Alexander O. Pasternak

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

ABSTRACT 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.

Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA

Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2 = 0.97, msRNA: R2 = 0.92) and patient-derived samples (usRNA: R2 = 0.51, msRNA: R2 = 0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used in clinical studies aimed at HIV eradication, but should be cross-validated by multiple laboratories prior to wider use.

Cellular Levels of HIV Unspliced RNA from Patients on Combination Antiretroviral Therapy with Undetectable Plasma Viremia Predict the Therapy Outcome

Background Combination antiretroviral therapy (cART), the standard of care for HIV-1 infection, is considered to be successful when plasma viremia remains below the detection limit of commercial assays. Yet, cART fails in a substantial proportion of patients after the apparent success. No laboratory markers are known that are predictive of cART outcome in initial responders during the period of undetectable plasma viremia. Methodology/Principal Findings Here, we report the results of a retrospective longitudinal study of twenty-six HIV-infected individuals who initially responded to cART by having plasma viremia suppressed to <50 copies/ml. Eleven of these patients remained virologically suppressed, whereas fifteen experienced subsequent cART failure. Using sensitive methods based on seminested real-time PCR, we measured the levels of HIV-1 proviral (pr) DNA, unspliced (us) RNA, and multiply spliced RNA in the peripheral blood mononuclear cells (PBMC) of these patients at multiple time points during the period of undetectable plasma viremia on cART. Median under-therapy level of usRNA was significantly higher (0.43 log10 difference, P = 0.0015) in patients who experienced subsequent cART failure than in successfully treated patients. In multivariate analysis, adjusted for baseline CD4+ counts, prior ART experience, and particular cART regimens, the maximal usRNA level under therapy was the best independent predictor of subsequent therapy failure (adjusted odds ratio [95% CI], 24.4 [1.5–389.5], P = 0.024). The only other factor significantly associated with cART failure was prior ART experience (adjusted odds ratio [95% CI], 12.3 [1.1–138.4], P = 0.042). Levels of usRNA under cART inversely correlated with baseline CD4+ counts (P = 0.0003), but did not correlate with either baseline usRNA levels or levels of prDNA under therapy. Conclusion Our data demonstrate that the level of HIV-1 usRNA in PBMC, measured in cART-treated patients with undetectable plasma viremia, is a strong predictive marker for the outcome of therapy.

Cell-associated HIV RNA: a dynamic biomarker of viral persistence

In most HIV-infected individuals adherent to modern antiretroviral therapy (ART), plasma viremia stays undetectable by clinical assays and therefore, additional virological markers for monitoring and predicting therapy responses and for measuring the degree of HIV persistence in patients on ART should be identified. For the above purposes, quantitation of cell-associated HIV biomarkers could provide a useful alternative to measurements of viral RNA in plasma. This review concentrates on cell-associated (CA) HIV RNA with the emphasis on its use as a virological biomarker. We discuss the significance of CA HIV RNA as a prognostic marker of disease progression in untreated patients and as an indicator of residual virus replication and the size of the dynamic viral reservoir in ART-treated patients. Potential value of this biomarker for monitoring the response to ART and to novel HIV eradication therapies is highlighted.

The Stability of the Duplex between Sense and Antisense Transcription-Regulating Sequences Is a Crucial Factor in Arterivirus Subgenomic mRNA Synthesis

ABSTRACT Subgenomic mRNAs of nidoviruses (arteriviruses and coronaviruses) are composed of a common leader sequence and a “body” part of variable size, which are derived from the 5′- and 3′-proximal part of the genome, respectively. Leader-to-body joining has been proposed to occur during minus-strand RNA synthesis and to involve transfer of the nascent RNA strand from one site in the template to another. This discontinuous step in subgenomic RNA synthesis is guided by short transcription-regulating sequences (TRSs) that are present at both these template sites (leader TRS and body TRS). Sense-antisense base pairing between the leader TRS in the plus strand and the body TRS complement in the minus strand is crucial for strand transfer. Here we show that extending the leader TRS-body TRS duplex beyond its wild-type length dramatically enhanced the subgenomic mRNA synthesis of the arterivirus Equine arteritis virus (EAV). Generally, the relative amount of a subgenomic mRNA correlated with the calculated stability of the corresponding leader TRS-body TRS duplex. In addition, various leader TRS mutations induced the generation of minor subgenomic RNA species that were not detected upon infection with wild-type EAV. The synthesis of these RNA species involved leader-body junction events at sites that bear only limited resemblance to the canonical TRS. However, with the mutant leader TRS, but not with the wild-type leader TRS, these sequences could form a duplex that was stable enough to direct subgenomic RNA synthesis, again demonstrating that the stability of the leader TRS-body TRS duplex is a crucial factor in arterivirus subgenomic mRNA synthesis.

