Many positive-stranded RNA viruses use subgenomic mRNAs to express part of their genetic information. To produce structural and accessory proteins, members of the order Nidovirales (corona-, toro-, arteri- and roniviruses) generate a 3′ co-terminal nested set of at least three and often seven to nine mRNAs. Coronavirus and arterivirus subgenomic transcripts are not only 3′ co-terminal but also contain a common 5′ leader sequence, which is derived from the genomic 5′ end. Their synthesis involves a process of discontinuous RNA synthesis that resembles similarity-assisted RNA recombination. Most models proposed over the past 25 years assume co-transcriptional fusion of subgenomic RNA leader and body sequences, but there has been controversy over the question of whether this occurs during plus- or minus-strand synthesis. In the latter model, which has now gained considerable support, subgenomic mRNA synthesis takes place from a complementary set of subgenome-size minus-strand RNAs, produced by discontinuous minus-strand synthesis. Sense–antisense base-pairing interactions between short conserved sequences play a key regulatory role in this process. In view of the presumed common ancestry of nidoviruses, the recent finding that ronivirus and torovirus mRNAs do not contain a common 5′ leader sequence is surprising. Apparently, major mechanistic differences must exist between nidoviruses, which raises questions about the functions of the common leader sequence and nidovirus transcriptase proteins and the evolution of nidovirus transcription. In this review, nidovirus transcription mechanisms are compared, the experimental systems used are critically assessed and, in particular, the impact of recently developed reverse genetic systems is discussed.

After initiating antiretroviral therapy (ART), a rapid decline in HIV viral load is followed by a long period of undetectable viremia. Viral outgrowth assay suggests the reservoir continues to decline slowly. Here, we use full-length sequencing to longitudinally study the proviral landscape of four subjects on ART to investigate the selective pressures influencing the dynamics of the treatment-resistant HIV reservoir. We find intact and defective proviruses that contain genetic elements favoring efficient protein expression decrease over time. Moreover, proviruses that lack these genetic elements, yet contain strong donor splice sequences, increase relatively to other defective proviruses, especially among clones. Our work suggests that HIV expression occurs to a significant extent during ART and results in HIV clearance, but this is obscured by the expansion of proviral clones. Paradoxically, clonal expansion may also be enhanced by HIV expression that leads to splicing between HIV donor splice sites and downstream human exons.

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.

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.

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.

Nidovirus subgenomic mRNAs contain a leader sequence derived from the 5′ end of the genome fused to different sequences (‘bodies’) derived from the 3′ end. Their generation involves a unique mechanism of discontinuous subgenomic RNA synthesis that resembles copy‐choice RNA recombination. During this process, the nascent RNA strand is transferred from one site in the template to another, during either plus or minus strand synthesis, to yield subgenomic RNA molecules. Central to this process are transcription‐regulating sequences (TRSs), which are present at both template sites and ensure the fidelity of strand transfer. Here we present results of a comprehensive co‐variation mutagenesis study of equine arteritis virus TRSs, demonstrating that discontinuous RNA synthesis depends not only on base pairing between sense leader TRS and antisense body TRS, but also on the primary sequence of the body TRS. While the leader TRS merely plays a targeting role for strand transfer, the body TRS fulfils multiple functions. The sequences of mRNA leader–body junctions of TRS mutants strongly suggested that the discontinuous step occurs during minus strand synthesis.