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 39 co-terminal nested set of at least three and often seven to nine mRNAs. Coronavirus and arterivirus subgenomic transcripts are not only 39 coterminal but also contain a common 59 leader sequence, which is derived from the genomic 59 end. Their synthesis involves a process of discontinuous RNA synthesis that resembles similarityassisted 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 59 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. The increasing complexity of the nidovirus groupNidoviruses are a group of enveloped positive-stranded RNA viruses. Currently known representatives mostly infect mammals (coronaviruses, toroviruses and arteriviruses), but do also have avian (coronaviruses) or invertebrate (roniviruses) hosts. Nidoviruses cause a variety of diseases, the outcome of which can range from an asymptomatic, persistent carrier-state to a sometimes fatal infection. The severity of coronavirus infection is exemplified by severe acute respiratory syndrome (SARS) in humans, which was caused by a newly emerged coronavirus that gripped worldwide attention in (Drosten et al., 2003Ksiazek et al., 2003;Peiris et al., 2003). In the wake of the SARS outbreak, several other novel coronaviruses, including two that infect humans (van der Hoek et al., 2004; Fouchier et al., 2004;Woo et al., 2005), were identified and added to the growing list of nidoviruses that were first characterized during the past two decades.During that same period of time, the systematic sequence analysis of virus genomes has changed the face of virus taxonomy. With the rise of virus genetics and molecular virology, it has become clear that comparative sequence analysis will provide the most solid basis for future systems for virus classifi...

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.

A.O.Pasternak and E.van den Born contributed equally to this workNidovirus 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 ®delity 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 ful®ls multiple functions. The sequences of mRNA leader±body junctions of TRS mutants strongly suggested that the discontinuous step occurs during minus strand synthesis.

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.