Total Viral DNA Research Articles

Non-Detection of HCMV Total Genomic DNA in Human Glioma Cells Genome.

To demonstrate if the human cytomegalovirus (HCMV) genome, that is involved in the pathogenesis of gliomas, is part of the genomic DNA of glioma cells or not. The study included U87MG glioblastoma cell culture and tumor samples from glioma patients. The genomic DNA of tumor samples and U87MG cells were extracted and real-time quantitative PCR was used to assess the presence of the human cytomegalovirus genomic DNA. Consequently, HCMV positivity was not detected in the tumor and cell line genomic DNA under the aforementioned experimental conditions. We found that the genomic DNA of all the samples was negative for HCMV genomic DNA. Thus, HCMV could not be detected in human glioma tumors and we put forward that HCMV genomic DNA was not incorporated into the genomic DNA of glioma cells. Thus, total viral DNA is not involved in the pathogenesis of glioma; however, small viral particles or specific genes might be incorporated into the genomic DNA of glioma cells, leading to cancer development. This prompts further studies for verification.

DNA ultra-sensitive quantification, a technology for studying HIV unintegrated linear DNA

Unintegrated HIV DNA represents between 20% and 35% of the total viral DNA in infected patients. Only the linear forms (unintegrated linear DNAs [ULDs]) can be substrates for integration and for the completion of a full viral cycle. In quiescent cells, these ULDs may be responsible for pre-integrative latency. However, their detection remains difficult due to the lack of specificity and sensitivity of existing techniques. We developed an ultra-sensitive, specific, and high-throughput technology for ULD quantification called DUSQ (DNA ultra-sensitive quantification) combining linker-mediated PCR and next-generation sequencing (NGS) using molecular barcodes. Studying cells with different activity levels, we determined that the ULD half-life goes up to 11days in resting CD4+ Tcells. Finally, we were able to quantify ULDs in samples from patients infected with HIV-1, providing a proof of concept for the use of DUSQ invivo to track pre-integrative latency. DUSQ can be adapted to the detection of other rare DNA molecules.

CD8+ lymphocytes do not impact SIV reservoir establishment under ART

Persistence of the human immunodeficiency virus type-1 (HIV-1) latent reservoir in infected individuals remains a problem despite fully suppressive antiretroviral therapy (ART). While reservoir formation begins during acute infection, the mechanisms responsible for its establishment remain unclear. CD8+ T cells are important during the initial control of viral replication. Here we examined the effect of CD8+ T cells on formation of the latent reservoir in simian immunodeficiency virus (SIV)-infected macaques by performing experimental CD8+ depletion either before infection or before early (that is, day 14 post-infection) ART initiation. We found that CD8+ depletion resulted in slower decline of viremia, indicating that CD8+ lymphocytes reduce the average lifespan of productively infected cells during acute infection and early ART, presumably through SIV-specific cytotoxic T lymphocyte (CTL) activity. However, CD8+ depletion did not change the frequency of infected CD4+ T cells in the blood or lymph node as measured by the total cell-associated viral DNA or intact provirus DNA assay. In addition, the size of the persistent reservoir remained the same when measuring the kinetics of virus rebound after ART interruption. These data indicate that during early SIV infection, the viral reservoir that persists under ART is established largely independent of CTL control.

Raltegravir Inclusion Decreases CD4 T-Cells Intra-Cellular Viral Load and Increases CD4 and CD28 Positive T-Cells in Selected HIV Patients.

