Human Immunodeficiency Virus Type 1 DNA Research Articles

High Natural Permissivity of Primary Rabbit Cells for HIV-1, with a Virion Infectivity Defect in Macrophages as the Final Replication Barrier

An immunocompetent, permissive, small-animal model would be valuable for the study of human immunodeficiency virus type 1 (HIV-1) pathogenesis and for the testing of drug and vaccine candidates. However, the development of such a model has been hampered by the inability of primary rodent cells to efficiently support several steps of the HIV-1 replication cycle. Although transgenesis of the HIV receptor complex and human cyclin T1 have been beneficial, additional late-phase blocks prevent robust replication of HIV-1 in rodents and limit the range of in vivo applications. In this study, we explored the HIV-1 susceptibility of rabbit primary T cells and macrophages. Envelope-specific and coreceptor-dependent entry of HIV-1 was achieved by expressing human CD4 and CCR5. A block of HIV-1 DNA synthesis, likely mediated by TRIM5, was overcome by limited changes to the HIV-1 gag gene. Unlike with mice and rats, primary cells from rabbits supported the functions of the regulatory viral proteins Tat and Rev, Gag processing, and the release of HIV-1 particles at levels comparable to those in human cells. While HIV-1 produced by rabbit T cells was highly infectious, a macrophage-specific infectivity defect became manifest by a complex pattern of mutations in the viral genome, only part of which were deamination dependent. These results demonstrate a considerable natural HIV-1 permissivity of the rabbit species and suggest that receptor complex transgenesis combined with modifications in gag and possibly vif of HIV-1 to evade species-specific restriction factors might render lagomorphs fully permissive to infection by this pathogenic human lentivirus.

Novel application of Locked Nucleic Acid chemistry for a Taqman assay for measuring diverse human immunodeficiency virus type 1 subtypes

There remains a need for sensitive and cost-effective assays to monitor therapy in human immunodeficiency virus type-1 (HIV-1) infection. However, the genetic diversity of HIV poses difficulties for traditional real-time PCR assays that require long oligonucleotides probes. LNA™ probes may be useful in overcoming these limits to traditional probe design. A new application of LNA™ chemistry in a Taqman assay applicable to a wide range of HIV-1 subtypes is described. This assay, based on a 13-mer LNA™ probe that matches the majority of HIV-1 sequences in the Los Alamos database, exhibited a wide dynamic range (101–107 copies of HIV DNA), high sensitivity (limit of detection of 1 copy of HIV DNA in 105 cells), and broad applicability to a range of HIV-1 subtypes (including A, B, C, D, F, H, B/C, and A/E CRFs). Using the LNA™ probe assay, HIV-1 DNA was detected in all dried blood spots (DBS) from treatment naïve HIV-1 positive Ugandan children, and HIV DNA levels significantly correlated with viral RNA levels in plasma (r=0.765, p<0.0001). This approach to Taqman probe design should be explored further for use in diagnosis and monitoring of HIV in resource-limited settings, especially where several subtypes co-circulate.

Remarkable Lethal G-to-A Mutations in vif- Proficient HIV-1 Provirus by Individual APOBEC3 Proteins in Humanized Mice

Genomic hypermutation of RNA viruses, including human immunodeficiency virus type 1 (HIV-1), can be provoked by intrinsic and extrinsic pressures, which lead to the inhibition of viral replication and/or the progression of viral diversity. Human APOBEC3G was identified as an HIV-1 restriction factor, which edits nascent HIV-1 DNA by inducing G-to-A hypermutations and debilitates the infectivity of vif-deficient HIV-1. On the other hand, HIV-1 Vif protein has the robust potential to degrade APOBEC3G protein. Although subsequent investigations have revealed that lines of APOBEC3 family proteins have the capacity to mutate HIV-1 DNA, it remains unclear whether these endogenous APOBEC3s, including APOBEC3G, contribute to mutations of vif-proficient HIV-1 provirus in vivo and, if so, what is the significance of these mutations. In this study, we use a human hematopoietic stem cell-transplanted humanized mouse (NOG-hCD34 mouse) model and demonstrate the predominant accumulation of G-to-A mutations in vif-proficient HIV-1 provirus displaying characteristics of APOBEC3-mediated mutagenesis. Notably, the APOBEC3-associated G-to-A mutation of HIV-1 DNA that leads to the termination of translation was significantly observed. We further provide a novel insight suggesting that HIV-1 G-to-A hypermutation is independently induced by individual APOBEC3 proteins. In contrast to the prominent mutation in intracellular proviral DNA, viral RNA in plasma possessed fewer G-to-A mutations. Taken together, these results provide the evidence indicating that endogenous APOBEC3s are associated with G-to-A mutation of HIV-1 provirus in vivo, which can result in the abrogation of HIV-1 infection.

