20.109(F09): John and Ashley HIV Rev

= Engineering a Peptide against HIV-1 Rev Protein = John K. And Ashley F. (Blue W/F)

Project Overview
There has been significant study on the interaction between Rev and IN. There are two regions of IN that appear to bind to two different regions of Rev. These four areas have been identified and their protein sequence and position in the protein as a whole is known. It may be possible to create a library of mutagenized peptides using PCR of one of the known peptides. Using a Rev or IN inhibitor needs to be decided. Rev inhibitor does not seem to be focused on as of yet. Creating a Rev inhibitor which would increase the integration events might destabilize the cells enough in combination with other HIV-1 drugs to actually decrease HIV viability noticeably. Need to look up HIV drugs and determine if Rev is a target (does not seem to be for any currently on the market, but unsure about others in pipeline), and if so, how targeted.

Research Problem and Goals
Rev inhibition may or may not reduce the ability of HIV-1 to proliferate. This project aims to find a peptide that inhibits Rev to test whether knockdown of Rev could be used as a future drug target.

Review of existing literature
Daelemans, D., Afonina, E., Nilsson, J., Werner, G., Kjems, J., De, C. E., Pavlakis, G. N. and Vandamme, A. M. (2002). A synthetic HIV-1 Rev inhibitor interfering with the CRM1-mediated nuclear export. Proc. Natl. Acad. Sci. USA 99, 14440-14445.[Abstract/Free Full Text]

This paper goes through Rev-Nes interaction with CRM1 = toxicity, and suggests Rev-RRE has been ineffective in past

Novel regulation of HIV-1 replication and pathogenicity: Rev inhibition of integration  Levin et al, 2009

Brief: This paper establishes relationship b/w Rev and IN, and proposes second role for Rev as inhibitor of IN

This paper was published extremely recently (10/29/09) and focuses on establishing the role of the Rev protein in HIV infected cells. Rev has been previously established as a protein that exports viral RNA from the nucleus. Before this study it was also established that Rev has an additional role of binding the protein Integrase (IN). IN is carried by the HIV virus and is responsible for integrating the viral genome into the host's. This study begins by establishing that these two proteins bind (via co-IP). These researchers also establish that Rev is expressed 6 hours post-insertion (PI) meaning that it is translated without being integrated into the genome. When Rev is expressed in wt cells, it inhibits the activity of integrase and results in 1-2 HIV genome integrations per cell. When Rev is absent the cells undergo as many as 13 integrations and have an increased rate of lysis which may indicate that there is genomic instability from so may insertions. Other retro viruses do insert many more copies than HIV, so it may turn out that HIV is able to still infect without Rev, but it might be weaker. So it remains to be determined whether HIV can recover, and if so how well, from Rev knowckout. When Rev is overexpressed there are 0-1 insertions. This data implies that Rev is responsible for maintaining a viable amount of virus integration so that HIV can prosper with in an organism.

Peptides Derived from HIV-1 Integrase that Bind Rev Stimulate Viral Genome Integration  Levin et al, 2009

This paper identified the 2 regions of Rev and In that bind and determines the sequence and position of these peptides. It appears as if it might be more effective to block IN through this interaction, because blocking Rev does not impact HIV efficacy in vitro. Of the two regions in IN identified, each binds to a different peptide identified from Rev. This paper would be useful in determining how to create peptides to mimic the binding sites because it identifies the target sites.

Peptides derived from HIV-1 Rev inhibit HIV-1 integrase in a shiftide mechanism  Hayouka et al, 2008

This paper relates information found about peptides that could be used to create drugs to inhibit IN. Apparently IN drug inhibitors are becoming more popular, with the first recently (in 2008) coming onto the market. This paper identifies to potential areas of interest, both IN's interaction with LEDGF/p75 and with Rev-like peptides. It creates the idea of 'shiftides' which it defines as peptides based on the binding areas of the molecules that IN binds with that somehow disrupt the active dimers of IN creating inactive tetramers. The actual pathway is unclear, but they lay the basis for some drug inhibitors of IN.

Interaction between HIV-1 Rev and Integrase Proteins  Rosenbluh et al, 2007

This paper is a precursor to the one above, but it focuses on the discovery of short peptides that inhibit IN activity by creating peptides that mimic Rev. They create two peptides based on IN-Rev binding sites and test their effect in vivo by using a galactosidase indicator and other assays. They found that both peptides inhibited HIV-1 replication in cultured cells.

The HIV-1 Rev Protein  Pollard and Malim, 1998

This review gives an introduction to the basic mechanism of HIV and then goes into great detail about the first known function of Rev. It details the domains of Rev and diagrams how Rev controls the splicing and export of the viral genome. The paper also goes into a structural analysis of Rev and lists lots of basic information (such as sequence, and how many amino acids (116)). While some of this paper is outdated (especially with regard to the single known function of Rev at that time) it does give a basis for structural knowledge of Rev, and it helps to introduce HIV-1 in general terms in the introduction.

Management of Newly Diagnosed HIV Infection  Scott M. Hammer, M.D., 2005

This paper talks generally about different HIV cocktail mixes that are in use and which ones are being combined. It also explains the different groupings of treatments, and is useful background information on treatment of HIV and what appears to be more effective.