Germline von Hippel-Lindau tumour suppressor gene (VHL) mutations cause renal cell carcinomas, haemangioblastomas and phaeochromocytomas in humans. Mutations in VHL also occur in sporadic renal cell carcinomas. The protein encoded by VHL, VHL, is part of the ubiquitin ligase that downregulates the heterodimeric transcription factor Hif under well-oxygenated conditions. Here we show that acute VHL inactivation causes a senescent-like phenotype in vitro and in vivo. This phenotype was independent of p53 and Hif but dependent on the retinoblastoma protein (Rb) and the SWI2/SNF2 chromatin remodeller p400. Rb activation occurred through a decrease in Skp2 messenger RNA, which resulted in the upregulation of p27 in a Hif-independent fashion. Our results suggest that senescence induced by VHL inactivation is a tumour-suppressive mechanism that must be overcome to develop VHL-associated neoplasias.

Rta, mainly encoded by open reading frame 50 (ORF50), is the product of an immediate-early gene of human herpesvirus-8 (HHV-8)/Kaposi's sarcoma-associated herpesvirus. Rta is a transcriptional activator that is both necessary and sufficient to disrupt viral latency and activate the expression of downstream viral lytic genes. We report that ectopically expressed Rta protein could also activate the rta promoter on a reporter plasmid up to 144-fold, both in latently infected B cells and in uninfected epithelial cells, and that this activation was dose-dependent. Furthermore, by analysing the 5h untranslated region using ribonuclease protection assays, we demonstrated that transfection of an Rta expression plasmid into latently infected cells activated the expression of rta transcripts from endogenous viral genomes. We propose that autoactivation of the immediate-early gene, rta, is an important strategy for HHV-8 to effectively respond to environmental stimuli and maximally activate the virus lytic cycle.

Genetic research on influenza virus biology has been informed in large part by nucleotide variants present in seasonal or pandemic samples, or individual mutants generated in the laboratory, leaving a substantial part of the genome uncharacterized. Here, we have developed a single-nucleotide resolution genetic approach to interrogate the fitness effect of point mutations in 98% of the amino acid positions in the influenza A virus hemagglutinin (HA) gene. Our HA fitness map provides a reference to identify indispensable regions to aid in drug and vaccine design as targeting these regions will increase the genetic barrier for the emergence of escape mutations. This study offers a new platform for studying genome dynamics, structure-function relationships, virus-host interactions, and can further rational drug and vaccine design. Our approach can also be applied to any virus that can be genetically manipulated.

The E2F family of transcription factors are critical regulators of the cell cycle and have also been implicated in apoptosis, development, DNA damage checkpoints, and differentiation. Retinoblastoma (Rb) proteins interact with E2F to regulate transcription, and several mechanisms have been proposed for Rb-E2F transcriptional regulation. We designed microarray-based experiments to characterize the relative contributions of each mechanism, and unexpectedly, we found that distinct functional gene groups show preference for one mechanism over the others. We propose that such a distribution may provide signaling specificity to enable regulatory proteins to turn on or off entire pathways that determine cell fate.

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Using the Delphi technique, this paper continues to develop a set of attributes that ARL directors of today and the near future (next ten years) will need to possess. The research reported here drew upon the view-points of both directors and their immediate deputies. The questions remaining are: How does the list of attributes change in other organizational settings? and Where can each attribute best be acquired?

Viral proteins often display several functions which require multiple assays to dissect their genetic basis. Here, we describe a systematic approach to screen for loss-of-function mutations that confer a fitness disadvantage under a specified growth condition. Our methodology was achieved by genetically monitoring a mutant library under two growth conditions, with and without interferon, by deep sequencing. We employed a molecular tagging technique to distinguish true mutations from sequencing error. This approach enabled us to identify mutations that were negatively selected against, in addition to those that were positively selected for. Using this technique, we identified loss-of-function mutations in the influenza A virus NS segment that were sensitive to type I interferon in a high-throughput fashion. Mechanistic characterization further showed that a single substitution, D92Y, resulted in the inability of NS to inhibit RIG-I ubiquitination. The approach described in this study can be applied under any specified condition for any virus that can be genetically manipulated. IMPORTANCETraditional genetics focuses on a single genotype-phenotype relationship, whereas high-throughput genetics permits phenotypic characterization of numerous mutants in parallel. High-throughput genetics often involves monitoring of a mutant library with deep sequencing. However, deep sequencing suffers from a high error rate (ϳ0.1 to 1%), which is usually higher than the occurrence frequency for individual point mutations within a mutant library. Therefore, only mutations that confer a fitness advantage can be identified with confidence due to an enrichment in the occurrence frequency. In contrast, it is impossible to identify deleterious mutations using most next-generation sequencing techniques. In this study, we have applied a molecular tagging technique to distinguish true mutations from sequencing errors. It enabled us to identify mutations that underwent negative selection, in addition to mutations that experienced positive selection. This study provides a proof of concept by screening for loss-of-function mutations on the influenza A virus NS segment that are involved in its anti-interferon activity.

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