Single-cell analysis of population context advances RNAi screening at multiple levels.

Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single-cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome-wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell-based screens at this depth reveals widespread RNAi-induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell-to-cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large-scale RNAi screens are increasingly performed to reach a systems-level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single-cell microenvironment.

The C type lectins DC-SIGN and L-SIGN: receptors for viral glycoproteins.

DC-SIGN and L-SIGN are C-type lectins that recognize carbohydrate structures present on viral glycoproteins and function as attachment factors for several enveloped viruses. DC-SIGN and L-SIGN enhance viral entry and facilitate infection of cells that express the cognate entry receptor (cis-infection). They are also able to capture viruses and transfer viral infections to other target cells (trans-infection). In this chapter, we will give an overview of protocols used to produce soluble viral glycoproteins at high levels and to study the molecular basis of viruses/DC-SIGN and L-SIGN binding and internalization. We will also describe techniques to investigate the molecular mechanisms by which DC-SIGN or L-SIGN spread viral infections.

Viral shedding and susceptibility to oseltamivir in hospitalized immunocompromised patients with influenza in the Influenza Resistance Information Study (IRIS).

BACKGROUND: Immunocompromised patients are at greater risk of complicated influenza and may be more likely to develop antiviral resistance. This observational substudy of the Influenza Resistance Information Study (NCT00884117) aimed to study antiviral resistance in immunocompromised patients with influenza and characterize its effect on clinical and virological outcomes. METHODS: Eligible immunocompromised patients were aged ≥1 year with a local rapid diagnostic or PCR test positive for influenza ≤96 h after diagnosis and with influenza symptoms. Nasal and throat swabs were taken for RT-PCR analysis on day 1 and then every 3 days until patients were virus-free. Resistance was assessed by mutation-specific RT-PCR, phenotypic susceptibility analysis and Sanger sequencing. RESULTS: Of 42 patients enrolled, 29 (69%) were influenza-positive (RT-PCR) on day 1: 18 adults and 11 children aged 1-12 years. Six patients were severely immunocompromised. On days 3, 6 and 9, most patients tested (18/24, 9/15 and 6/9, respectively) had not cleared the virus. Two of five patients assessed after day 9 continued shedding virus until day 15. H1N1pdm09 viruses harbouring H275Y mutations were detected in post-baseline samples from four patients (aged 52-61 years), one of whom had prolonged viral shedding. No genotypic antiviral resistance was detected in the other 20 treated patients (prevalence of resistance, 17%). Correlation between level of immune compromise and resistance or outcomes could not be assessed. Ten patients (seven influenza-positive) were admitted to intensive care and three died. CONCLUSIONS: In these patients with mild/moderate immunocompromise, emergence of oseltamivir-resistant viruses was not common. Severity of influenza symptoms ranged from mild to moderate, but correlation with level of compromise could not be determined.

Infection of dendritic cells (DCs), not DC-SIGN-mediated internalization of human immunodeficiency virus, is required for long-term transfer of virus to T cells.

The C-type lectin DC-SIGN expressed on immature dendritic cells (DCs) captures human immunodeficiency virus (HIV) particles and enhances the infection of CD4+ T cells. This process, known as trans-enhancement of T-cell infection, has been related to HIV endocytosis. It has been proposed that DC-SIGN targets HIV to a nondegradative compartment within DCs and DC-SIGN-expressing cells, allowing incoming virus to persist for several days before infecting target cells. In this study, we provide several lines of evidence suggesting that intracellular storage of intact virions does not contribute to HIV transmission. We show that endocytosis-defective DC-SIGN molecules enhance T-cell infection as efficiently as their wild-type counterparts, indicating that DC-SIGN-mediated HIV internalization is dispensable for trans-enhancement. Furthermore, using immature DCs that are genetically resistant to infection, we demonstrate that several days after viral uptake, HIV transfer from DCs to T cells requires viral fusion and occurs exclusively through DC infection and transmission of newly synthesized viral particles. Importantly, our results suggest that DC-SIGN participates in this process by cooperating with the HIV entry receptors to facilitate cis-infection of immature DCs and subsequent viral transfer to T cells. We suggest that such a mechanism, rather than intracellular storage of incoming virus, accounts for the long-term transfer of HIV to CD4+ T cells and may contribute to the spread of infection by DCs.

Influenza a viruses with mutations in the m1 helix six domain display a wide variety of morphological phenotypes.

Several functions required for the replication of influenza A viruses have been attributed to the viral matrix protein (M1), and a number of studies have focused on a region of the M1 protein designated "helix six." This region contains an exposed positively charged stretch of amino acids, including the motif 101-RKLKR-105, which has been identified as a nuclear localization signal, but several studies suggest that this domain is also involved in functions such as binding to the ribonucleoprotein genome segments (RNPs), membrane association, interaction with the viral nuclear export protein, and virus assembly. In order to define M1 functions in more detail, a series of mutants containing alanine substitutions in the helix six region were generated in A/WSN/33 virus. These were analyzed for RNP-binding function, their capacity to incorporate into infectious viruses by using reverse genetics, the replication properties of rescued viruses, and the morphological phenotypes of the mutant virus particles. The most notable effect that was identified concerned single amino acid substitution mutants that caused significant alterations to the morphology of budded viruses. Whereas A/WSN/33 virus generally forms particles that are predominantly spherical, observations made by negative stain electron microscopy showed that several of the mutant virions, such as K95A, K98A, R101A, and K102A, display a wide range of shapes and sizes that varied in a temperature-dependent manner. The K102A mutant is particularly interesting in that it can form extended filamentous particles. These results support the proposition that the helix six domain is involved in the process of virus assembly.

Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN)-mediated enhancement of dengue virus infection is independent of DC-SIGN internalization signals.

Dengue virus (DV) is a mosquito-borne flavivirus that causes hemorrhagic fever in humans. In the natural infection, DV is introduced into human skin by an infected mosquito vector where it is believed to target immature dendritic cells (DCs) and Langerhans cells (LCs). We found that DV productively infects DCs but not LCs. We show here that the interactions between DV E protein, the sole mannosylated glycoprotein present on DV particles, and the C-type lectin dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) are essential for DV infection of DCs. Binding of mannosylated N-glycans on DV E protein to DC-SIGN triggers a rapid and efficient internalization of the viral glycoprotein. However, we observed that endocytosis-defective DC-SIGN molecules allow efficient DV replication, indicating that DC-SIGN endocytosis is dispensable for the internalization step in DV entry. Together, these results argue in favor of a mechanism by which DC-SIGN enhances DV entry and infection in cis. We propose that DC-SIGN concentrates mosquito-derived DV particles at the cell surface to allow efficient interaction with an as yet unidentified entry factor that is ultimately responsible for DV internalization and pH-dependent fusion into DCs.