HIV-1 DNA proviral load in treated and untreated HIV-1 sero-positive patients

As proviral human immunodeficiency virus type 1 (HIV-1) DNA can replenish and revive viral infection upon activation, its detection might offer significant therapeutic information, complementing the input provided by plasma RNA determination in the follow-up of infected individuals. A selected group of acutely infected subjects was studied to verify both total and 2-long terminal repeat (2-LTR) DNA proviral load during the acute phase of infection and thereafter. Patients were divided in two sex- and age-matched groups: 19 naive individuals who did not receive antiretroviral therapy during the observation period and 20 subjects treated according to current guidelines. Total and 2-LTR HIV-1 DNA proviral load, in addition to RNA viral load and CD4 cell count, were determined in peripheral blood mononuclear cells (PBMC) at baseline, 6 and 12 months after the first sampling. Total and 2-LTR HIV-1 DNA proviral load exhibited no significant variation at any time in the naive patients (total HIV-1 DNA ranging from 896 ± 731 to 715 ± 673 copies/105 PBMC and 2-LTR HIV-1 DNA ranging from 94 ± 105 to 65 ± 44 copies/105 PBMC), whereas a significant reduction in both total HIV-1 DNA (ranging from 997 ± 676 to 262 ± 174 copies/105 PBMC) and 2-LTR HIV-1 DNA proviral load (ranging from 116 ± 55 to 26 ± 35 copies/105 PBMC) was detected in highly active antiretroviral therapy (HAART) patients, together with a CD4+ T cell count increase and RNA load decrease. HAART negatively affects both the labile HIV burden and the integrated proviral DNA, at least in the initial period of successful treatment, suggesting that quantification of HIV-1 DNA proviral load may be an important parameter in monitoring HIV infection.

Nucleoprotein Intermediates in HIV-1 DNA Integration Visualized by Atomic Force Microscopy

Integration of HIV-1 (human immunodeficiency virus type 1) DNA into the genome of the host cell is an essential step in the viral replication cycle that is mediated by the virally encoded integrase protein. We have used atomic force microscopy to study stable complexes formed between HIV-1 integrase and viral DNA and their interaction with host DNA. A tetramer of integrase stably bridges a pair of viral DNA ends, consistent with previous analysis by gel electrophoresis. The intasome, composed of a tetramer of integrase bridging a pair of viral DNA ends, is highly stable to high ionic strength that would strip more loosely associated integrase from internal regions of the viral DNA. We also observed tetramers of integrase associated with single viral DNA ends; time-course experiments suggest that these may be intermediates in intasome assembly. Strikingly, integrase tetramers are only observed in tight association with viral DNA ends. The self-association properties of intasomes suggest that the integrase tetramer within the intasome is different from the integrase tetramer formed at high concentration in solution in the absence of viral DNA. Finally, the integration product remains tightly bound by the integrase tetramer, but the 3′ ends of the target DNA in the complex are not restrained and are free to rotate, resulting in relaxation of initially supercoiled target DNA.

APOBEC3F and APOBEC3G inhibit HIV-1 DNA integration by different mechanisms.