Raltegravir (RLT) prevents the integration of HIV DNA in the nucleus, but published studies remain controversial, suggesting that it does not decrease proviral DNA. However, there are only a few studies focused on virus-targeted cells. We aimed our study on the impact of RLT inclusion on total intra-cellular viral DNA (TID) in cellular subsets and immune effects in patients with newly acquired undetectable plasmatic viral load (UVL). Six patients having UVL using an antiretroviral combination for 6 months and CD4 T-cells > 350/mL and <500/mL were selected to receive RLT for 3 months from M0 to M3. Patients had 7 sequential viro-immunological determinations from M-1 to M5. Immune phenotypes were determined by flow cytometry and TID quantification was performed using PCR assay on purified cells. TID (median values) at the initiation of RLT in CD4 T-cells was 117 copies/millions of cells, decreased to 27.5 on M3, and remained thereafter permanently under the cut-off (<10 copies/millions of cells) in 4 out of 6 patients. This was associated with an increase of CD4 and CD4 + CD28+ T-cells and a decrease of HLA-DR expression and apoptosis of CD4 T-cells. RLT inclusion led to decreases in the viral load along with positive immune reconstitution, mainly for CD4 T-cells in HIV patients.

Herpes Infections in Suspected Cases of Yellow Fever in the Democratic Republic of the Congo.

In the battle to quickly identify potential yellow fever arbovirus outbreaks in the Democratic Republic of the Congo, active syndromic surveillance of acute febrile jaundice patients across the country is a powerful tool. However, patients who test negative for yellow fever virus infection are too often left without a diagnosis. By retroactively screening samples for other potential viral infections, we can both try to find sources of patient disease and gain information on how commonly they may occur and co-occur. Several human arboviruses have previously been identified, but there remain many other viral families that could be responsible for acute febrile jaundice. Here, we assessed the prevalence of human herpes viruses (HHVs) in these acute febrile jaundice disease samples. Total viral DNA was extracted from serum of 451 patients with acute febrile jaundice. We used real-time quantitative PCR to test all specimens for cytomegalovirus (CMV), herpes simplex virus (HSV), human herpes virus type 6 (HHV-6) and varicella-zoster virus (VZV). We found 21.3% had active HHV replication (13.1%, 2.4%, 6.2% and 2.4% were positive for CMV, HSV, HHV-6 and VZV, respectively), and that nearly half (45.8%) of these infections were characterized by co-infection either among HHVs or between HHVs and other viral infection, sometimes associated with acute febrile jaundice previously identified. Our results show that the role of HHV primary infection or reactivation in contributing to acute febrile jaundice disease identified through the yellow fever surveillance program should be routinely considered in diagnosing these patients.

Modified Adenovirus Prime-Protein Boost Clade C HIV Vaccine Strategy Results in Reduced Viral DNA in Blood and Tissues Following Tier 2 SHIV Challenge.

Designing immunogens and improving delivery methods eliciting protective immunity is a paramount goal of HIV vaccine development. A comparative vaccine challenge study was performed in rhesus macaques using clade C HIV Envelope (Env) and SIV Gag antigens. One group was vaccinated using co-immunization with DNA Gag and Env expression plasmids cloned from a single timepoint and trimeric Env gp140 glycoprotein from one of these clones (DNA+Protein). The other group was a prime-boost regimen composed of two replicating simian (SAd7) adenovirus-vectored vaccines expressing Gag and one Env clone from the same timepoint as the DNA+Protein group paired with the same Env gp140 trimer (SAd7+Protein). The env genes were isolated from a single pre-peak neutralization timepoint approximately 1 year post infection in CAP257, an individual with a high degree of neutralization breadth. Both DNA+Protein and SAd7+Protein vaccine strategies elicited significant Env-specific T cell responses, lesser Gag-specific responses, and moderate frequencies of Env-specific TFH cells. Both vaccine modalities readily elicited systemic and mucosal Env-specific IgG but not IgA. There was a higher frequency and magnitude of ADCC activity in the SAd7+Protein than the DNA+Protein arm. All macaques developed moderate Tier 1 heterologous neutralizing antibodies, while neutralization of Tier 1B or Tier 2 viruses was sporadic and found primarily in macaques in the SAd7+Protein group. Neither vaccine approach provided significant protection from viral acquisition against repeated titered mucosal challenges with a heterologous Tier 2 clade C SHIV. However, lymphoid and gut tissues collected at necropsy showed that animals in both vaccine groups each had significantly lower copies of viral DNA in individual tissues compared to levels in controls. In the SAd7+Protein-vaccinated macaques, total and peak PBMC viral DNA were significantly lower compared with controls. Taken together, this heterologous Tier 2 SHIV challenge study shows that combination vaccination with SAd7+Protein was superior to combination DNA+Protein in reducing viral seeding in tissues in the absence of protection from infection, thus emphasizing the priming role of replication-competent SAd7 vector. Despite the absence of correlates of protection, because antibody responses were significantly higher in this vaccine group, we hypothesize that vaccine-elicited antibodies contribute to limiting tissue viral seeding.

