confirming some candidates through real-time PCR. Using in silico and PCR analyses, we identified a novel ERV proviral sequence derived from a gag region, designated bovine endogenous retroviruses (BERV)-K3, containing Gag_p10 and Gag_p24, zinc finger domain. Initial expression of this ERV in bovine conceptuses was on day 20 (day 0 = day of estrus), soon after conceptus attachment to the endometrial epithelium, and its high placental expression was maintained up to the middle of pregnancy. The BERV-K3 transcript was also found in the uterine luminal and glandular epithelia, liver, kidney, intestine, and skin. BERV-K3 is located on chromosome 7 and integrated within LOC100848658, from which noncoding RNA could be transcribed. Furthermore, the expression of endogenous BERV-K3 in bovine trophoblast cell lines was induced by a WNT agonist, a signaling system common to genes expressed in placentas. These data support the argument that during the evolutionary process, mammals incorporated not only similar ERV sequences, but also ERVs unique to individual species. BERV-K3 is in the latter case, likely providing functions unique to ruminant gestation.

Viral Blips Were Infrequent in HIV-1-Infected Virologically-Suppressed Adults Treated with Tenofovir Alafenamide or Tenofovir DF Rilpivirine-Containing Regimens

BackgroundDifferences between regimens in the frequency of transient episodes of viremia (viral blips) as well as the impact of these viral blips on the risk of virologic failure and resistance development is not fully understood. Here we investigate the frequency of viral blips in virologically-suppressed subjects switching to rilpivirine/emtricitabine/tenofovir alafenamide (R/F/TAF) vs. maintaining R/F/tenofovir disoproxil fumarate (TDF) and the association of viral blips with clinical outcome through Week 48 of study GS-US-366-1216.MethodsGS-US-366-1216 is a randomized, double-blind, phase 3b study evaluating the safety and efficacy of switching to R/F/TAF from R/F/TDF in HIV-1-infected virologically-suppressed subjects. For the viral blip analysis, treated subjects with ≥1 post-baseline HIV-1 RNA value were included. All on-drug HIV-1 RNA data points and FDA snapshot outcome data through Week 48 were utilized. Plasma HIV-1 RNA was measured using the Roche Taqman 2.0 assay. A viral blip was defined as any post-baseline HIV-1 RNA value ≥50 c/mL preceded and followed by HIV-1 RNA <50 c/mL.ResultsOf the 627 subjects included in the analysis, 23 (3.7%) experienced ≥1 blip through Week 48 and were distributed similarly between treatment groups (10/315, 3.2% R/F/TAF; 13/312, 4.2% R/F/TDF; P = 0.53; median 6 viral load measurements per subject). Twenty subjects had single blips (8 R/F/TAF, 12 R/F/TDF) and 3 subjects experienced 2 blips each (2 R/F/TAF, 1 R/F/TDF). Of 26 total blip events, 19 (73%) were low-level at 50–199 c/mL. Among subjects with blips, 22/23 (96%) were virologic successes at Week 48 (9/10, 90% R/F/TAF; 13/13, 100% R/F/TDF), similar to those subjects without blips (568/604, 94% overall; 287/305, 94% R/F/TAF; 281/299, 94% R/F/TDF). One subject in the R/F/TAF group had 2 blips prior to experiencing virologic rebound with mutations also detected at baseline (determined by retrospective proviral DNA sequencing).ConclusionViral blips were infrequent among subjects switching to R/F/TAF or maintaining R/F/TDF through Week 48 of study GS-US-366-1216. No differences in blip frequency or virologic failure post-blip were observed between treatment groups. Most blips were low-level (<200 c/mL) and most subjects with blips remained suppressed through Week 48.Disclosures D. A. Wohl, Gilead Sciences Inc.: Consultant and Investigator, Consulting fee and Research grant; ViiV: Consultant and Investigator, Consulting fee and Research grant; Janssen: Consultant, Consulting fee; Bristol Myers Squibb: Consultant, Consulting fee; R. Kulkarni, Gilead Sciences, Inc.: Employee and Shareholder, Salary; W. Garner, Gilead Sciences, Inc.: Employee and Shareholder, Salary; K. White, Gilead Sciences, Inc.: Employee and Shareholder, Salary; D. Porter, Gilead Sciences, Inc.: Employee and Shareholder, Salary

Identification of Genetically Intact HIV-1 Proviruses in Specific CD4+ T Cells from Effectively Treated Participants

Latent replication-competent HIV-1 persists in individuals on long-term antiretroviral therapy (ART). We developed the Full-Length Individual Proviral Sequencing (FLIPS) assay to determine the distribution of latent replication-competent HIV-1 within memory CD4+ Tcell subsets in six individuals on long-term ART. FLIPS is an efficient, high-throughput assay that amplifies and sequences near full-length (∼9 kb) HIV-1 proviral genomes and determines potential replication competency through genetic characterization. FLIPS provides a genome-scale perspective that addresses the limitations of other methods that also genetically characterize the latent reservoir. Using FLIPS, we identified 5% of proviruses as intact and potentially replication competent. Intact proviruses were unequally distributed between Tcell subsets, with effector memory cells containing the largest proportion of genetically intact HIV-1 proviruses. We identified multiple identical intact proviruses, suggesting a role for cellular proliferation in the maintenance of the latent HIV-1 reservoir.

Zoonotic infection of Brazilian primate workers with New World simian foamy virus.