APOBEC3F (A3F) and APBOBEC3G (A3G) both are host restriction factors that can potently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Their antiviral activities are at least partially mediated by cytidine deamination, which causes lethal mutations of the viral genome. We recently showed that A3G blocks viral plus-strand DNA transfer and inhibits provirus establishment in the host genome (J. L. Mbisa, R. Barr, J. A. Thomas, N. Vandegraaff, I. J. Dorweiler, E. S. Svarovskaia, W. L. Brown, L. M. Mansky, R. J. Gorelick, R. S. Harris, A. Engelman, and V. K. Pathak, J. Virol. 81:7099-7110, 2007). Here, we investigated whether A3F similarly interferes with HIV-1 provirus formation. We observed that both A3F and A3G inhibit viral DNA synthesis and integration, but A3F is more potent than A3G in preventing viral DNA integration. We further investigated the mechanisms by which A3F and A3G block viral DNA integration by analyzing their effects on viral cDNA processing using Southern blot analysis. A3G generates a 6-bp extension at the viral U5 end of the 3' long terminal repeat (3'-LTR), which is a poor substrate for integration; in contrast, A3F inhibits viral DNA integration by reducing the 3' processing of viral DNA at both the U5 and U3 ends. Furthermore, we demonstrated that a functional C-terminal catalytic domain is more critical for A3G than A3F function in blocking HIV-1 provirus formation. Finally, we showed that A3F has a greater binding affinity for a viral 3'-LTR double-stranded DNA (dsDNA) oligonucleotide template than A3G. Taking these results together, we demonstrated that mechanisms utilized by A3F to prevent HIV-1 viral DNA integration were different from those of A3G, and that their target specificities and/or their affinities for dsDNA may contribute to their distinct mechanisms.

Structure-Function Analysis of the HIV-1 Integrase in Complex With Two Cellular Proteins: Ledgf and INI1

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA into the human genome is catalyzed by the viral integrase protein (IN) that requires cellular cofactors for viral infectivity. Recently, we solved a cryo-EM structure at 14 A resolution of the HIV-1 integrase in complex with the lens epithelium-derived growth factor (LEDGF), a cellular transcriptional coactivator, in presence and absence of DNA (1). This structure revealed the molecular mechanism of DNA integration in the human genome. Another cellular co-factor, the integrase interactor 1 protein (INI1/SNF5) which is a part of the SWI/SNF complex, an ATP dependant chromatin remodeler, has been shown to binds directly to integrase. Its function in the viral DNA integration process is not well characterized, but its presence is critical for viral infectivity. We stably formed, in vitro, a complex comprising IN, LEDGF and a fragment of INI1. In vitro functional assays have been performed and a 15 A resolution cryo-EM structure of the ternary complex has been solved. The structure function analysis and the effect of INI1 on the DNA binding, 3′ processing and integration reaction will be presented.(1) Michel, F., Crucifix, C., Granger, F., Eiler, S., Mouscadet, J.F., Korolev, S., Agapkina, J., Ziganshin, R., Gottikh , M., Nazabal, A., Emiliani, S., Benarous, R., Moras, D., Schultz, P. and Ruff, M. (2009). Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor. EMBO J., 28, 980-991.

Integration of HIV-1 DNA Is Regulated by Interplay between Viral Rev and Cellular LEDGF/p75 Proteins

The present work describes a novel interaction between the human immunodeficiency virus type 1 (HIV-1) Rev protein and the cellular lens epithelium-derived growth factor p75 (LEDGF/p75) protein in vitro and in virus-infected cells. Here we show, for the first time, that formation of an Rev-LEDGF/p75 complex is a crucial step in regulating viral cDNA integration. Coimmunoprecipitation experiments at various times after virus infection revealed that, first, an integrase enzyme (IN)-LEDGF/p75 complex is formed, which is then replaced by a Rev-LEDGF/p75 and Rev-IN complexes. This was supported by in vitro experiments showing that Rev promotes dissociation of the IN-LEDGF/p75 complex. Combination of the viral IN and the cellular LEDGF/p75 is required for proper integration of the viral cDNA into the host chromosomal DNA. Our findings demonstrate that integration of HIV-1 cDNA is regulated by an interplay between viral Rev and the host-cell LEDGF/p75 proteins.