Concurrent administration of IFN\u03b114 and cART in TKO-BLT mice enhances suppression of HIV-1 viremia but does not eliminate the latent reservoir

Combination antiretroviral therapy (cART) prevents HIV-1 replication but does not eliminate the latent reservoir and cure the infection. Type I interferons (IFN) mediate antiviral effects through different mechanisms than cART. We previously showed that IFNα14 is the most potent IFNα subtype against HIV-1 and that it can significantly reduce the HIV-1 proviral reservoir. This study sought to determine whether combining cART with IFNα14 therapy would produce greater reductions in HIV-1 viral and proviral loads than ART alone. Immunodeficient Rag2−/−γc−/−CD47−/− C57BL/6 mice were humanized by the BLT method, infected with HIV-1JR-CSF and the in vivo efficacy of cART was compared with combined cART/IFNα14 therapy. Infection was allowed to establish for 6 weeks prior to 4 weeks of treatment with oral cART either with or without IFNα14. Plasma viral RNA and splenic CD4+ T cell viral DNA levels were measured immediately after treatment and after 2 weeks of therapy interruption. Augmentation of cART with IFNα14 resulted in significantly enhanced suppression of HIV-1 plasma viremia. However, no significant reduction in total viral DNA was detectable. Furthermore, virus rebounded after treatment interruption to similar levels in both groups. Thus, augmentation of cART with IFNα14 resulted in a more pronounced reduction of HIV viremia levels over cART alone, but the effect was not potent enough to be detected at the viral DNA level or to prevent virus rebound following therapy interruption in immune system-humanized mice.

Potential impact of the antirheumatic agent auranofin on proviral HIV-1 DNA in individuals under intensified antiretroviral therapy: Results from a randomised clinical trial

Antiretroviral therapy (ART) is typically composed of a combination of three antiretroviral drugs and is the treatment of choice for people with human immunodeficiency virus type 1/acquired immune deficiency syndrome (HIV-1/AIDS). However, it is unable to impact on viral reservoirs, which harbour latent HIV-1 genomes that are able to reignite the infection upon treatment suspension. The aim of this study was to provide an estimate of the safety of the disease-modifying antirheumatic agent auranofin and its impact on the HIV-1 reservoir in humans under intensified ART. For this purpose, an interim analysis was conducted of three of the six arms of the NCT02961829 clinical trial (five patients each) with: no intervention, i.e. continuation of first-line ART; intensified ART (ART + dolutegravir and maraviroc); and intensified ART plus auranofin. Auranofin treatment was found to be well tolerated. No major adverse events were detected apart from a transient decrease in CD4+ T-cell counts at Weeks 8 and 12. Auranofin decreased total viral DNA in peripheral blood mononuclear cells compared with ART-only regimens at Week 20 (P = 0.036) and induced a decrease in integrated viral DNA as quantified by Alu PCR. Despite the limited number of patient-derived sequences available in this study, phylogenetic analyses of nef sequences support the idea that auranofin may impact on the viral reservoir. [ClinicalTrials.gov ID: NCT02961829].

