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

Aktar SJ, Vivet-Boudou V, Ali LM, Jabeen A, Kalloush RM, Richer D, Mustafa F, Marquet R, Rizvi TA - Retrovirology (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology & Immunology, College of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666, Al Ain, United Arab Emirates. sjaktar@uaeu.ac.ae.

ABSTRACT

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.

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Structural analysis of pal II and PBS pal deletion-mutant homodimers. Predicted homodimer structures of (A) MMTV wild type (SA035), (B) pal II deletion (SA042), (C) deletion of the pal sequence within PBS (SA051) and (D) double deletion of both pal II and pal sequence within PBS (SA046). The dimers were predicted using RNAstructure and the contact point (point of dimerization) of the sequences between two RNAs leading to dimerization are boxed.
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Fig4: Structural analysis of pal II and PBS pal deletion-mutant homodimers. Predicted homodimer structures of (A) MMTV wild type (SA035), (B) pal II deletion (SA042), (C) deletion of the pal sequence within PBS (SA051) and (D) double deletion of both pal II and pal sequence within PBS (SA046). The dimers were predicted using RNAstructure and the contact point (point of dimerization) of the sequences between two RNAs leading to dimerization are boxed.

Mentions: These results indicated that although pal II deletion mutants could reduce RNA dimerization, they could not completely abrogate it. Such an observation suggested the presence of another sequence that could facilitate dimerization in addition to pal II, though not to the wild type levels. Therefore, the pal II deletion mutant (SA042) sequence was folded as dimer using the RNAstructure software, which predicted that in the absence of pal II, the MMTV RNA packaging signal could potentially dimerize via the two overlapping pals in the PBS (5’ CAGCUGGCGCC 3’; the first pal is in italics and the second pal is in bold (Figure 4). To determine the role of PBS pal, if any, on dimerization, a mutant (SA051) was generated containing a complete deletion of the 11 nt overlapping pals within the PBS. Along the same lines, a double mutant (SA046) was created containing the deletion of pal II sequence as well as the overlapping pals within the PBS (Figure 3A). Deletion of the 11 nt PBS pal alone in SA051 resulted in 60% reduction in RNA dimerization levels (P ≤0.05; Figure 3B and Figure 3C). Deletion of both sequences (SA046) resulted in almost a complete abrogation of dimerization in TBM condition (18-fold; P <0.002) and to a 3.5-fold decrease in TB condition (P <0.006; Figure 3B and Figure 3C). These results suggest that the MMTV genome may require two points of contact during intermolecular interactions to augment efficient gRNA dimerization.Figure 4


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

Aktar SJ, Vivet-Boudou V, Ali LM, Jabeen A, Kalloush RM, Richer D, Mustafa F, Marquet R, Rizvi TA - Retrovirology (2014)

Structural analysis of pal II and PBS pal deletion-mutant homodimers. Predicted homodimer structures of (A) MMTV wild type (SA035), (B) pal II deletion (SA042), (C) deletion of the pal sequence within PBS (SA051) and (D) double deletion of both pal II and pal sequence within PBS (SA046). The dimers were predicted using RNAstructure and the contact point (point of dimerization) of the sequences between two RNAs leading to dimerization are boxed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4264320&req=5

Fig4: Structural analysis of pal II and PBS pal deletion-mutant homodimers. Predicted homodimer structures of (A) MMTV wild type (SA035), (B) pal II deletion (SA042), (C) deletion of the pal sequence within PBS (SA051) and (D) double deletion of both pal II and pal sequence within PBS (SA046). The dimers were predicted using RNAstructure and the contact point (point of dimerization) of the sequences between two RNAs leading to dimerization are boxed.
Mentions: These results indicated that although pal II deletion mutants could reduce RNA dimerization, they could not completely abrogate it. Such an observation suggested the presence of another sequence that could facilitate dimerization in addition to pal II, though not to the wild type levels. Therefore, the pal II deletion mutant (SA042) sequence was folded as dimer using the RNAstructure software, which predicted that in the absence of pal II, the MMTV RNA packaging signal could potentially dimerize via the two overlapping pals in the PBS (5’ CAGCUGGCGCC 3’; the first pal is in italics and the second pal is in bold (Figure 4). To determine the role of PBS pal, if any, on dimerization, a mutant (SA051) was generated containing a complete deletion of the 11 nt overlapping pals within the PBS. Along the same lines, a double mutant (SA046) was created containing the deletion of pal II sequence as well as the overlapping pals within the PBS (Figure 3A). Deletion of the 11 nt PBS pal alone in SA051 resulted in 60% reduction in RNA dimerization levels (P ≤0.05; Figure 3B and Figure 3C). Deletion of both sequences (SA046) resulted in almost a complete abrogation of dimerization in TBM condition (18-fold; P <0.002) and to a 3.5-fold decrease in TB condition (P <0.006; Figure 3B and Figure 3C). These results suggest that the MMTV genome may require two points of contact during intermolecular interactions to augment efficient gRNA dimerization.Figure 4

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology & Immunology, College of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666, Al Ain, United Arab Emirates. sjaktar@uaeu.ac.ae.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus