The relative contributions of infectious and mitotic spread to HTLV-1 persistence.

Human T-lymphotropic virus type-1 (HTLV-1) persists within hosts via infectious spread (de novo infection) and mitotic spread (infected cell proliferation), creating a population structure of multiple clones (infected cell populations with identical genomic proviral integration sites). The relative contributions of infectious and mitotic spread to HTLV-1 persistence are unknown, and will determine the efficacy of different approaches to treatment. The prevailing view is that infectious spread is negligible in HTLV-1 persistence beyond early infection. However, in light of recent high-throughput data on the abundance of HTLV-1 clones, and recent estimates of HTLV-1 clonal diversity that are substantially higher than previously thought (typically between 104 and 105 HTLV-1+ T cell clones in the body of an asymptomatic carrier or patient with HTLV-1-associated myelopathy/tropical spastic paraparesis), ongoing infectious spread during chronic infection remains possible. We estimate the ratio of infectious to mitotic spread using a hybrid model of deterministic and stochastic processes, fitted to previously published HTLV-1 clonal diversity estimates. We investigate the robustness of our estimates using three alternative estimators. We find that, contrary to previous belief, infectious spread persists during chronic infection, even after HTLV-1 proviral load has reached its set point, and we estimate that between 100 and 200 new HTLV-1 clones are created and killed every day. We find broad agreement between all estimators. The risk of HTLV-1-associated malignancy and inflammatory disease is strongly correlated with proviral load, which in turn is correlated with the number of HTLV-1-infected clones, which are created by de novo infection. Our results therefore imply that suppression of de novo infection may reduce the risk of malignant transformation.

Clinical features and human T-cell leukemia virus type-1 (HTLV-1) proviral load in HTLV-1-positive patients with rheumatoid arthritis: Baseline data in a single center cohort study.

Objective: Recently, Human T-cell leukemia virus type-1 proviral load (HTLV-1 PVL) has been evaluated as an important predictor of adult T-cell leukemia/lymphoma (ATL) in HTLV-1 carriers. We aimed to evaluate whether HTLV-1 PVL is also important for the development of ATL among HTLV-1-positive patients with rheumatoid arthritis (RA).Methods: We established a cohort of 82 HTLV-1-positive RA patients between 2017 and 2018. Of those, 27 (32.9%) were treated with biological disease-modifying anti-rheumatic drugs (bDMARDs) with/without methotrexate. We measured HTLV-1 PVL in peripheral blood mononuclear cells (PBMCs) at study entry and compared the value by clinical status and treatment options.Results: The median PVL for all was 9.6 copies per 1000 PBMCs without sex difference (male 17.2 and female 8.6; p = .24). The median PVL was significantly higher for patient's comorbid bronchiectasis, malignancies, and opportunistic infectious diseases, compared with patients without comorbidity. There were no significant differences in PVL levels among types of bDMARDs, although the level was tended to be higher for patients treated with JAK inhibitor.Conclusions: HTLV-1 seropositive RA patients comorbid for any diseases having higher HTLV-1 PVLs will be a higher risk for developing ATL. Careful follow-up of these patients is necessary to detect ATL development.

Decellularized pig pulmonary heart valves-Depletion of nucleic acids measured by proviral PERV pol.

BACKGROUND: Decellularized human pulmonary heart valve (dhHV) scaffolds have been shown to be the gold standard especially for younger, adolescent patients. However, human heart valves are limited in availability. Xenogeneic decellularized pig heart valves (dpHV) may serve as alternative. METHODS: The efficacy of DNA reduction processes upon decellularization of heart valves from German Landrace pigs was analyzed by measurements of remaining nucleic acids including proviral porcine endogenous retrovirus (PERV) sequences. Porcine pulmonary heart valves (pPHV) were decellularized by three different protocols and further treated with DNaseI or Benzonase, at varying incubation times. DNA isolated from valve associated muscle (m), valve cusp (c), and pulmonary artery (pa) was monitored by PCR and qRT-PCR using GAPDH and the PERV polymerase (pol) for read-out. RESULTS: Decellularization of pPHV led to a significant reduction of DNA (>99%) which could be further significantly increased for (m) and (pa) by nuclease treatment, reducing proviral PERV pol from approximately 5 × 107 to 5 × 103  copies/mg in nuclease treated tissues. CONCLUSIONS: Both nucleases demonstrated comparable activities. But DNaseI revealed to be less consistent for PERV, especially at muscular tissue. Noteworthy, remaining proviral sequences are still detectable by PCR; however, due to the absence of the cellular replication machinery the production of infectious particles is not expected. Decellularization and nuclease treatment of pPHV is an efficient procedure to reduce the DNA content including PERV, thus represents a valuable option to increase virus safety independently from the source animal background.

Characterization of porcine endogenous retrovirus particles released by the CRISPR/Cas9 inactivated cell line PK15 clone 15.

The infection of human transplant recipients by porcine endogenous retrovirus (PERV) is a safety issue for xenotransplantation (XTx). CRISPR/Cas9 technology has enabled the generation of pigs free of functional PERVs, and the susceptibility of these animals to reinfection by PERVs remains unclear. To assess virological safety, we characterized a cell line in which PERVs have been inactivated by CRISPR/Cas9 (PK15 clone 15) for its susceptibility to infectious PERV. First, basal expression of PERV pol, the porcine PERV-A receptor (POPAR), and reverse transcriptase (RT) activity of PERV were determined. PK15 clone 15 cells were inoculated with PERV and monitored post infection for virus expression and RT activity. Particles were visualized by electron microscopy. Our data show that PK15 clone 15 cells still produce viral proteins that assemble to produce impaired viral particles. These virions have an irregular morphology that diverges from that of mature wild type. The particles are no longer infectious when tested in a downstream infection assay using supernatants of PK15 clone 15 cells to infect susceptible swine testis-IOWA (ST-IOWA) cells. The expression of POPAR was quantified to exclude the possibility that lack of susceptibility to reinfection, for PERV-A, is caused by absence of viral host receptor(s). PK15 and PK15 clone 15 cells do, in fact, express POPAR equally. PERV RT inactivation mediated by CRISPR/Cas9 does not compromise virus assembly but affects virion structure and proviral integration. The constitutive virion production seems to maintain cellular resistance to superinfection and possibly indicates a protective side effect of this specific CRISPR/Cas9 mediated RT inactivation.

Targeting the proviral host kinase, FAK, limits influenza a virus pathogenesis and NFkB-regulated pro-inflammatory responses.

Influenza A virus (IAV) infections result in ∼500,000 global deaths annually. Host kinases link multiple signaling pathways at various stages of infection and are attractive therapeutic target. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, regulates several cellular processes including NFkB and antiviral responses. We investigated how FAK kinase activity regulates IAV pathogenesis. Using a severe infection model, we infected IAV-susceptible DBA/2 J mice with a lethal dose of H1N1 IAV. We observed reduced viral load and pro-inflammatory cytokines, delayed mortality, and increased survival in FAK inhibitor (Y15) treated mice. In vitro IAV-induced NFkB-promoter activity was reduced by Y15 or a dominant negative kinase-dead FAK mutant (FAK-KD) independently of the viral immune modulator, NS1. Finally, we observed reduced IAV-induced nuclear localization of NFkB in FAK-KD expressing cells. Our data suggest a novel mechanism where IAV hijacks FAK to promote viral replication and limit its ability to contribute to innate immune responses.

Farnesoid X receptor-α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo.

Hepatitis B virus (HBV) infection and bile acid (BA) metabolism are interdependent: infection modifies the expression of the BA nuclear receptor farnesoid X receptor (FXR)-α, and modulation of FXRα activity by ligands alters HBV replication. Mechanisms of HBV control by FXRα remain to be unveiled. FXRα silencing in HBV-infected HepaRG cells decreased the viral covalently closed circular (ccc)DNA pool size and transcriptional activity. Treatment with the FXRα agonist GW4064 inhibited FXRα proviral effect on cccDNA similarly for wild-type and hepatitis B viral X protein (HBx)-deficient virus, whereas agonist-induced inhibition of pregenomic and precore RNA transcription and viral DNA secretion was HBx dependent. These data indicated that FXRα acts as a proviral factor by 2 different mechanisms, which are abolished by FXRα stimulation. Finally, infection of C3H/HeN mice by a recombinant adeno-associated virus-2/8-HBV vector induced a sustained HBV replication in young mice in contrast with the transient decline in adult mice. Four-week GW4064 treatment of infected C3H/HeN mice decreased secretion of HBV DNA and HB surface antigen in adult mice only. These results suggest that the physiologic balance of FXRα expression and activation by bile acid is a key host metabolic pathway in the regulation of HBV infection and that FXRα can be envisioned as a target for HBV treatment.-Mouzannar, K., Fusil, F., Lacombe, B., Ollivier, A., Ménard, C., Lotteau, V., Cosset, F.-L., Ramière, C., André, P. Farnesoid X receptor α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo.

