Post-transcriptional regulatory elements (PTREs) effect various aspects of RNA function: transport of RNAs from the nucleus to the cytoplasm, localization of the RNA in specific cellular compartments, association with ribosomes, and translation. Retroviruses and lentiviruses have provided a fertile ground for the discovery of such elements. In the case of human immunodeficiency virus (HIV), the viral Rev protein, in conjunction with an RNA element (Rev-response element or RRE), has been shown to assist in the nucleo-cytoplasmic transport, as well as, the translation of intron containing HIV mRNAs. The constitutive transport element (CTE), a structured RNA element, performs an analogous function to that of Rev and RRE in Mason-Pfizer monkey virus (MPMV). We have previously described novel HIV type 1 (HIV-1) based packaging systems that use various combinations of these transport elements for the expression of the packaging and gene transfer vector RNAs. In this proposal, we wish to improve and extend this idea, by investigating the function of these and other heterologous PTREs in the context of HIV-1 packaging and gene transfer vectors.
The specific aims of this proposal are:
Aim 1. Improve performance of CTE-based lentivirus packaging systems. Our previously described combination packaging system, that used CTE for expression of helper proteins and the Rev/RRE for expression of gene transfer vector RNA in packaging cells, was found to be hampered by an unexpected inhibitory effect of Rev on CTE-function. In this aim we wish to investigate how Rev proteins of HIV-1 and other lentiviruses impact on CTE function with a view towards improving the combination packaging system. To this end, we propose studies on the effect of Rev on nucleocytoplasmic transport and polysomal association of CTE-containing messages, and the identification of domains of Rev that are involved in the inhibition of CTE function. We will compare this activity of HIV-1 Rev with similar proteins derived from other lentiviruses such as HIV type 2 (HIV-2) Rev and human T-cell leukemia virus type 1 (HTLV-1) Rex. Once we have established the possible mechanism(s) by which Rev interferes with CTE function, we will try and modulate this inhibition by over expression of Rev-RRE or CTE-interacting proteins.
Aim 2. Evaluate PTREs from other viruses in the context of HIV-1 packaging and gene transfer vectors. As part of this aim, we will test the effect of Rev proteins of different viruses on the transport of homologous and heterologous RTE containing expression vectors. One of the goals of this aim is to find PTREs that are quite dissimilar to each other sequence-wise but do not interfere with each other's function. Such PTREs will be ideal for the creation of packaging cell systems for lentivirus vectors. We will also evaluate the use of multiple PTREs to try and improve gene expression by complementation of attributes of single elements.
Aim 3. Create packaging cell lines using the best constructs identified in Aim 2 and test the RNA elements in the context of integrated HIV-1 packaging and gene transfer vectors. These packaging cell lines will be useful for the production of large amounts of vector stocks. The results of these studies will : 1) provide a better understanding of the function of post-transcriptional RNA regulatory elements, 2) highlight potential interactions between various elements, and 3) identify RNA elements that provide high levels of gene expression in the context of lentivirus-based packaging and gene transfer vectors. This, in turn, will allow us to devise high-titer lentivirus-based packaging systems with a reduced chance of generating replication competent virus.
| Srinivasakumar, Narasimhachar (2011) Rev-free HIV-1 gene delivery system for targeting Rev-RRE-Crm1 nucleocytoplasmic RNA transport pathway. PLoS One 6:e28462 |