Simian foamy viruses (SFVs) are retroviruses present in nearly all nonhuman primates (NHPs), including Old World primates (OWP) and New World primates (NWP). While all confirmed human infections with SFV are from zoonotic transmissions originating from OWP, little is known about the zoonotic transmission potential of NWP SFV. We conducted a longitudinal, prospective study of 56 workers occupationally exposed to NWP in Brazil. Plasma from these workers was tested using Western blot (WB) assays containing NWP SFV antigens. Genomic DNA from blood and buccal swabs was analyzed for the presence of proviral SFV sequences by three nested PCR tests and a new quantitative PCR assay. Exposure histories were obtained and analyzed for associations with possible SFV infection. Ten persons (18%) tested seropositive and two persons were seroindeterminate (3.6%) for NWP SFV. Six persons had seroreactivity over 2–3 years suggestive of persistent infection. All SFV NWP WB-positive workers reported at least one incident involving NWP, including six reporting NWP bites. NWP SFV viral DNA was not detected in the blood or buccal swabs from all 12 NWP SFV seroreactive workers. We also found evidence of SFV seroreversion in three workers suggestive of possible clearance of infection. Our findings suggest that NWP SFV can be transmitted to occupationally-exposed humans and can elicit specific humoral immune responses but infection remains well-controlled resulting in latent infection and may occasionally clear.

Ongoing HIV Replication During ART Reconsidered.

Lorenzo-Redondo et al. recently analyzed HIV RNA sequences in plasma virus and proviral DNA sequences in lymph nodes (LN) and peripheral blood mononuclear cells (PBMC) from samples collected over a 6-month period from 3 individuals following initiation of antiretroviral therapy (ART) and concluded that ongoing HIV replication occurred in LN despite ART and that this replication maintained the HIV reservoir. We analyzed the same sequences and found that the dataset was very limited (median of 5 unique RNA or DNA sequences per sample) after accounting for polymerase chain reaction resampling and hypermutation and that the few remaining DNA sequences after 3 and 6 months on ART were not more diverse or divergent from those in pre-ART in any of the individuals studied. These findings, and others, lead us to conclude that the claims of ongoing replication on ART made by Lorenzo-Redondo et al. are not justified from the dataset analyzed in their publication.

Abstract 5757: Study of human mammary tumor virus (HMTV) in human breast cancer by NanoString nCounter and FISH analysis

Abstract Human Mammary Tumor Virus (HMTV), a retrovirus 90-98% homologous to Mouse Mammary tumor Virus (MMTV), the etiological agent of murine mammary cancer, has been detected by PCR in 40% of American women’s breast cancers, but not in healthy tissues of the same breast (Cancer Research 1995, 55:5173-79; Clin. Cancer Research 2001, 7-283-4). A complete 9.9 kb HMTV proviral sequence has been detected in breast cancer genomic DNA (Cancer Res 2001,61:1754-9). HMTV viral particles with betaretrovirus characteristics have been isolated from metastatic breast cancer cells in effusions (MSSM cells). Expression of HMTV proteins has been detected in MSSM cells by western blot, fluorescence-activated cell sorting analysis, and immunofluorescence assays but not in normal mammary epithelial cells (J Virol Methods 2010, 163(1): 157-61). PCR is a sensitive technique susceptible of contamination, which due to amplification, could result in false positive detection. Although murine DNA was not found by PCR, the possibility still exists that HMTV in human DNA could be a result of laboratory contamination. Despite the substantial evidence supporting the presence of HMTV in breast cancer the controversy continues because the chance of contamination has led to doubt that murine-like viruses are human pathogens. We now report additional analyses of breast cancer specimens employing other methods for HMTV detection that reinforce previous findings. NanoString nCounter, a new technology using specific 100 mer oligonucleotides as probes bound to reporter molecules that can detect HMTV gag, env and LTR sequences as well as two murine specific probes in a single reaction without amplification. Flourescence in situ hybridization (FISH) assays with a HMTV probe is a technology that permits visualization of HMTV proviral DNA in the nuclei of surgical samples of breast cancer as well as in MSSM cells. Results from Nanostring nCounter, and FISH analysis show the presence of HMTV DNA in surgical cancer specimens and in MSSM cells. The data exclude contamination and confirm the authenticity of HMTV in human breast cancer. Next-generation sequencing and other studies are in progress to determine the role of HMTV in the pathogenesis of breast carcinoma. Citation Format: Stella M. Melana, Joseph Tripodi, Digna Nosike, Jaffer Shabnam, Polly Etkind, Beatriz GT Pogo, James F. Holland. Study of human mammary tumor virus (HMTV) in human breast cancer by NanoString nCounter and FISH analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5757. doi:10.1158/1538-7445.AM2017-5757

Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells.

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.

Live cell imaging of single genomic loci with quantum dot-labeled TALEs

Single genomic loci are often related to specific cellular functions, genetic diseases, or pathogenic infections. Visualization of single genomic loci in live human cells is currently of great interest, yet it remains challenging. Here, we describe a strategy for live cell imaging of single genomic loci by combining transcription activator-like effectors (TALEs) with a quantum dot labelling technique. We design and select a pair of TALEs that specifically target HIV-1 proviral DNA sequences, and use bioorthogonal ligation reactions to label them with different colour quantum dots (QDs). These QD-labelled TALEs are able to enter the cell nucleus to provide fluorescent signals to identify single gene loci. Based on the co-localization of the pair of different coloured QD-labelled TALEs, we determine and map single-copy HIV-1 provirus loci in human chromosomes in live host cells.