Porcine endogenous retroviruses: Quantification of the copy number in cell lines, pig breeds, and organs.

BACKGROUND: Porcine endogenous retroviruses (PERVs) may pose a risk of xenotransplantation using porcine cells, tissues, or organs. PERVs are integrated in the genome of all pigs, and some can infect certain human cells. The copy number of PERVs in different pig breeds has been determined by using different methods, with varying results. METHODS: To determine the PERV copy number in pig cell lines and in animals, a new method, droplet digital polymerase chain reaction (ddPCR) was used. DNA was isolated from pig cell lines (PK15 and PTK75 cells), from Aachen, Göttingen, and Black minipigs, and from genetically modified and non-modified German landrace pigs. Primers specific for the polymerase gene (pol) were used for the ddPCR. RESULTS: The median copy number of integrated proviruses was found between 46 and 70 copies in three different PK15 cell lines, 49 copies in PTK75 cells, 64 copies in Göttingen minipigs, 69 copies in Aachen minipigs, 117 copies in Black minipigs, and 59 copies in genetically modified pigs generated for xenotransplantation. PERV copy numbers varied between different organs from a single pig, indicating proviral amplification. The study also revealed that different PK15 cell lines from different laboratories which had been used as virus source for infection experiments carry different PERV copies. Furthermore, different copy numbers of cellular reference genes (GAPDH, ACTB) were detected in different cell lines and pigs. CONCLUSION: The determination of the PERV copy number using ddPCR extended previous data showing differences between the pig breeds and between different organs of a single animal. The determination of PERV copy numbers can be used to select animals less likely to transmit PERVs during xenotransplantation. In addition, this method will be of special value when PERV proviruses are to be inactivated by CRISPR/Cas9.

Interferon Lambda Family along with HTLV-1 Proviral Load, Tax, and HBZ Implicated in the Pathogenesis of Myelopathy/Tropical Spastic Paraparesis.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic neuroinflammatory disease related to human T lymphotropic virus type 1 (HTLV-1) infection. Interferon type III (IFN-λ), which includes IL28, IL29, and IL28R, and affects the outcome of viral infections, might be complicated in the progression of HAM/TSP. Here, we investigated the host-virus interactions in the manifestation of HAM/TSP, using IL28B, IL29, IL28R, HTLV-1 Tax, HTLV-1 basic leucine zipper factor (HBZ), and proviral load (PVL). The study groups consisted of 20 patients with HAM/TSP, 20 asymptomatic HTLV-1 carriers (ACs), and 20 healthy controls (HCs). The means of PVL, Tax, and HBZ gene expressions in the HAM/TSP group (p = 0.004, 0.006, and < 0.0001, respectively) were significantly higher than in the AC group. The comparison of IL28B, IL29, and IL28R expression in the HAM/TSP, AC, and HC groups revealed no significant difference between the first 2, but lower concentrations in the HCs (IL28B: p = 0.03, 0.01; IL29: p = 0.07, 0.01; and IL28R: p < 0.0001, respectively). In the HAM/TSP group, correlations were seen between Tax and HBZ (R = 0.61, p = 0.004) and between Tax and IL29 (R = 0.45, p = 0.04). Negative correlations were observed between Tax and IL28B (R = -0.49, p = 0.02) and between HBZ and IL28R (R = -0.43, p = 0.06). In the ACs, an inverse correlation was found between Tax and IL28B (R = -0.42, p = 0.06). These findings suggest that IL29, IL28B, and IL28R interfere in the infection of HAM/TSP, mainly via Tax activation.

