Isolation of MMTV and MLV mRNA regulatory proteins

Retroviruses depend on specific mechanisms to efficiently export their unspliced and incompletely spliced RNA from the nucleus. In complex retroviruses this is achieved by a trans-acting virally encoded regulatory protein which binds to responsive elements in the viral RNA. In simple retroviruses, a cis-acting structure called the cytoplasmic transport element (CTE), has evolved to interact with a host cell mRNA transport receptor. Hence, in both Gases an interaction between an RNA structure in the viral unspliced and incompletely spliced mRNA and a viral or cellular transport protein takes place. Mouse mammary tumour virus (MMTV) has been recently been proposed as a complex retrovirus. Data from our lab and others strongly supports the existence of a protein termed regulator of expression of the virion for MMTV (Rem). A role for Rem in the regulation of MMTV gene expression is proposed, however, it remains to clearly identify an RNA responsive region or indeed to isolate the wild-type protein from MMTV infected cells. It has long been considered that the simple retrovirus murine leukaemia virus (MLV) uses a CTE to achieve nuclear export of its unspliced RNA species, however, neither a CTE nor the cellular export mechanism have yet been clearly identified. Here we propose to isolate and identify Rem and Rem associated host cell factors and to screen for the MLV CTE and cellular binding partner(s) using an adaptation of the StreptoTag method. The StreptoTag method is an affinity chromatography purification procedure for the isolation of RNA binding proteins from complex protein mixtures in vilro. Recently we have optimised this method by developing a novel protocol to express and purify StreptoTag labelled RNA in/from eukaryotic cells. We next aim to isolate StreptoTag labelled Rem responsive MMTV RNA attached with its natural binding partner, the wild-type Rem protein, from infected human and murine cells. The new method will also allow us to identify the cellular binding partners of the MLV CTE by tagging the genomic RNA of a replication competent MLV vector. The association of tagged viral mRNAs with specific cellular proteins will further the understanding of the mechanisms for regulation of MMTV and MLV mRNA and host cell interactions during virus replication.

RNA-protein interactions play a key role in fundamental cellular processes in living organisms. As this type of interaction is a common regulator in pathogenic networks such as virus-host interactions, it is of special interest to be investigated. A number of in vitro methods have been established to isolate RNA binding proteins. However, these in vitro approaches may result in artificial interactions that would not occur under physiological conditions within a given cell. In the course of the funded project the so called `StreptoTag` method was successfully established for isolation of RNA binding proteins in vivo, i.e. from living cells. This method is based on RNA/protein interactions occurring within cells followed by affinity chromatography-assisted isolation and purification of the formed complexes and mass spectrometric analysis of the RNA-bound protein. For a proof-of- principle, the well investigated interaction of the human immunodeficiency virus-1 (HIV-1) RNA binding protein Rev and its respective target RNA was applied. Having demonstrated functionality of the system the standardised method was used to investigate less investigated RNA/protein interactions of the mouse mammary tumor virus (MMTV) which causes breast tumors in mice and was also shown to infect human cells. By these means the supposed binding of the MMTV protein Rem to a defined region within the viral RNA genome could be proven and a functional role of this interaction in RNA export was clearly demonstrated. In addition, the established in vivo StreptoTag method facilitated isolation and identification of a cellular protein so far unknown to interact with MMTV RNA. The potential role of this interesting protein in MMTV regulation will now be further investigated.