Structural basis for HIV-1 DNA integration in the human genome

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA into the human genome is catalyzed by the viral integrase protein that requires the lens epithelium-derived growth factor (LEDGF), a cellular transcriptional coactivator. In the presence of LEDGF, integrase forms a stable complex in vitro and importantly becomes soluble by contrast with integrase alone which aggregates and precipitates. Using cryo-electron microscopy (EM) and single-particle reconstruction, we obtained three-dimensional structures of the wild type full length integrase-LEDGF complex with and without DNA [1]. The stoichiometry of the complex was found to be (integrase)4-(LEDGF)2 by mass spectrometry analysis and existing atomic structures were unambiguous positioned in the EM map. In vitro functional assays reveal that LEDGF increases integrase activity likely in maintaining a stable and functional integrase structure. DNA-Protein cross-linking experiments show specific interaction between viral DNA and the C-terminal domain of integrase. Upon DNA binding, IN undergoes large conformational changes. Cryo-EM structure underlines the path of viral and target DNA and a model for DNA integration in human DNA is proposed (see fig. ​fig.1,1, overleaf). Figure 1 Proposed mechanism for thei ntegration of viral cDNA into the host genome: The LEDGF envelope is represented in blue; the integrase tetramer is shown as atomic structures. The viral DNA is in orange and the target DNA in red. On target DNA binding, there ...

The Remarkable Frequency of Human Immunodeficiency Virus Type 1 Genetic Recombination

The genetic diversity of human immunodeficiency virus type 1 (HIV-1) results from a combination of point mutations and genetic recombination, and rates of both processes are unusually high. This review focuses on the mechanisms and outcomes of HIV-1 genetic recombination and on the parameters that make recombination so remarkably frequent. Experimental work has demonstrated that the process that leads to recombination--a copy choice mechanism involving the migration of reverse transcriptase between viral RNA templates--occurs several times on average during every round of HIV-1 DNA synthesis. Key biological factors that lead to high recombination rates for all retroviruses are the recombination-prone nature of their reverse transcription machinery and their pseudodiploid RNA genomes. However, HIV-1 genes recombine even more frequently than do those of many other retroviruses. This reflects the way in which HIV-1 selects genomic RNAs for coencapsidation as well as cell-to-cell transmission properties that lead to unusually frequent associations between distinct viral genotypes. HIV-1 faces strong and changeable selective conditions during replication within patients. The mode of HIV-1 persistence as integrated proviruses and strong selection for defective proviruses in vivo provide conditions for archiving alleles, which can be resuscitated years after initial provirus establishment. Recombination can facilitate drug resistance and may allow superinfecting HIV-1 strains to evade preexisting immune responses, thus adding to challenges in vaccine development. These properties converge to provide HIV-1 with the means, motive, and opportunity to recombine its genetic material at an unprecedented high rate and to allow genetic recombination to serve as one of the highest barriers to HIV-1 eradication.

Ultra-High-Throughput, Automated Nucleic Acid Detection of Human Immunodeficiency Virus (HIV) for Infant Infection Diagnosis Using the Gen-Probe Aptima HIV-1 Screening Assay

The early diagnosis of human immunodeficiency virus (HIV) infection in infants is critical to ensure the initiation of treatment before significant immunological compromise. Each year an estimated 300,000 HIV-exposed infants in South Africa require access to tests for the diagnosis of HIV infection. Currently, testing is performed at several facilities by using PCR amplification of HIV DNA at 6 weeks of age by the use of dried blood spots (DBSs) and whole blood (WB). The Gen-Probe Aptima HIV type 1 (HIV-1) screening assay (the Aptima assay) is a qualitative nucleic acid test based on transcription-mediated amplification (TMA), a technology routinely used in blood banks in South Africa. The performance characteristics of Gen-Probe's TMA technology compared well to those of the Roche Amplicor HIV-1 DNA (version 1.5) assay. The sensitivity of the assay with WB and DBS samples was 100%, and the specificities were 99.4% and 99.5% for DBSs and WB, respectively. The detection of HIV by the Aptima assay at greater levels of dilution in samples negative by the comparator assay indicates an improvement in sensitivity by the use of the TMA technology. The ability to process 1,900 samples in a 24-h period on the Tigris instrument makes the Aptima assay an attractive option for high-volume, centralized laboratories.