Nucleic acid polymer REP 2139 and nucleos(T)ide analogues act synergistically against chronic hepadnaviral infection in vivo in Pekin ducks.

Nucleic acid polymer (NAP) REP 2139 treatment was shown to block the release of viral surface antigen in duck HBV (DHBV)‐infected ducks and in patients with chronic HBV or HBV/hepatitis D virus infection. In this preclinical study, a combination therapy consisting of REP 2139 with tenofovir disoproxil fumarate (TDF) and entecavir (ETV) was evaluated in vivo in the chronic DHBV infection model. DHBV‐infected duck groups were treated as follows: normal saline (control); REP 2139 TDF; REP 2139 + TDF; and REP 2139 + TDF + ETV. After 4 weeks of treatment, all animals were followed for 8 weeks. Serum DHBsAg and anti‐DHBsAg antibodies were monitored by enzyme‐linked immunosorbent assay and viremia by qPCR. Total viral DNA and covalently closed circular DNA (cccDNA) were quantified in autopsy liver samples by qPCR. Intrahepatic DHBsAg was assessed at the end of follow‐up by immunohistochemistry. On‐treatment reduction of serum DHBsAg and viremia was more rapid when REP 2139 was combined with TDF or TDF and ETV, and, in contrast to TDF monotherapy, no viral rebound was observed after treatment cessation. Importantly, combination therapy resulted in a significant decrease in intrahepatic viral DNA (>3 log) and cccDNA (>2 log), which were tightly correlated with the clearance of DHBsAg in the liver. Conclusion: Synergistic antiviral effects were observed when REP 2139 was combined with TDF or TDF + ETV leading to control of infection in blood and liver, associated with intrahepatic viral surface antigen elimination that persisted after treatment withdrawal. Our findings suggest the potential of developing such combination therapy for treatment of chronically infected patients in the absence of pegylated interferon. (Hepatology 2018;67:2127‐2140).

Modeling brain lentiviral infections during antiretroviral therapy in AIDS.

Understanding HIV-1 replication and latency in different reservoirs is an ongoing challenge in the care of patients with HIV/AIDS. A mathematical model was created to describe and predict the viral dynamics of HIV-1 and SIV infection within the brain during effective combination antiretroviral therapy (cART). The mathematical model was formulated based on the biology of lentiviral infection of brain macrophages and used to describe the dynamics of transmission and progression of lentiviral infection in brain. Based on previous reports quantifying total viral DNA levels in brain from HIV-1 and SIV infections, estimates of integrated proviral DNA burden were made, which were used to calibrate the mathematical model predicting viral accrual in brain macrophages from primary infection. The annual rate at which susceptible brain macrophages become HIV-1 infected was estimated to be 2.90×10-7-4.87×10-6 per year for cART-treated HIV/AIDS patients without comorbid neurological disorders. The transmission rate for SIV infection among untreated macaques was estimated to be 5.30×10-6-1.37×10-5 per year. An improvement in cART effectiveness (1.6-48%) would suppress HIV-1 infection in patients without neurological disorders. Among patients with advanced disease, a substantial improvement in cART effectiveness (70%) would eradicate HIV-1 provirus from the brain within 3-32 (interquartile range 3-9) years in patients without neurological disorders, whereas 4-51 (interquartile range 4-16) years of efficacious cART would be required for HIV/AIDS patients with comorbid neurological disorders. HIV-1 and SIV provirus burdens in the brain increase over time. A moderately efficacious antiretroviral therapy regimen could eradicate HIV-1 infection in the brain that was dependent on brain macrophage lifespan and the presence of neurological comorbidity.

Histones Are Rapidly Loaded onto Unintegrated Retroviral DNAs Soon after Nuclear Entry.