Risk factors associated with HTLV-1 vertical transmission in Brazil: longer breastfeeding, higher maternal proviral load and previous HTLV-1-infected offspring.

HTLV-1 is transmitted primarily either through sexual intercourse or from mother to child. The mother/child pairs were classified as seroconcordant or serodiscordant. We analyzed mother to child transmission (MTCT) according to sociodemographic, clinical and epidemiological characteristics of the mother, child's gender and duration of breastfeeding. Between June 2006 and August 2016 we followed 192 mothers with HTLV-1 infection (mean age 41 years old), resulting in 499 exposed offspring, 288 (57.7%) of whom were tested for HTLV-1, making up the final sample for the study, along with their 134 respective mothers. Among the tested mother/child pairs, 41 (14.2%) were HTLV-1 positive, highlighted that seven of 134 family clusters concentrated 48.8% of positive cases. Variables associated with a positive child: breastfeeding duration ≥12 months, maternal PVL ≥100 copies/104 PBMC, mother's age at delivery >26 years old, and HTLV-1 in more than one child of the same mother. In a multiple logistic regression, breastfeeding ≥12 months, higher maternal PVL and ≥2 previous HTLV-1-infected children remained independently associated with the outcome. Thus, high maternal PVL and breastfeeding beyond 12 months were independently associated with MTCT of the HTLV-1 infection. Our results reinforce the need for both prenatal HTLV screening in endemic areas and for advising mothers on breastfeeding.

The impact of HTLV-1 on the cellular genome.

Human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of adult T-cell leukaemia/lymphoma (ATL), an aggressive CD4+ T-cell malignancy. The mechanisms of leukaemogenesis in ATL are incompletely understood. Insertional mutagenesis has not previously been thought to contribute to the pathogenesis of ATL. However, the recent discovery that HTLV-1 binds the key chromatin architectural protein CTCF raises the hypothesis that HTLV-1 deregulates host gene expression by causing abnormal chromatin looping, bringing the strong HTLV-1 promoter-enhancer near to host genes that lie up to 2Mb from the integrated provirus. Here we review current opinion on the mechanisms of oncogenesis in ATL, with particular emphasis on the local and distant impact of HTLV-1 on the structure and expression of the host genome.

Advancements in Developing Strategies for Sterilizing and Functional HIV Cures.

Combined antiretroviral therapy (cART) has been successful in prolonging lifespan and reducing mortality of patients infected with human immunodeficiency virus (HIV). However, the eradication of latent HIV reservoirs remains a challenge for curing HIV infection (HIV cure) because of HIV latency in primary memory CD4+ T cells. Currently, two types of HIV cures are in development: a "sterilizing cure" and a "functional cure." A sterilizing cure refers to the complete elimination of replication-competent proviruses in the body, while a functional cure refers to the long-term control of HIV replication without treatment. Based on these concepts, significant progress has been made in different areas. This review focuses on recent advancements and future prospects for HIV cures.

Deficiency in DNA damage response, a new characteristic of cells infected with latent HIV-1.

Viruses can interact with host cell molecules responsible for the recognition and repair of DNA lesions, resulting in dysfunctional DNA damage response (DDR). Cells with inefficient DDR are more vulnerable to therapeutic approaches that target DDR, thereby raising DNA damage to a threshold that triggers apoptosis. Here, we demonstrate that 2 Jurkat-derived cell lines with incorporated silent HIV-1 provirus show increases in DDR signaling that responds to formation of double strand DNA breaks (DSBs). We found that phosphorylation of histone H2AX on Ser139 (gamma-H2AX), a biomarker of DSBs, and phosphorylation of ATM at Ser1981, Chk2 at Thr68, and p53 at Ser15, part of signaling pathways associated with DSBs, are elevated in these cells. These results indicate a DDR defect even though the virus is latent. DDR-inducing agents, specifically high doses of nucleoside RT inhibitors (NRTIs), caused greater increases in gamma-H2AX levels in latently infected cells. Additionally, latently infected cells are more susceptible to long-term exposure to G-quadruplex stabilizing agents, and this effect is enhanced when the agent is combined with an inhibitor targeting DNA-PK, which is crucial for DSB repair and telomere maintenance. Moreover, exposing these cells to the cancer drug etoposide resulted in formation of DSBs at a higher rate than in un-infected cells. Similar effects of etoposide were also observed in population of primary memory T cells infected with latent HIV-1. Sensitivity to these agents highlights a unique vulnerability of latently infected cells, a new feature that could potentially be used in developing therapies to eliminate HIV-1 reservoirs.