Development and Assessment of a Multiplex Real-Time PCR Assay for Quantification of Human Immunodeficiency Virus Type 1 DNA

Previous studies showed that high levels of human immunodeficiency virus type 1 (HIV-1) DNA are associated with a faster progression to AIDS, an increased risk of death, and a higher risk of HIV RNA rebound in patients on highly active antiretroviral therapy. Our objective was to develop and assess a highly sensitive real-time multiplex PCR assay for the quantification of HIV-1 DNA (RTMP-HIV) based on molecular beacons. HIV-1 DNA quantification was carried out by RTMP in a LightCycler 2.0 apparatus. HIV-1 DNA was quantified in parallel with CCR5 as a reference gene, and reported values are numbers of HIV-1 DNA copies/10(6) peripheral blood mononuclear cells (PBMCs). The clinical sensitivity of the assay was assessed for 115 newly diagnosed HIV-1-infected individuals. The analytical sensitivity was estimated to be 12.5 copies of HIV-1 DNA per 10(6) PBMCs, while the clinical sensitivity was 100%, with levels ranging from 1.23 to 4.25 log(10) HIV-1 DNA copies/10(6) PBMCs. In conclusion, we developed and assessed a new RTMP-HIV assay based on molecular beacons, using a LightCycler 2.0 instrument. This multiplex assay has comparable sensitivity, reproducibility, and accuracy to single real-time PCR assays.

Exosomes Packaging APOBEC3G Confer Human Immunodeficiency Virus Resistance to Recipient Cells

The human cytidine deaminase APOBEC3G (A3G) is a part of a cellular defense system against human immunodeficiency virus type 1 (HIV-1) and other retroviruses. Antiretroviral activity of A3G can be severely blunted in the presence of the HIV-1 protein Vif. However, in some cells expressing the enzymatically active low-molecular-mass form of A3G, HIV-1 replication is restricted at preintegration steps, before accumulation of Vif. Here, we show that A3G can be secreted by cells in exosomes that confer resistance to both vif-defective and wild-type HIV-1 in exosome recipient cells. Our results also suggest that A3G is the major exosomal component responsible for the anti-HIV-1 activity of exosomes. However, enzymatic activity of encapsidated A3G does not correlate with the observed limited cytidine deamination in HIV-1 DNA, suggesting that A3G-laden exosomes restrict HIV-1 through a nonenzymatic mechanism. Real-time PCR quantitation demonstrated that A3G exosomes reduce accumulation of HIV-1 reverse transcription products and steady-state levels of HIV-1 Gag and Vif proteins. Our findings suggest that A3G exosomes could be developed into a novel class of anti-HIV-1 therapeutics.

Performance of a Novel Human Immunodeficiency Virus (HIV) Type 1 Total Nucleic Acid-Based Real-Time PCR Assay Using Whole Blood and Dried Blood Spots for Diagnosis of HIV in Infants

The new Cobas AmpliPrep/Cobas TaqMan HIV-1 Qual test offers advanced automation for the detection of human immunodeficiency virus type 1 (HIV-1) RNA and DNA in dried blood spots (DBS) and whole blood. An analytical evaluation using an HIV-1 secondary standard yielded limits of detection of 514, 710, and 1,090 HIV RNA copies/ml for EDTA plasma, whole blood, and DBS, respectively. The precision and reproducibility of HIV-1 detection was equivalent for DBS and whole blood. Inclusivity was demonstrated for a reference panel of HIV-1 subtypes A to N. A clinical evaluation of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qual test was performed at a center for routine diagnostics in Johannesburg, South Africa, using 1,013 clinical specimens from HIV-1 exposed children. The Amplicor HIV-1 DNA test v1.5 with the MagNApure DNA isolation procedure was used as the reference method. A total of 995 valid results for whole blood with both methods yielded 691 and 303 concordant negative and positive results for the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qual test, respectively. For the 800 valid DBS specimen results, 495 and 300 concordant negative and positive results were obtained, respectively. The resulting clinical specificities and sensitivities of the new test were 100% and 99.7% for whole blood and DBS, respectively. The new test was characterized by its robustness, enhanced automation, and improved sample throughput. The Cobas AmpliPrep/Cobas TaqMan HIV-1 Qual test will support early, reliable diagnosis of HIV in children in routine laboratory settings.