Chromosomal structure of nuclear DNA is usually maintained by insertion of nucleosomes into preexisting chromatin, both on newly synthesized DNA atreplication forks and at sites of DNA damage. But during retrovirus infection, a histone-free DNA copy of the viral genome is synthesized that must be loaded with nucleosomes de novo. Here, we show that core histones are rapidly loaded onto unintegrated Moloney murine leukemia virus DNAs. Loading of nucleosomes requires nuclear entry, but does not require viral DNA integration. The histones associated with unintegrated DNAs become marked by covalent modifications, with a delay relative to thetime of core histone loading. Expression from unintegrated DNA can be enhanced by modulation of the histone-modifying machinery. The data show that histone loading onto unintegrated DNAs occurs very rapidly after nuclear entry and does not require prior establishment of an integrated provirus.

Human stem cell-derived hepatocytes as a model for hepatitis B virus infection, spreading and virus-host interactions

One major obstacle of hepatitis B virus (HBV) research is the lack of efficient cell culture system permissive for viral infection and replication. The aim of our study was to establish a robust HBV infection model by using hepatocyte-like cells (HLCs) derived from human pluripotent stem cells. HLCs were differentiated from human embryonic stem cells and induced pluripotent stem cells. Maturation of hepatocyte functions was determined. After HBV infection, total viral DNA, cccDNA, total viral RNA, pgRNA, HBeAg and HBsAg were measured. More than 90% of the HLCs expressed strong signals of human hepatocyte markers, like albumin, as well as known host factors required for HBV infection, suggesting that these cells possessed key features of mature hepatocytes. Notably, HLCs expressed the viral receptor sodium-taurocholate cotransporting polypeptide more stably than primary human hepatocytes (PHHs). HLCs supported robust infection and some spreading of HBV. Finally, by using this model, we identified two host-targeting agents, genistin and PA452, as novel antivirals. Stem cell-derived HLCs fully support HBV infection. This novel HLC HBV infection model offers a unique opportunity to advance our understanding of the molecular details of the HBV life cycle; to further characterize virus-host interactions and to define new targets for HBV curative treatment. Our study used human pluripotent stem cells to develop hepatocyte-like cells (HLCs) capable of expressing hepatocyte markers and host factors important for HBV infection. These cells fully support HBV infection and virus-host interactions, allowing for the identification of two novel antiviral agents. Thus, stem cell-derived HLCs provide a highly physiologically relevant system to advance our understanding of viral life cycle and provide a new tool for antiviral drug screening and development.

Lack of concordance between residual viremia and viral variants driving de novo infection of CD4(+) T cells on ART.

BackgroundIn most patients, current antiretroviral therapy (ART) regimens can rapidly reduce plasma viral load. However, even after years of effective treatment, a significant proportion of patients show residual plasma viremia below the clinical detection limit. Although residual viremia might be associated with increased chronic immune activation and morbidity, its origin and its potential role in the replenishment of the viral reservoir during suppressive ART is not completely understood. We performed an in-depth genetic analysis of the total and episomal cell-associated viral DNA (vDNA) repertoire in purified CD4+ T cell subsets of three HIV-infected individuals, and used phylogenetic analysis to explore its relationship with plasma viruses.ResultsThe predominant proviral reservoir was established in naïve or memory (central and transitional) CD4+ T cell subsets in patients harboring X4- or R5-tropic viruses, respectively. Regardless of the viral tropism, most plasma viruses detected under suppressive ART resembled the proviral reservoir identified in effector and transitional memory CD4+ T-cell subsets in blood, suggesting that residual viremia originates from these cells in either blood or lymphoid tissue. Most importantly, sequences in episomal vDNA in CD4+ T-cells were not well represented in residual viremia.ConclusionsViral tropism determines the differential distribution of viral reservoir among CD4+ T-cell subsets. In spite of viral tropism, the effector and transitional memory CD4+ T-cells subsets are the main source of residual viremia during suppressive ART, even though their contribution to the total proviral pool is small. However, the lack of concordance between residual viremia and viral variants driving de novo infection of CD4+ T cells on ART may reflect the predominance of defective plasma HIV RNA genomes. These findings highlight the need for monitoring the multiple viral RNA/DNA persistence markers, based on their differential contribution to viral persistence.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0282-9) contains supplementary material, which is available to authorized users.

