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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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Effects of pal II substitution mutations on MMTV gRNA dimerization, RNA packaging and viral propagation. (A) Description of the pal II substitution mutants. (B) Typical RNA dimerization in TBM and TB gels of wild type and mutant MMTV RNAs. The last two lanes correspond to the co-incubation of trans-complementary mutants SA044 and SA045. M: monomer lane or monomer conformer; D: dimer lane or dimer conformer. (C) Quantification of the relative RNA dimerization (see table below the histogram). Table above the histogram represent the P values of the respective mutants in TBM and TB gels. The experiments were repeated 2-6 times. (D) Relative packaging efficiency of transfer vector RNAs. (E) RNA propagation efficiency expressed as relative Hygr colony forming unit per ml (CFU/ml). In (C), (D), and (E), the histograms represent data from at least three independent experiments (± SD). The P values for all mutants in panels (D) and (E) were significant (<0.001).
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Fig5: Effects of pal II substitution mutations on MMTV gRNA dimerization, RNA packaging and viral propagation. (A) Description of the pal II substitution mutants. (B) Typical RNA dimerization in TBM and TB gels of wild type and mutant MMTV RNAs. The last two lanes correspond to the co-incubation of trans-complementary mutants SA044 and SA045. M: monomer lane or monomer conformer; D: dimer lane or dimer conformer. (C) Quantification of the relative RNA dimerization (see table below the histogram). Table above the histogram represent the P values of the respective mutants in TBM and TB gels. The experiments were repeated 2-6 times. (D) Relative packaging efficiency of transfer vector RNAs. (E) RNA propagation efficiency expressed as relative Hygr colony forming unit per ml (CFU/ml). In (C), (D), and (E), the histograms represent data from at least three independent experiments (± SD). The P values for all mutants in panels (D) and (E) were significant (<0.001).

Mentions: Results in Figure 3 and Figure 4 showed that one of the points of contact between the two RNAs is pal II. To ascertain whether the primary sequence or the palindromic nature of pal II is important to initiate intermolecular interactions between the two RNAs during MMTV gRNA dimerization, we generated several additional mutants. The wild type pal II was substituted with HIV-1 pal flanked by additional purines (5’ AAGCGCGCA 3’; Figure 5A, mutant SA047). Two additional mutants were also created in which the pal II sequence was substituted by non-palindromic trans-complementary sequences (Figure 5A, mutants SA044 and SA045). In all of these mutants, the pal sequence within the PBS was maintained. Consistent with the effect of the purines flanking the pal sequence on HIV-1 RNA dimerization [26], replacing pal II by the HIV-1 pal and the flanking purines (SA047) preserved MMTV RNA dimerization to wild type levels (Figure 5B and Figure 5C). The trans-complementary mutants SA044 and SA045 RNAs were incubated either separately or together, allowing them to interact to initiate intermolecular interactions due to the trans-complementary nature of the sequences. When incubated separately, the mutants displayed a ~ two-fold decrease in dimerization (P <0.001; Figure 5B and Figure 5C), as expected similar to other pal II substitution and deletion mutants (Figures 3B and Figure 3C). Surprisingly, RNA dimerization was not restored when these trans-complementary mutants were co-incubated since the level of dimerization observed for the individual mutants was the same as when the equal amounts of two mutant RNAs were incubated together (two-fold decrease in dimerization; P <0.001; Figure 5B and Figure 5C). This is in sharp contrast with other retroviral systems in which dimerization has been restored to wild type levels in the mutants containing trans-complementary sequences [21,53-55].Figure 5


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)

Effects of pal II substitution mutations on MMTV gRNA dimerization, RNA packaging and viral propagation. (A) Description of the pal II substitution mutants. (B) Typical RNA dimerization in TBM and TB gels of wild type and mutant MMTV RNAs. The last two lanes correspond to the co-incubation of trans-complementary mutants SA044 and SA045. M: monomer lane or monomer conformer; D: dimer lane or dimer conformer. (C) Quantification of the relative RNA dimerization (see table below the histogram). Table above the histogram represent the P values of the respective mutants in TBM and TB gels. The experiments were repeated 2-6 times. (D) Relative packaging efficiency of transfer vector RNAs. (E) RNA propagation efficiency expressed as relative Hygr colony forming unit per ml (CFU/ml). In (C), (D), and (E), the histograms represent data from at least three independent experiments (± SD). The P values for all mutants in panels (D) and (E) were significant (<0.001).
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Related In: Results  -  Collection

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Fig5: Effects of pal II substitution mutations on MMTV gRNA dimerization, RNA packaging and viral propagation. (A) Description of the pal II substitution mutants. (B) Typical RNA dimerization in TBM and TB gels of wild type and mutant MMTV RNAs. The last two lanes correspond to the co-incubation of trans-complementary mutants SA044 and SA045. M: monomer lane or monomer conformer; D: dimer lane or dimer conformer. (C) Quantification of the relative RNA dimerization (see table below the histogram). Table above the histogram represent the P values of the respective mutants in TBM and TB gels. The experiments were repeated 2-6 times. (D) Relative packaging efficiency of transfer vector RNAs. (E) RNA propagation efficiency expressed as relative Hygr colony forming unit per ml (CFU/ml). In (C), (D), and (E), the histograms represent data from at least three independent experiments (± SD). The P values for all mutants in panels (D) and (E) were significant (<0.001).
Mentions: Results in Figure 3 and Figure 4 showed that one of the points of contact between the two RNAs is pal II. To ascertain whether the primary sequence or the palindromic nature of pal II is important to initiate intermolecular interactions between the two RNAs during MMTV gRNA dimerization, we generated several additional mutants. The wild type pal II was substituted with HIV-1 pal flanked by additional purines (5’ AAGCGCGCA 3’; Figure 5A, mutant SA047). Two additional mutants were also created in which the pal II sequence was substituted by non-palindromic trans-complementary sequences (Figure 5A, mutants SA044 and SA045). In all of these mutants, the pal sequence within the PBS was maintained. Consistent with the effect of the purines flanking the pal sequence on HIV-1 RNA dimerization [26], replacing pal II by the HIV-1 pal and the flanking purines (SA047) preserved MMTV RNA dimerization to wild type levels (Figure 5B and Figure 5C). The trans-complementary mutants SA044 and SA045 RNAs were incubated either separately or together, allowing them to interact to initiate intermolecular interactions due to the trans-complementary nature of the sequences. When incubated separately, the mutants displayed a ~ two-fold decrease in dimerization (P <0.001; Figure 5B and Figure 5C), as expected similar to other pal II substitution and deletion mutants (Figures 3B and Figure 3C). Surprisingly, RNA dimerization was not restored when these trans-complementary mutants were co-incubated since the level of dimerization observed for the individual mutants was the same as when the equal amounts of two mutant RNAs were incubated together (two-fold decrease in dimerization; P <0.001; Figure 5B and Figure 5C). This is in sharp contrast with other retroviral systems in which dimerization has been restored to wild type levels in the mutants containing trans-complementary sequences [21,53-55].Figure 5

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