Diminished Human Immunodeficiency Virus Type 1 DNA Yield from Dried Blood Spots after Storage in a Humid Incubator at 37°C Compared to −20°C

ABSTRACT Collecting whole blood on filter paper simplifies the processing, transport, and storage of specimens used for the diagnosis of human immunodeficiency virus type 1 (HIV-1) and other tests. Specimens may be collected in tropical or rural areas with minimal facilities for handling specimens. To compare simulated tropical conditions with freezer storage, we examined the stability of HIV-1 DNA in dried blood spots (DBS) stored in humid heat and at −20°C. DBS were created by spotting 50-μl aliquots of whole blood on 903 filter paper. DNA was extracted from DBS at baseline and after 2, 6, or 12 months of storage at −20°C or at 37°C with ∼85% humidity. The DNA was tested undiluted or diluted using the Amplicor HIV-1 DNA PCR (Roche), version 1.5. Each reaction was scored positive, negative, or indeterminate based on optical density. Results were compared between storage conditions and over time. A total of 1,832 reactions from 916 DBS were analyzed, including 100 DBS at baseline, 418 stored at −20°C, and 398 stored at 37°C. A chi-square test showed fewer positive reactions for DBS stored at 37°C (55%) than for those stored at −20°C (78%) ( P &lt; 0.0001). Samples stored at −20°C showed little change in the probability of detection of HIV-1 DNA over time; the odds ratio (OR) was 0.93 after storage for 1 year. Samples stored at 37°C demonstrated a significant change in detection at 1 year (OR, 0.29). We conclude that exposure of DBS to 37°C and high humidity impaired the recovery of HIV-1 DNA from DBS, whereas DNA recovery was preserved when DBS were stored frozen.

Highly Sensitive Methods Based on Seminested Real-Time Reverse Transcription-PCR for Quantitation of Human Immunodeficiency Virus Type 1 Unspliced and Multiply Spliced RNA and Proviral DNA

The effectiveness of highly active antiretroviral therapy (HAART), the standard of care for the treatment of human immunodeficiency virus type 1 (HIV-1) infection, is assessed by measuring the viral RNA load in plasma. A patient is considered to be successfully treated when the HIV-1 load in plasma stays below the detection limit of commercial assays. However, virus replication and evolution do continue in patients under HAART, which may eventually result in the development of drug-resistant HIV-1 strains and therapy failure. To monitor this low-level virus replication in peripheral blood mononuclear cells (PBMC), sensitive methods are required to measure HIV-1 molecular markers. We report the development of highly sensitive methods for the quantitation of unspliced and multiply spliced HIV-1 RNA and proviral DNA in PBMC. The methods are based on innovative seminested real-time reverse transcription-PCR (RT-PCR) that combines the accuracy and precision of real-time PCR and the sensitivity of nested PCR. We show that the newly developed methods are superior to the conventional single-step real-time RT-PCR in their sensitivity, accuracy, dynamic range, and the power of quantitative detection of HIV-1 RNA and DNA in clinical samples. These easy-to-perform methods can be widely used in research, including clinical studies, to monitor intracellular processes of virus replication.

Dynamics of Total, Linear Nonintegrated, and Integrated HIV‐1 DNA In Vivo and In Vitro

In patients infected with human immunodeficiency virus type 1 (HIV-1), HIV-1 DNA persists during highly active antiretroviral treatment, reflecting long-lived cellular reservoirs of HIV-1. Recent studies report an association between HIV-1 DNA levels, disease progression, and treatment outcome. However, HIV-1 DNA exists as distinct molecular forms that are not distinguished by conventional assays. We analyzed HIV-1 RNA levels in plasma, CD4 cell counts, and levels of integrated and nonintegrated HIV-1 DNA in peripheral blood mononuclear cells (PBMCs) from patients with early or chronic infection before and during antiretroviral treatment. We also studied HIV-1 DNA decay in primary CD4 T cells infected in vitro. HIV-1 DNA was analyzed using an assay that is unaffected by the location of HIV-1 integration sites. HIV-1 RNA levels and total HIV-1 DNA levels decayed rapidly in patients during receipt of suppressive antiretroviral therapy. Ratios of total HIV-1 DNA levels to integrated HIV-1 DNA levels were high before initiation of therapy but diminished during therapy. Levels of linear nonintegrated HIV-1 DNA decayed rapidly in vitro (t (1/2) = 1- 4.8 days). Total HIV-1 DNA decays rapidly with suppression of virus replication in vivo. Clearance of HIV-1 DNA during the first 6 months of therapy reflects a disproportionate loss of nonintegrated HIV-1 DNA genomes, suggesting that levels of total HIV-1 DNA in PBMCs after prolonged virus suppression largely represent integrated HIV-1 genomes.

