SummaryNuclear transcribed genes produce mRNA transcripts destined to travel from the site of transcription to the cytoplasm for protein translation. Certain transcripts can be further localized to specific cytoplasmic regions. We examined the life cycle of a transcribed -actin mRNA throughout gene expression and localization, in a cell system that allows the in vivo detection of the gene locus, the transcribed mRNAs and the cytoplasmic -actin protein that integrates into the actin cytoskeleton. Quantification showed that RNA polymerase II elongation progressed at a rate of 3.3 kb/minute and that transactivator binding to the promoter was transient (40 seconds), and demonstrated the unique spatial structure of the coding and non-coding regions of the integrated gene within the transcription site. The rates of gene induction were measured during interphase and after mitosis, demonstrating that daughter cells were not synchronized in respect to transcription initiation of the studied gene. Comparison of the spatial and temporal kinetics of nucleoplasmic and cytoplasmic mRNA transport showed that the -actin-localization response initiates from the existing cytoplasmic mRNA pool and not from the newly synthesized transcripts arising after gene induction. It was also demonstrated that mechanisms of random movement were predominant in mediating the efficient translocation of mRNA in the eukaryotic cell.

Genetic diversity is the fuel of evolution and facilitates adaptation to novel environments. However, our understanding of what drives differences in the genetic diversity during the early stages of viral infection is somewhat limited. Here, we use ultra-deep sequencing to interrogate 43 clinical samples taken from early infections of the human-infecting viruses HIV, RSV and CMV. Hundreds to thousands of virus templates were sequenced per sample, allowing us to reveal dramatic differences in within-host genetic diversity among virus populations. We found that increased diversity was mostly driven by presence of multiple divergent genotypes in HIV and CMV samples, which we suggest reflect multiple transmitted/founder viruses. Conversely, we detected an abundance of low frequency hyper-edited genomes in RSV samples, presumably reflecting defective virus genomes (DVGs). We suggest that RSV is characterized by higher levels of cellular co-infection, which allow for complementation and hence elevated levels of DVGs.

The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein-pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism.pathogen-host interactions | membrane protein array | receptor discovery | integrated microfluidics

Viral-host interactions represent potential drug targets for novel antiviral strategies (Flisiak et al., Hepatology, 2008, 47, 817-26). Hence, it is important to establish an adequate platform for identifying and analyzing such interactions. In this review, we discuss bottlenecks in conventional protein-protein interaction methodologies and present the contribution of innovative microfluidic-based technologies towards a solution to these problems with respect to viral-host proteomics.

1Mutations fuel evolution and facilitate adaptation to novel environments. However, 2 characterizing the spectrum of mutations in a population is obscured by high error rates of next 3 generation sequencing. Here, we present AccuNGS, a novel in vivo sequencing approach that 4 detects variants as rare as 1:10,000. Applying it to 46 clinical samples taken from early infections 5 of the human-infecting viruses HIV, RSV and CMV, revealed large differences in within-host 6 genetic diversity among virus populations. Haplotype reconstruction revealed that increased 7 diversity was mostly driven by multiple transmitted/founder viruses in HIV and CMV samples. 8Conversely, we detected an abundance of defective virus genomes (DVGs) in RSV samples, 9including hyper-edited genomes, nonsense mutations and single point deletions. Higher 10 proportions of DVGs correlated with increased viral loads, suggesting increased cellular co-11 infection rates, which enable DVG persistence. AccuNGS establishes a general platform that 12 allows detecting DVGs, and in general, rare variants that drive evolution. 13 2016; Wang, et al. 2017); error reduction by overlapping paired reads in paired-end sequencing 46 (Chen-Harris, et al. 2013;Schirmer, et al. 2015;Preston, et al. 2016); and usage of improved 47 polymerases (Imashimizu, et al. 2013). However, most experimental methods described above 48 are designed for samples with high biomass and are inapplicable for sequencing of clinical 49 samples, where the biomass may be extremely low. Furthermore, these experimental protocols 50 may introduce their own artifacts to the sequencing process (Lou, et al. 2013; Brodin, et al. 51 2015). On the computational side, it has been suggested that well-established variant callers do 52 not perform well on clinical virus samples (McCrone and Lauring 2016). Here, we sought to 53 develop a simple and rapid approach that can tackle the problem of accurate sequencing of 54 clinical samples, and applied it to study the early stages of virus infection. 55We describe AccuNGS, a simple yet powerful approach for accurate population sequencing and 56 bioinformatics variant calling. We extensively optimize all stages of the method to ensure high 57 accuracy and maximal yield. We use AccuNGS to perform in-depth sequencing of 46 samples 58 from three different major human pathogenic viruses: human immunodeficiency virus (HIV), 59 respiratory syncytial virus (RSV), and cytomegalovirus (CMV), all sampled during the acute 60 infection stage. We compare the within-host genetic diversity among and within different virus 61 populations, and find patterns characteristic of each virus. We demonstrate the role of multiple 62 transmitted/founder viruses as major contributors to the genetic diversity in HIV and CMV. 63 Furthermore, we identify and quantify the impact of various host editing enzymes on the 64 mutational spectrum of viral genomes/populations in vivo. Intriguingly, we find that RSV 65 samples bear much higher levels of potentially defective virus genome (DVGs) ...