Biochemical and Functional Characterization of Mouse Mammary Tumor Virus Full-Length Pr77Gag Expressed in Prokaryotic and Eukaryotic Cells

The mouse mammary tumor virus (MMTV) Pr77Gag polypeptide is an essential retroviral structural protein without which infectious viral particles cannot be formed. This process requires specific recognition and packaging of dimerized genomic RNA (gRNA) by Gag during virus assembly. Most of the previous work on retroviral assembly has used either the nucleocapsid portion of Gag, or other truncated Gag derivatives—not the natural substrate for virus assembly. In order to understand the molecular mechanism of MMTV gRNA packaging process, we expressed and purified full-length recombinant Pr77Gag-His6-tag fusion protein from soluble fractions of bacterial cultures. We show that the purified Pr77Gag-His6-tag protein retained the ability to assemble virus-like particles (VLPs) in vitro with morphologically similar immature intracellular particles. The recombinant proteins (with and without His6-tag) could both be expressed in prokaryotic and eukaryotic cells and had the ability to form VLPs in vivo. Most importantly, the recombinant Pr77Gag-His6-tag fusion proteins capable of making VLPs in eukaryotic cells were competent for packaging sub-genomic MMTV RNAs. The successful expression and purification of a biologically active, full-length MMTV Pr77Gag should lay down the foundation towards performing RNA–protein interaction(s), especially for structure-function studies and towards understanding molecular intricacies during MMTV gRNA packaging and assembly processes

Cross- and Co-Packaging of Retroviral RNAs and Their Consequences

Retroviruses belong to the family Retroviridae and are ribonucleoprotein (RNP) particles that contain a dimeric RNA genome. Retroviral particle assembly is a complex process, and how the virus is able to recognize and specifically capture the genomic RNA (gRNA) among millions of other cellular and spliced retroviral RNAs has been the subject of extensive investigation over the last two decades. The specificity towards RNA packaging requires higher order interactions of the retroviral gRNA with the structural Gag proteins. Moreover, several retroviruses have been shown to have the ability to cross-/co-package gRNA from other retroviruses, despite little sequence homology. This review will compare the determinants of gRNA encapsidation among different retroviruses, followed by an examination of our current understanding of the interaction between diverse viral genomes and heterologous proteins, leading to their cross-/co-packaging. Retroviruses are well-known serious animal and human pathogens, and such a cross-/co-packaging phenomenon could result in the generation of novel viral variants with unknown pathogenic potential. At the same time, however, an enhanced understanding of the molecular mechanisms involved in these specific interactions makes retroviruses an attractive target for anti-viral drugs, vaccines, and vectors for human gene therapy

Structural basis of genomic RNA (gRNA) dimerization and packaging determinants of mouse mammary tumor virus (MMTV)

BACKGROUND: One of the hallmarks of retroviral life cycle is the efficient and specific packaging of two copies of retroviral gRNA in the form of a non-covalent RNA dimer by the assembling virions. It is becoming increasingly clear that the process of dimerization is closely linked with gRNA packaging, and in some retroviruses, the latter depends on the former. Earlier mutational analysis of the 5' end of the MMTV genome indicated that MMTV gRNA packaging determinants comprise sequences both within the 5' untranslated region (5' UTR) and the beginning of gag. RESULTS: The RNA secondary structure of MMTV gRNA packaging sequences was elucidated employing selective 2'hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE analyses revealed the presence of a U5/Gag long-range interaction (U5/Gag LRI), not predicted by minimum free-energy structure predictions that potentially stabilizes the global structure of this region. Structure conservation along with base-pair covariations between different strains of MMTV further supported the SHAPE-validated model. The 5' region of the MMTV gRNA contains multiple palindromic (pal) sequences that could initiate intermolecular interaction during RNA dimerization. In vitro RNA dimerization, SHAPE analysis, and structure prediction approaches on a series of pal mutants revealed that MMTV RNA utilizes a palindromic point of contact to initiate intermolecular interactions between two gRNAs, leading to dimerization. This contact point resides within pal II (5' CGGCCG 3') at the 5' UTR and contains a canonical "GC" dyad and therefore likely constitutes the MMTV RNA dimerization initiation site (DIS). Further analyses of these pal mutants employing in vivo genetic approaches indicate that pal II, as well as pal sequences located in the primer binding site (PBS) are both required for efficient MMTV gRNA packaging. CONCLUSIONS: Employing structural prediction, biochemical, and genetic approaches, we show that pal II functions as a primary point of contact between two MMTV RNAs, leading to gRNA dimerization and its subsequent encapsidation into the assembling virus particles. The results presented here enhance our understanding of the MMTV gRNA dimerization and packaging processes and the role of structural motifs with respect to RNA-RNA and possibly RNA-protein interactions that might be taking place during MMTV life cycle

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

<p>Packaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5ʹ untranslated region (UTR) in conjunction with the first 120 nucleotides of the <i>gag</i> gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation <i>versus</i> packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.</p