Evaluation of HTLV-1 HBZ and proviral load, together with host IFN λ3, in pathogenesis of HAM/TSP.

Human T-cell lymphotropic virus 1 (HTLV-1) is associated with two progressive diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia/lymphoma (ATLL). Although HTLV-1 proviral load (PVL) has been introduced as a risk factor for these diseases' progression, it is not sufficient on its own to yield an accurate estimation of the outcome of the infection. In the present study, PVL and HTLV-1 basic leucine zipper factor (HBZ) expression level as viral factors, and IFN λ3 as a host factor, were evaluated in HAM/TSP patients and HTLV-1 asymptomatic carriers (ACs). During 2014-2015, 12 HAM/TSP patients and 18 ACs who had been referred to the HTLV-1 Clinic, Ghaem Hospital, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran, were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) were isolated and the DNA and mRNA were extracted for quantification of HBZ, IFN λ3 expression, and PVL using real-time PCR (TaqMan method). Although the PVL was higher in the HAM/TSP group, with a 94% confidence interval, there were no considerable differences in terms of HBZ mRNA and PVL between ACs and HAM patients. IFN λ3 expression in the HAM/TSP group was significantly higher than in the ACs (P = 0.02). To the best of our knowledge, no study has evaluated the expression level of IFN λ3 in HTLV-1 positive patients. The immune response against HTLV-1 viral antigens and virulent factors will therefore further refine our knowledge of interactions between the virus and host in the pathogenesis of HTLV-1-related disorders. The virus PVL and the host IFN λ3 can be used as pathogenic factors of HTLV-1 infected patients at risk of HAM/TSP manifestation. J. Med. Virol. 89:1102-1107, 2017. © 2016 Wiley Periodicals, Inc.

Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome.

The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics.

HTLV-1 subgroups associated with the risk of HAM/TSP are related to viral and host gene expression in peripheral blood mononuclear cells, independent of the transactivation functions of the viral factors.

Among human T cell leukemia virus type 1 (HTLV-1)-infected individuals, the risk of developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) across lifetime differs between ethnic groups. There is an association between HTLV-1 tax gene subgroups (subgroup-A or subgroup-B) and the risk of HAM/TSP in the Japanese population. In this study, we investigated the full-length proviral genome sequences of various HTLV-1-infected cell lines and patient samples. The functional differences in the viral transcriptional regulators Tax and HTLV-1 bZIP factor (HBZ) between each subgroup and the relationships between subgroups and the clinical and laboratory characteristics of HAM/TSP patients were evaluated. The results of these analyses indicated the following: (1) distinct nucleotide substitutions corresponding to each subgroup were associated with nucleotide substitutions in viral structural, regulatory, and accessory genes; (2) the HBZ messenger RNA (mRNA) expression in HTLV-1-infected cells was significantly higher in HAM/TSP patients with subgroup-B than in those with subgroup-A; (3) a positive correlation was observed between the expression of HBZ mRNA and its target Foxp3 mRNA in HAM/TSP patients with subgroup-B, but not in patients with subgroup-A; (4) no clear differences were noted in clinical and laboratory characteristics between HAM/TSP patients with subgroup-A and subgroup-B; and (5) no functional differences were observed in Tax and HBZ between each subgroup based on reporter gene assays. Our results indicate that although different HTLV-1 subgroups are characterized by different patterns of viral and host gene expression in HAM/TSP patients via independent mechanisms of direct transcriptional regulation, these differences do not significantly affect the clinical and laboratory characteristics of HAM/TSP patients.

Cell Association in Rous Sarcoma Virus (RSV) Rescue and Cell Infection.

In my article I tried to present the results of early experiments suggesting a significant role for cell association in Rous sarcoma virus transformation of non-permissive cells and revealing that infectious virus can be efficiently rescued from such cells by their fusion with permissive chicken fibroblasts.

Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting.

