A competitive cell growth assay for the detection of subtle effects of gene transduction on cell proliferation.

RNA interference (RNAi) is a sequence-specific gene silencing mechanism with therapeutic potential against many human pathogens. To obtain a durable therapeutic effect, stable transduction of target cells with for instance a lentiviral vector that expresses a short hairpin (shRNA) inducer of the RNAi pathway is necessary. Apart from the intended therapeutic effect, this treatment can induce negative effects on cell proliferation via off-target effects. A careful evaluation of the transduced cells is required to develop a safe gene therapy approach. Stably transduced cells are usually selected by expression of the enhanced green fluorescent protein (GFP) marker. In this study we show that the mixed transduction culture, containing both transduced GFP(+) and untransduced GFP(-) cells, can simply be passaged to score the GFP(+)/GFP(-) ratio by longitudinal flow cytometric analysis as a measure of the negative impact of the RNAi treatment on the cellular proliferation rate. We show that this assay is sensitive, easy to use and internally controlled for assessing subtle effects on cell proliferation of lentiviral transduction and transgene expression.

[Molecular epidemiology of HIV-1 strains in the Moscow Region].

The Moscow Region is one of the HIV-1-affected subjects of the Russian Federation; there were 34613 HIV-1-infected subjects as of October 31, 2009. To characterize the molecular epidemiology of HIV-1 in the Moscow Region, the investigators obtained and studied HIV-1 variants from 61 infected subjects of the region, who were major risk groups: intravenous drug users (IDUs) and hetero- and homosexually infected persons. Genetic analysis of HIV-1 variants was carried out by sequencing the gag genes (729 nucleotides in length, including full-length protein p17 and partial p24) andlor env (270 nucleotides in length, V3 region) with further phylogenetic analysis. The findings demonstrated that HIV-1 subtype A variants are dominant in the Moscow Region and detectable in 93.5% of IDUs and 100% of heterosexually infected persons. Phylogenetically (and accordingly epidemiologically) unrelated HIV-1 subtype B strains were revealed in 4 patients, including 2 IDUs.

Sequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis.

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.

Genetic manipulation of arterivirus alternative mRNA leader-body junction sites reveals tight regulation of structural protein expression.

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.