A Systems Survey of Progressive Host-Cell Reorganization during Rotavirus Infection.

Pathogen invasion is often accompanied by widespread alterations in cellular physiology, which reflects the hijacking of host factors and processes for pathogen entry and replication. Although genetic perturbation screens have revealed the complexity of host factors involved for numerous pathogens, it has remained challenging to temporally define the progression of events in host cell reorganization during infection. We combine high-confidence genome-scale RNAi screening of host factors required for rotavirus infection in human intestinal cells with an innovative approach to infer the trajectory of virus infection from fixed cell populations. This approach reveals a comprehensive network of host cellular processes involved in rotavirus infection and implicates AMPK in initiating the development of a rotavirus-permissive environment. Our work provides a powerful approach that can be generalized to order complex host cellular requirements along a trajectory of cellular reorganization during pathogen invasion.

Impact of sustained RNAi-mediated suppression of cellular cofactor Tat-SF1 on HIV-1 replication in CD4+ T cells.

BACKGROUND: Conventional anti-HIV drug regimens targeting viral enzymes are plagued by the emergence of drug resistance. There is interest in targeting HIV-dependency factors (HDFs), host proteins that the virus requires for replication, as drugs targeting their function may prove protective. Reporter cell lines provide a rapid and convenient method of identifying putative HDFs, but this approach may lead to misleading results and a failure to detect subtle detrimental effects on cells that result from HDF suppression. Thus, alternative methods for HDF validation are required. Cellular Tat-SF1 has long been ascribed a cofactor role in Tat-dependent transactivation of viral transcription elongation. Here we employ sustained RNAi-mediated suppression of Tat-SF1 to validate its requirement for HIV-1 replication in a CD4+ T cell-derived line and its potential as a therapeutic target. RESULTS: shRNA-mediated suppression of Tat-SF1 reduced HIV-1 replication and infectious particle production from TZM-bl reporter cells. This effect was not a result of increased apoptosis, loss of cell viability or an immune response. To validate its requirement for HIV-1 replication in a more relevant cell line, CD4+ SupT1 cell populations were generated that stably expressed shRNAs. HIV-1 replication was significantly reduced for two weeks (~65%) in cells with depleted Tat-SF1, although the inhibition of viral replication was moderate when compared to SupT1 cells expressing a shRNA targeting the integration cofactor LEDGF/p75. Tat-SF1 suppression was attenuated over time, resulting from decreased shRNA guide strand expression, suggesting that there is a selective pressure to restore Tat-SF1 levels. CONCLUSIONS: This study validates Tat-SF1 as an HDF in CD4+ T cell-derived SupT1 cells. However, our findings also suggest that Tat-SF1 is not a critical cofactor required for virus replication and its suppression may affect cell growth. Therefore, this study demonstrates the importance of examining HIV-1 replication kinetics and cytotoxicity in cells with sustained HDF suppression to validate their therapeutic potential as targets.

Small RNA-induced transcriptional gene regulation in mammals mechanisms, therapeutic applications, and scope within the genome.

Argonaute-bound small RNAs, derived from RNA interference and related pathways, are well-known effectors of posttranscriptional gene silencing (PTGS). Yet, these complexes also play an important role in affecting gene expression at the transcriptional level, either by transcriptional gene silencing (TGS) or activation (TGA). Our current understanding of how small RNAs are able to both activate and suppress transcription is unclear. In this review, we briefly outline the biogenesis of small RNAs and explore the mechanisms behind the various phenomena attributed to AGO-bound small RNA-mediated transcriptional regulation. The therapeutic potential of TGS and TGA is examined, emphasizing the distinct advantages over PTGS approaches with examples of application to cancer and diseases associated with viruses, aberrant splicing, and dysregulated heterochromatin. Finally, the influence of promoter architecture on gene susceptibility to transcriptional regulation is discussed in the light of how this impacts the scope of small RNA-induced transcriptional regulation within the genome.

FOXP3 expression is upregulated in CD4T cells in progressive HIV-1 infection and is a marker of disease severity.

BACKGROUND: Understanding the role of different classes of T cells during HIV infection is critical to determining which responses correlate with protective immunity. To date, it is unclear whether alterations in regulatory T cell (Treg) function are contributory to progression of HIV infection. METHODOLOGY: FOXP3 expression was measured by both qRT-PCR and by flow cytometry in HIV-infected individuals and uninfected controls together with expression of CD25, GITR and CTLA-4. Cultured peripheral blood mononuclear cells were stimulated with anti-CD3 and cell proliferation was assessed by CFSE dilution. PRINCIPAL FINDINGS: HIV infected individuals had significantly higher frequencies of CD4(+)FOXP3(+) T cells (median of 8.11%; range 1.33%-26.27%) than healthy controls (median 3.72%; range 1.3-7.5%; P = 0.002), despite having lower absolute counts of CD4(+)FOXP3(+) T cells. There was a significant positive correlation between the frequency of CD4(+)FOXP3(+) T cells and viral load (rho = 0.593 P = 0.003) and a significant negative correlation with CD4 count (rho = -0.423 P = 0.044). 48% of our patients had CD4 counts below 200 cells/microl and these patients showed a marked elevation of FOXP3 percentage (median 10% range 4.07%-26.27%). Assessing the mechanism of increased FOXP3 frequency, we found that the high FOXP3 levels noted in HIV infected individuals dropped rapidly in unstimulated culture conditions but could be restimulated by T cell receptor stimulation. This suggests that the high FOXP3 expression in HIV infected patients is likely due to FOXP3 upregulation by individual CD4(+) T cells following antigenic or other stimulation. CONCLUSIONS/SIGNIFICANCE: FOXP3 expression in the CD4(+) T cell population is a marker of severity of HIV infection and a potential prognostic marker of disease progression.