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Stabilizing role of structural elements within the 5´ Untranslated Region (UTR) and gag sequences in Mason-Pfizer monkey virus (MPMV) genomic RNA packaging.

ABSTRACT: The Mason-Pfizer monkey virus (MPMV) genomic RNA (gRNA) packaging signal is a highly-structured element with several stem-loops held together by two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences. These LRIs play a critical role in maintaining the structure of the 5´ end of the MPMV gRNA. Thus, one could hypothesize that the overall RNA secondary structure of this region is further architecturally held together by three other stem loops (SL3, Gag SL1, and Gag SL2) comprising of sequences from the distal parts of the 5´untranslated region (5' UTR) to ~ 120 nucleotides into gag, excluding gag sequences involved in forming the U5-Gag LRIs. To provide functional evidence for the biological significance of these stem loops during gRNA encapsidation, these structural motifs were mutated and their effects on MPMV RNA packaging and propagation were tested in a single round trans-complementation assay. The mutant RNA structures were further studied by high throughput SHAPE (hSHAPE) assay. Our results reveal that sequences involved in forming these three stem loops do not play crucial roles at an individual level during MPMV gRNA packaging or propagation. Further structure-function analysis indicates that the U5-Gag LRIs have a more important architectural role in stabilizing the higher order structure of the 5´ UTR than the three stem loops which have a more secondary and perhaps indirect role in stabilizing the overall RNA secondary structure of the region. Our work provides a better understanding of the molecular interactions that take place during MPMV gRNA packaging.

SUBMITTER: Kalloush RM

PROVIDER: S-EPMC6546405 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Stabilizing role of structural elements within the 5´ Untranslated Region (UTR) and gag sequences in Mason-Pfizer monkey virus (MPMV) genomic RNA packaging.

Kalloush Rawan M RM Vivet-Boudou Valérie V Ali Lizna M LM Pillai Vineeta N VN Mustafa Farah F Marquet Roland R Rizvi Tahir A TA

RNA biology 20190217 5

The Mason-Pfizer monkey virus (MPMV) genomic RNA (gRNA) packaging signal is a highly-structured element with several stem-loops held together by two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences. These LRIs play a critical role in maintaining the structure of the 5´ end of the MPMV gRNA. Thus, one could hypothesize that the overall RNA secondary structure of this region is further architecturally held together by three other stem loops (SL3, ...[more]

PMID: 30773097

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Project description:Retroviral envelope glycoprotein (Env) is essential for the specific recognition of the host cell and the initial phase of infection. As reported for human immunodeficiency virus (HIV), the recruitment of Env into a retroviral membrane envelope is mediated through its interaction with a Gag polyprotein precursor of structural proteins. This interaction, occurring between the matrix domain (MA) of Gag and the cytoplasmic tail (CT) of the transmembrane domain of Env, takes place at the host cell plasma membrane. To determine whether the MA of Mason-Pfizer monkey virus (M-PMV) also interacts directly with the CT of Env, we mimicked the in vivo conditions in an in vitro experiment by using a CT in its physiological trimeric conformation mediated by the trimerization motif of the GCN4 yeast transcription factor. The MA protein was used at the concentration shifting the equilibrium to its trimeric form. The direct interaction between MA and CT was confirmed by a pulldown assay. Through the combination of nuclear magnetic resonance (NMR) spectroscopy and protein cross-linking followed by mass spectrometry analysis, the residues involved in mutual interactions were determined. NMR has shown that the C terminus of the CT is bound to the C-terminal part of MA. In addition, protein cross-linking confirmed the close proximity of the N-terminal part of CT and the N terminus of MA, which is enabled in vivo by their location at the membrane. These results are in agreement with the previously determined orientation of MA on the membrane and support the already observed mechanisms of M-PMV virus-like particle transport and budding.IMPORTANCE By a combination of nuclear magnetic resonance (NMR) and mass spectroscopy of cross-linked peptides, we show that in contrast to human immunodeficiency virus type 1 (HIV-1), the C-terminal residues of the unstructured cytoplasmic tail of Mason-Pfizer monkey virus (M-PMV) Env interact with the matrix domain (MA). Based on biochemical data and molecular modeling, we propose that individual cytoplasmic tail (CT) monomers of a trimeric complex bind MA molecules belonging to different neighboring trimers, which may stabilize the MA orientation at the membrane by the formation of a membrane-bound net of interlinked Gag and CT trimers. This also corresponds with the concept that the membrane-bound MA of Gag recruits Env through interaction with the full-length CT, while CT truncation during maturation attenuates the interaction to facilitate uncoating. We propose a model suggesting different arrangements of MA-CT complexes between a D-type and C-type retroviruses with short and long CTs, respectively.

Dataset Information

Stabilizing role of structural elements within the 5´ Untranslated Region (UTR) and gag sequences in Mason-Pfizer monkey virus (MPMV) genomic RNA packaging.

Publications

Stabilizing role of structural elements within the 5´ Untranslated Region (UTR) and gag sequences in Mason-Pfizer monkey virus (MPMV) genomic RNA packaging.

Similar Datasets

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