Comparison of Results Obtained with Amplicor HIV-1 DNA PCR Test Version 1.5 Using 100 versus 500 Microliters of Whole Blood

The Amplicor HIV-1 DNA PCR assay (Roche Diagnostics, Branchburg, NJ) requires 500 microl of whole blood for a diagnosis of human immunodeficiency virus type 1 (HIV-1) infection, and this amount is often difficult to obtain from infants. A comparison was performed using 100 and 500 microl of whole blood from infants less than 18 months of age. The concordance rate for HIV DNA PCR-negative and -positive samples was 100% for the two different volumes.

Early Infant Human Immunodeficiency Virus Type 1 Detection Suitable for Resource-Limited Settings with Multiple Circulating Subtypes by Use of Nested Three-Monoplex DNA PCR and Dried Blood Spots

The early detection of human immunodeficiency virus type 1 (HIV-1) infection in infants is complicated by the persistence of maternal antibodies and by diverse HIV-1 subtypes. We developed a nested, three-monoplex HIV-1 DNA PCR (N3M-PCR) assay to detect diverse HIV-1 subtypes in infants born to infected mothers. We optimized the test for use with dried blood spot (DBS) samples for ease of storage and transport from rural China to central laboratories. Six pairs of primers were designed that targeted env, gag, and pol genes, and the test was run in three reactions with an analytical sensitivity of 10 copies DNA per reaction to cover nine HIV-1 subtypes, A, B, C, D, F, G, CRF01-AE, CRF08-BC, and CRF07-BC. The assay performance was evaluated on 347 DBS specimens from 151 exposed infants in four diverse provinces of China in which multiple subtypes were circulating. The results of this test were compared to those of HIV antibody enzyme immunoassay and Western blotting confirmation for the infants at > or =18 months of age or to convincing clinical and epidemiologic data for deceased infants. The sensitivity of the N3M-PCR assay was 30.0% (3/10) for infants at 48 h after birth, 91.7% (11/12) at 1 to 2 months of age, and 93.7% (15/16) at 3 to 6 months of age. The specificity was 100% (94/94) at all three time points. The PCR reproducibility in the three DNA regions was 100% for samples at 48 h after birth, 96.7% at 1 to 2 months, and 100% at 3 to 6 months of age. The HIV-1 DNA N3M-PCR assay on DBSs offers a simple and affordable approach for early infant HIV-1 diagnosis in regions with diverse HIV-1 circulating subtypes.

Investigating the Mechanism of the Nucleocapsid Protein Chaperoning of the Second Strand Transfer during HIV-1 DNA Synthesis

Conversion of the human immunodeficiency virus type 1 (HIV-1) genomic RNA into the proviral DNA by reverse transcriptase involves two obligatory strand transfers that are chaperoned by the nucleocapsid protein (NC). The second strand transfer relies on the annealing of the (−) and (+) copies of the primer binding site, (−)PBS and (+) PBS, which fold into complementary stem–loops (SLs) with terminal single-stranded overhangs. To understand how NC chaperones their hybridization, we investigated the annealing kinetics of fluorescently labelled (+)PBS with various (−)PBS derivatives. In the absence of NC, the (+)/(−)PBS annealing was governed by a second-order pathway nucleated mainly by the single-stranded overhangs of the two PBS SLs. The annealing reaction appeared to be rate-limited by the melting of the stable G·C-rich stem subsequent to the formation of the partially annealed intermediate. A second pathway nucleated through the loops could be detected, but was very minor. NC(11-55), which consists primarily of the zinc finger domain, increased the (−)/(+) PBS annealing kinetics by about sixfold, by strongly activating the interaction between the PBS loops. NC(11-55) also activated (−)/(+) PBS annealing through the single-strand overhangs, but by a factor of only 2. Full-length NC(1-55) further increased the (−)/(+)PBS annealing kinetics by tenfold. The NC-promoted (−)/(+)PBS mechanism proved to be similar with extended (−)DNA molecules, suggesting that it is relevant in the context of proviral DNA synthesis. These findings favour the notion that the ubiquitous role of NC in the viral life-cycle probably relies on the ability of NC to chaperone nucleic acid hybridization via different mechanisms.