Increased fecal viral content associated with obesity in mice.

To investigate the presence of total gut viral content in obese mice, and establish correlation with obesity associated metabolic measures and gut microbiome. Fresh fecal samples were collected from normal and obese (Leptin deficient: Lep(ob/ob)) mice. Total viral DNA and RNA was isolated and quantified for establishing the correlation with metabolic measures and composition of gut bacterial communities. In this report, we found that obese mice feces have higher viral contents in terms of total viral DNA and RNA (P < 0.001). Interestingly, these increased viral DNA and RNA content were tightly correlated with metabolic measures, i.e., body weight, fat mass and fasting blood glucose. Total viral content were positively correlated with firmicutes (R(2) > 0.6), whilst negatively correlated with bacteroidetes and bifidobacteria. This study suggests the strong correlation of increased viral population into the gut of obese mice and opens new avenues to explore the role of gut virome in pathophysiology of obesity.

Interleukin-37 Expression Is Increased in Chronic HIV-1-Infected Individuals and Is Associated with Inflammation and the Size of the Total Viral Reservoir.

Interleukin-37 (IL-37) is a recently identified cytokine with potent antiinflammatory and immunosuppressive functions. The objective of this study was to compare levels of IL-37 mRNA in immunological subgroups of chronic human immunodeficiency virus-1 (HIV-1)-infected individuals and noninfected controls, to determine IL-37's association with biomarkers of inflammation and reservoir size. This was a cross-sectional study. The HIV-1-infected patients were categorized in three subgroups depending on their combination antiretroviral therapy (cART) treatment status and CD4(+) T-cell count. Quantitative RT-PCR was used for the detection of IL-37 mRNA and HIV-1 DNA in peripheral blood mononuclear cells (PBMCs). Biomarkers in plasma were quantified by enzyme-linked immunosorbent assay (ELISA), whereas T-cell activation was determined by flow cytometry. Lastly, lipopolysaccharide (LPS) stimulations of patients PBMCs were carried out to determine differences in IL-37 mRNA response between the subgroups. Sixty HIV-1-infected patients and 20 noninfected controls were included in the study. Steady-state IL-37 mRNA levels in PBMCs were significantly higher in HIV-1-infected individuals compared with noninfected controls: 2.4-fold (p ≤ 0.01) cART-naïve subjects; 3.9-fold (p ≤ 0.0001) inadequate immunological responders; and 4.0-fold (p ≤ 0.0001) in immunological responders compared with non-infected controls. Additionally, levels of the monocyte inflammatory marker sCD14 correlated with IL-37 mRNA (p = 0.03), whereas there was no association with T-cell activation. Finally, we observed a significant correlation between total viral HIV-1 DNA and IL-37 mRNA in PBMCs (p < 0.0001). Collectively, our data shows that the level of IL-37 mRNA is affected by chronic HIV-1-infection. A relationship with the activation of the monocyte compartment is suggested by the correlation with sCD14 and, interestingly, IL-37 could be related to the size of the total viral HIV-1 reservoir.

Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease

Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in infected hepatocytes. Using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs (sgRNAs) specific for HBV, we observed effective inhibition of HBV DNA production in in vitro models of both chronic and de novo HBV infection. Cas9/sgRNA combinations specific for HBV reduced total viral DNA levels by up to ~1000-fold and HBV cccDNA levels by up to ~10-fold and also mutationally inactivated the majority of the residual viral DNA. Together, these data provide proof of principle for the hypothesis that CRISPR/Cas systems have the potential to serve as effective tools for the depletion of the cccDNA pool in chronically HBV infected individuals.