A fundamental feature of immune systems is the ability to distinguish pathogenic from self and commensal elements, and to attack the former but tolerate the latter. Prokaryotic CRISPR-Cas immune systems defend against phage infection by using Cas nucleases and small RNA guides that specify one or more target sites for cleavage of the viral genome. Temperate phages include viruses that can integrate into the bacterial chromosome, and they can carry genes that provide a fitness advantage to the lysogenic host. However, CRISPR-Cas targeting that relies strictly on DNA sequence recognition provides indiscriminate immunity both to lytic and lysogenic infection by temperate phages-compromising the genetic stability of these potentially beneficial elements altogether. Here we show that the Staphylococcus epidermidis CRISPR-Cas system can prevent lytic infection but tolerate lysogenization by temperate phages. Conditional tolerance is achieved through transcription-dependent DNA targeting, and ensures that targeting is resumed upon induction of the prophage lytic cycle. Our results provide evidence for the functional divergence of CRISPR-Cas systems and highlight the importance of targeting mechanism diversity. In addition, they extend the concept of 'tolerance to non-self' to the prokaryotic branch of adaptive immunity.

Protein kinase D3 is essential for prostratin-activated transcription of integrated HIV-1 provirus promoter via NF-κB signaling pathway.

Prostratin has been proposed as a promising reagent for eradicating the latent HIV-1 provirus by inducing HIV-1 transcription activation. The molecular mechanism of this activation, however, is far from clear. Here, we show that the protein kinase D3 (PKD3) is essential for prostratin-induced transcription activation of latent HIV-1 provirus. First, silencing PKD3, but not the other members of PKD family, blocked prostratin-induced transcription of HIV-1. Second, overexpressing the constitutively active form of PKD3, but not the wild-type or kinase-dead form of PKD3, augmented the expression of HIV-1. Consistent with this observation, we found that prostratin could trigger PKD3 activation by inducing the phosphorylation of its activation loop. In addition, we identified PKCε of the novel PKC subfamily as the upstream kinase for this phosphorylation. Finally, the activation effect of PKD3 on HIV-1 transcription was shown to depend on the presence of κB element and the prostratin-induced activation of NF-κB, as indicated by the fact that silencing PKD3 blocked prostratin-induced NF-κB activation and NF-κB-dependent HIV-1 transcription. Therefore, for the first time, PKD3 is implicated in the transcription activation of latent HIV-1 provirus, and our results revealed a molecular mechanism of prostratin-induced HIV-1 transcription via PKCε/PKD3/NF-κB signaling pathway.

Lifespan of effector memory CD4+ T cells determined by replication-incompetent integrated HIV-1 provirus.

OBJECTIVE: Determining the precise lifespan of human T-cell is challenging due to the inability of standard techniques to distinguish between dividing and dying cells. Here, we measured the lifespan of a pool of T cells that were derived from a single cell 'naturally' labelled with a single integrated clone of a replication-incompetent HIV-1 provirus. DESIGN/METHODS: Utilizing a combination of techniques, we were able to sequence/map an integration site of a unique provirus with a stop codon at position 42 of the HIV-1 protease. In-vitro reconstruction of this provirus into an infectious clone confirmed its inability to replicate. By combining cell separation and integration site-specific PCR, we were able to follow the fate of this single provirus in multiple T-cell subsets over a 20-year period. As controls, a number of additional integrated proviruses were also sequenced. RESULTS: The replication-incompetent HIV-1 provirus was solely contained in the pool of effector memory CD4 T cells for 17 years. The percentage of the total effector memory CD4 T cells containing the replication-incompetent provirus peaked at 1% with a functional half-life of 11.1 months. In the process of sequencing multiple proviruses, we also observed high levels of lethal mutations in the peripheral blood pool of proviruses. CONCLUSION: These data indicate that human effector memory CD4 T cells are able to persist in vivo for more than 17 years without detectably reverting to a central memory phenotype. A secondary observation is that the fraction of the pool of integrated HIV-1 proviruses capable of replicating may be considerably less than the 12% currently noted in the literature.

HIV integrase and the swan song of the CD4 T cells?

T cell apoptosis represents one pathophysiological mechanism associated with AIDS. Herein, we discuss the recent report published by A. Cooper et al. in Nature (June 2013) regarding HIV viral DNA integration-mediated apoptosis.