The HIV-1 RNA riboswitch

There is accumulating evidence that diverse biological processes are regulated by structural changes in an RNA molecule. We have identified such a riboswitch in the RNA genome of the human immunodeficiency virus type 1 (HIV-1). Many important replication signals are clustered in the untranslated leader of the viral genome, including signals for splicing, translation packaging of the RNA in new virion particles and reverse transcription of the RNA into DNA. Static RNA secondary structure models for the leader RNA have been proposed by us and others in the early 1990s, but we recently presented evidence that this RNA molecule can adopt two mutually exclusive conformations. It was demonstrated that this riboswitch regulates the dimerization of HIV-1 RNA, which is an essential step during packaging. Furthermore, we proposed that this riboswitch acts as an RNA-checkpoint to coordinate the diverse functions of this RNA molecule. We now propose a combination of in vitro (biophysics, biochemistry) and in vivo (molecular biology, virology) approaches to elaborate on this novel riboswitch concept. We hope to unravel the cellular RNA routing pathways, the role of this riboswitch in the use of distinct routes either towards translation on ribosomes or packaging into virion particles. Besides the study of HIV-1 RNA structure and function, we also propose a new project on the interaction between HIV-1 and the cellular RNA-interference (RNAi) machinery. Several distinct questions will be addressed, including whether HIV-1 encodes an RNAi-suppressor and whether HIV-1 can be inhibited by an introduced short-interfering (si)RNA. The RNAi field is currently booming and may be considered as extremely competitive. However, we will present several preliminary results to document that we have established a fruitful niche to study the interaction between the host RNAi machinery and the virus replication cycle. Several innovative approaches are presented to expand this new line of research, including the use of different RNAi-libraries to identify potent inhibitors that may be used in antiviral therapy approaches. We have divided this research proposal in two subprojects: 1. HIV-1 RNA structure and function and 2. RNA interference and HIV-1. Each subproject is further divided in three distinct sections as follows: 1. HIV-1 RNA structure and function: A. Mutants ? revertants B. Structure-specific antisense DNA C. Other viruses (HIV-1 subtypes, HIV-2, SIVs, other retroviruses) 2. RNA-interference and HIV-1: A. does HIV-1 encode an RNAi-suppressor B. does HIV-1 encode small regulatory RNA C. RNAi-mediated inhibition of HIV-1