Genetic Manipulation of Arterivirus Alternative mRNA Leader-Body Junction Sites Reveals Tight Regulation of Structural Protein Expression

ABSTRACT To express its structural proteins, the arterivirus Equine arteritis virus (EAV) produces a nested set of six subgenomic (sg) RNA species. These RNA molecules are generated by a mechanism of discontinuous transcription, during which a common leader sequence, representing the 5′ end of the genomic RNA, is attached to the bodies of the sg RNAs. The connection between the leader and body parts of an mRNA is formed by a short, conserved sequence element termed the transcription-regulating sequence (TRS), which is present at the 3′ end of the leader as well as upstream of each of the structural protein genes. With the exception of RNA3, only one body TRS was previously assumed to be used to join the leader and body of each EAV sg RNA. Here we show that for the synthesis of two other sg RNAs, RNA4 and RNA5, alternative leader-body junction sites that differ substantially in transcriptional activity are used. By site-directed mutagenesis of an EAV infectious cDNA clone, the alternative TRSs used to generate RNA3, -4, and -5 were inactivated, which strongly influenced the corresponding RNA levels and the production of infectious progeny virus. The relative amounts of RNA produced from alternative TRSs differed significantly and corresponded to the relative infectivities of the virus mutants. This strongly suggested that the structural proteins that are expressed from these RNAs are limiting factors during the viral life cycle and that the discontinuous step in sg RNA synthesis is crucial for the regulation of their expression. On the basis of a theoretical analysis of the predicted RNA structure of the 3′ end of the EAV genome, we propose that the local secondary RNA structure of the body TRS regions is an important factor in the regulation of the discontinuous step in EAV sg mRNA synthesis.

HIV-1 Dual Infection Is Associated With Faster CD4+ T-Cell Decline in a Cohort of Men With Primary HIV Infection

BACKGROUND  In vitro, animal, and mathematical models suggest that human immunodeficiency virus (HIV) co- or superinfection would result in increased fitness of the pathogen and, possibly, increased virulence. However, in patients, the impact of dual HIV type 1 (HIV-1) infection on disease progression is unclear, because parameters relevant for disease progression have not been strictly analyzed. The objective of the present study is to analyze the effect of dual HIV-1 infections on disease progression in a well-defined cohort of men who have sex with men. METHODS  Between 2000 and 2009, 37 men who had primary infection with HIV-1 subtype B, no indication for immediate need of combination antiretroviral therapy (cART), and sufficient follow-up were characterized with regard to dual infection or single infection and to coreceptor use. Patients were followed to estimate the effect of these parameters on clinical disease progression, as defined by the rate of CD4(+) T-cell decline and the time to initiation of cART. RESULTS  Four patients presented with HIV-1 coinfection; 6 patients acquired HIV-1 superinfection, on average 8.5 months from their primary infection; and 27 patients remained infected with a single strain. Slopes of longitudinal CD4(+) T-cell counts and time-weighted changes from baseline were significantly steeper for patients with dual infection compared with patients with single infection. Multivariate analysis showed that the most important parameter associated with CD4(+) T-cell decline over time was dual infection (P = .001). Additionally, patients with HIV-1 coinfection had a significantly earlier start of cART (P < .0001). CONCLUSIONS  Dual HIV-1 infection is the main factor associated with CD4(+) T-cell decline in men who have untreated primary infection with HIV-1 subtype B.