Growing a giant: Evaluation of the virological parameters for mimivirus production

Acanthamoeba polyphaga mimivirus (APMV) was described in 2003, and due to its unique structural and genetic complexity, the viral family Mimiviridae was created. APMV prompted the creation of an open field of study on the function of hundreds of never-before-seen open reading frames (ORFs) and their roles in virus–host interactions. In recent years, several giant viruses have been isolated from different environments and specimens. Although the scientific community has experienced a remarkable advancement in the comprehension of the mimivirus replication cycle in the last years, few studies have been devoted to the investigation of the methodological features and conditions for mimivirus cultivation. In this work, conditions for the cultivation of mimivirus isolates were investigated to obtain relevant information about the production of infectious particles, total viral particles and viral DNA. The results suggest that low viral doses are more efficient for the production of infectious particles, yielding up to 5000 TCID50 for each inoculated TCID50. Besides methodological information, these data also reveal, for the first time, the ratio between total and infectious particles (in TCID50) that are produced during mimivirus cultivation in laboratory conditions. All of this information can be used as a worldwide guide for the production of mimiviruses and can help prompt mimivirological studies in different fields.

Antiviral activity of methyl helicterate isolated from Helicteres angustifolia (Sterculiaceae) against hepatitis B virus

The anti-HBV effect of methyl helicterate (MH), a triterpenoid isolated from the Chinese herb Helicteres angustifolia, was explored both in vitro and in vivo. In the HBV-transfected cell line HepG2.2.15, the secretion of HBsAg/HBeAg, the levels of HBV DNA and cccDNA, and the amount of viral RNA were significantly decreased after treatment with MH for 144h. In addition, MH had no inhibitory effect on the mitochondrial DNA content. In DHBV-infected ducklings, MH significantly reduced the serum DHBV DNA, liver total viral DNA, and cccDNA levels. Furthermore, analysis of the liver pathological changes confirmed the hepatoprotective effect of MH. These results indicate that MH efficiently inhibits HBV replication both in vitro and in vivo and that MH may be a major bioactive ingredient in H. angustifolia.

Effects of Raltegravir on 2-Long Terminal Repeat Circle Junctions in HIV Type 1 Viremic and Aviremic Patients

Although 2-long terminal repeat (2-LTR) circles are only a fraction of the total viral DNA in infected cells, sequence analysis of 2-LTR circles reveals critical information regarding viral DNA synthesis and the nature of actively replicating virus. It was observed that a large proportion of the 2-LTR circular molecules in the peripheral blood mononuclear cell (PBMC) DNA of infected individuals are mutated at the circle junction. The integrase inhibitor raltegravir (RAL) blocks the strand transfer step of the integration of HIV-1; as a consequence of abortive integration a significant increase of episomal 2-LTR circles is observed. Moreover, it was demonstrated that in patients treated with highly active retroviral therapy (HAART) changes in 2-LTR concentration did not affect junction sequences and flanking regions of 2-LTR. Here we evaluated whether RAL therapy could have a differential impact on the 2-LTR circle junctional sequences in patients with different virological profiles at the time of starting RAL therapy. Sequence analysis indicates that RAL acts differently in the two populations.

Biochemical interactions between HIV-1 integrase and reverse transcriptase

Human immunodeficiency virus (HIV) infection yields a high level of non-integrated viral DNA in the infected cells and up to 99% of total viral DNA can be capable of transcription. This capability of non-integrated viral DNA is reducing the efficacy of anti-HIV drug development approaches that solely focus on the integration reactions of viral replication. Using kinetic modeling, we show the transient coordinated regulation of viral DNA production by retrovirus-encoded interacting enzymes reverse transcriptase (RT) and integrase (IN), and thus we identify a possible mechanism for reducing overall efficiency of viral replication. The results indicate that both IN·RT and IN·DNA complexes and their formation rates affect RT processivity and thereby the viral DNA expression level within the pre-integration complex.