Modest Nonadherence to Antiretroviral Therapy Promotes Residual HIV-1 Replication in the Absence of Virological Rebound in Plasma

BACKGROUND Modern antiretroviral therapy (ART) regimens are widely assumed to forgive modest nonadherence, because virological suppression in plasma is common at adherence levels of >70%. Yet, it is unknown whether human immunodeficiency virus type 1 (HIV-1) replication is completely suppressed at these levels of adherence. METHODS We longitudinally quantified levels of cell-associated HIV-1 RNA and DNA in 40 patients (median duration of successful ART before study initiation, 46 months), whose 1-week adherence to therapy prior to the sampling moments was measured electronically. RESULTS Patients were constantly 100% adherent (the optimal-adherence group), demonstrated improving adherence over time (the improving-adherence group), or neither of the above (the poor-adherence group). Adherence never decreased to <70% in any patient, and no rebound in plasma virological levels was observed. Nevertheless, poor adherence but not optimal or improving adherence caused a significant longitudinal increase in cell-associated HIV RNA levels (P = .006). Time-weighted changes and regression slopes of viral RNA load for the poor-adherence group were significantly higher than those for the optimal-adherence group (P < .01). CONCLUSIONS Because ART only blocks infection of new cells but not viral RNA transcription in cells infected before therapy initiation, the observed effects strongly suggest that modest nonadherence can cause new cycles of HIV-1 replication that are undetectable by commercial plasma viral load assays.

Regulation of Relative Abundance of Arterivirus Subgenomic mRNAs

ABSTRACT The subgenomic (sg) mRNAs of arteriviruses (order Nidovirales) form a 5′- and 3′-coterminal nested set with the viral genome. Their 5′ common leader sequence is derived from the genomic 5′-proximal region. Fusion of sg RNA leader and “body” segments involves a discontinuous transcription step. Presumably during minus-strand synthesis, the nascent RNA strand is transferred from one site in the genomic template to another, a process guided by conserved transcription-regulating sequences (TRSs) at these template sites. Subgenomic RNA species are produced in different but constant molar ratios, with the smallest RNAs usually being most abundant. Factors thought to influence sg RNA synthesis are size differences between sg RNA species, differences in sequence context between body TRSs, and the mutual influence (or competition) between strand transfer reactions occurring at different body TRSs. Using an Equine arteritis virus infectious cDNA clone, we investigated how body TRS activity affected sg RNA synthesis from neighboring body TRSs. Flanking sequences were standardized by head-to-tail insertion of several copies of an RNA7 body TRS cassette. A perfect gradient of sg RNA abundance, progressively favoring smaller RNA species, was observed. Disruption of body TRS function by mutagenesis did not have a significant effect on the activity of other TRSs. However, deletion of body TRS-containing regions enhanced synthesis of sg RNAs from upstream TRSs but not of those produced from downstream TRSs. The results of this study provide considerable support for the proposed discontinuous extension of minus-strand RNA synthesis as a crucial step in sg RNA synthesis.

Immune and Viral Correlates of "Secondary Viral Control" after Treatment Interruption in Chronically HIV-1 Infected Patients

Upon interruption of antiretroviral therapy, HIV-infected patients usually show viral load rebound to pre-treatment levels. Four patients, hereafter referred to as secondary controllers (SC), were identified who initiated therapy during chronic infection and, after stopping treatment, could control virus replication at undetectable levels for more than six months. In the present study we set out to unravel possible viral and immune parameters or mechanisms of this phenomenon by comparing secondary controllers with elite controllers and non-controllers, including patients under HAART. As candidate correlates of protection, virus growth kinetics, levels of intracellular viral markers, several aspects of HIV-specific CD4+ and CD8+ T cell function and HIV neutralizing antibodies were investigated. As expected all intracellular viral markers were lower in aviremic as compared to viremic subjects, but in addition both elite and secondary controllers had lower levels of viral unspliced RNA in PBMC as compared to patients on HAART. Ex vivo cultivation of the virus from CD4+ T cells of SC consistently failed in one patient and showed delayed kinetics in the three others. Formal in vitro replication studies of these three viruses showed low to absent growth in two cases and a virus with normal fitness in the third case. T cell responses toward HIV peptides, evaluated in IFN-γ ELISPOT, revealed no significant differences in breadth, magnitude or avidity between SC and all other patient groups. Neither was there a difference in polyfunctionality of CD4+ or CD8+ T cells, as evaluated with intracellular cytokine staining. However, secondary and elite controllers showed higher proliferative responses to Gag and Pol peptides. SC also showed the highest level of autologous neutralizing antibodies. These data suggest that higher T cell proliferative responses and lower replication kinetics might be instrumental in secondary viral control in the absence of treatment.