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Binding of the eukaryotic translation elongation factor 1A with the 5'UTR of HIV-1 genomic RNA is important for reverse transcription.

Li D, Wei T, Jin H, Rose A, Wang R, Lin MH, Spann K, Harrich D - Virol. J. (2015)

Bottom Line: Truncation and substitution mutations in the 5'UTR RNA demonstrated that a stem-loop formed by nucleotides 142 to 170, which encompass a reported tRNA anticodon-like-element, binds to eEF1A.Mutations that altered the stem-loop structure by changing two highly conserved sequence clusters in the stem-loop region result in reduction of the interaction with eEF1A in vitro.HIV-1 virus harbouring the same 5'UTR mutations significantly reduced the interaction of eEF1A with HIV-1 reverse transcription complex (RTC), reverse transcription and replication. eEF1A interacts with 5'UTR of HIV-1 genomic RNA and the interaction is important for late DNA synthesis in reverse transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Herston, QLD, 4029, Australia.

ABSTRACT

Background: The cellular protein eukaryotic translation elongation factor 1A (eEF1A) binds to aminoacylated transfer RNAs and delivers them to the ribosome during translation. eEF1A also binds to RNA secondary structures present in genomes of several RNA viruses and plays important roles in their replication. As a RNA binding protein, whether eEF1A can bind with HIV-1 genomic RNA has not been investigated and was the aim of the study.

Methods: RNA-protein interaction was determined by reversible crosslink co-immunoprecipitation (RC-Co-IP) and biolayer Interferometry assay (BLI). eEF1A binding region within RNA was mapped by deletion and mutation analysis. Virus with genomic RNA mutations were examined for eEF1A-RT interaction by proximity ligation assay, for reverse transcription by qPCR and for replication by CAp24 ELISA in cells.

Results: The interaction of eEF1A with 5'UTR of HIV-1 genomic RNA was detected in cells and in vitro. Truncation and substitution mutations in the 5'UTR RNA demonstrated that a stem-loop formed by nucleotides 142 to 170, which encompass a reported tRNA anticodon-like-element, binds to eEF1A. Mutations that altered the stem-loop structure by changing two highly conserved sequence clusters in the stem-loop region result in reduction of the interaction with eEF1A in vitro. HIV-1 virus harbouring the same 5'UTR mutations significantly reduced the interaction of eEF1A with HIV-1 reverse transcription complex (RTC), reverse transcription and replication.

Conclusion: eEF1A interacts with 5'UTR of HIV-1 genomic RNA and the interaction is important for late DNA synthesis in reverse transcription.

No MeSH data available.


Related in: MedlinePlus

The stem-loop formed by nucleotide 142 to 170 in 5’UTR of HIV-1 genomic RNA is important for interaction with eEF1A. a The predicted secondary structure of HIV-1 5’UTR is formed by TAR, polyA, tRNA anticodon-like element (TLE), PBS and stem-loop 1, 2, 3 (SL1, 2, 3). The arrows indicate the sites of truncated RNA. b Biotin labelled wild-type, truncated and mutated 5’UTR RNAs were immobilized on biosensors. The maximum responses (nm) during a 600 s incubation of each biosenor with 90 nM of eEF1A protein using OctetRed system are shown. c The local secondary structures of wild-type, loop and bulge mutated RNAs and their minimum free energy (ΔG) were predicted using Mfold [21]. All data sets are presented as mean ± SD from at least 3 independent experiments and * indicates p < 0.05
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Fig2: The stem-loop formed by nucleotide 142 to 170 in 5’UTR of HIV-1 genomic RNA is important for interaction with eEF1A. a The predicted secondary structure of HIV-1 5’UTR is formed by TAR, polyA, tRNA anticodon-like element (TLE), PBS and stem-loop 1, 2, 3 (SL1, 2, 3). The arrows indicate the sites of truncated RNA. b Biotin labelled wild-type, truncated and mutated 5’UTR RNAs were immobilized on biosensors. The maximum responses (nm) during a 600 s incubation of each biosenor with 90 nM of eEF1A protein using OctetRed system are shown. c The local secondary structures of wild-type, loop and bulge mutated RNAs and their minimum free energy (ΔG) were predicted using Mfold [21]. All data sets are presented as mean ± SD from at least 3 independent experiments and * indicates p < 0.05

Mentions: 5’UTR of HIV-1 genomic RNA contains several well-defined RNA elements with important roles in HIV-1 replication. These elements include TAR, PolyA loop, TLE, PBS, and the stem-loops 1 to 3 (SL1 to SL3) (Fig. 2a) [15, 18]. To better define the eEF1A binding region in the 5’UTR RNA, two truncations of the 5’ UTR RNAs were synthesized. The first truncation was made by removing the stem-loops SL1-SL3 (designated 5’UTR-ΔSL1-3). The second RNA contained only TAR and polyA regions (designated TAR + polyA). The interaction of the truncated 5’UTR RNAs, 5’UTR-ΔSL1-3 and TAR + polyA with eEF1A were examined using BLI assay by immobilizing biotinylated RNAs onto biosensors as described earlier. The 5’UTR-ΔSL1-3 RNA showed a similar binding profile with eEF1A as the intact 5’UTR RNA, and the maximum responses (nm) of the interaction with 90 nM eEF1A is 0.32 nm (no significant difference compared to the intact 5’UTR), whilst the interaction of TAR + polyA with eEF1A was significantly reduced (p < 0.05) (Fig. 2b). The results suggest that RNA sequences after polyA and before SL1-3, which includes the TLE and PBS (nt 106 to 224), are important for optimal interaction with eEF1A.Fig. 2


Binding of the eukaryotic translation elongation factor 1A with the 5'UTR of HIV-1 genomic RNA is important for reverse transcription.

Li D, Wei T, Jin H, Rose A, Wang R, Lin MH, Spann K, Harrich D - Virol. J. (2015)

The stem-loop formed by nucleotide 142 to 170 in 5’UTR of HIV-1 genomic RNA is important for interaction with eEF1A. a The predicted secondary structure of HIV-1 5’UTR is formed by TAR, polyA, tRNA anticodon-like element (TLE), PBS and stem-loop 1, 2, 3 (SL1, 2, 3). The arrows indicate the sites of truncated RNA. b Biotin labelled wild-type, truncated and mutated 5’UTR RNAs were immobilized on biosensors. The maximum responses (nm) during a 600 s incubation of each biosenor with 90 nM of eEF1A protein using OctetRed system are shown. c The local secondary structures of wild-type, loop and bulge mutated RNAs and their minimum free energy (ΔG) were predicted using Mfold [21]. All data sets are presented as mean ± SD from at least 3 independent experiments and * indicates p < 0.05
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4525723&req=5

Fig2: The stem-loop formed by nucleotide 142 to 170 in 5’UTR of HIV-1 genomic RNA is important for interaction with eEF1A. a The predicted secondary structure of HIV-1 5’UTR is formed by TAR, polyA, tRNA anticodon-like element (TLE), PBS and stem-loop 1, 2, 3 (SL1, 2, 3). The arrows indicate the sites of truncated RNA. b Biotin labelled wild-type, truncated and mutated 5’UTR RNAs were immobilized on biosensors. The maximum responses (nm) during a 600 s incubation of each biosenor with 90 nM of eEF1A protein using OctetRed system are shown. c The local secondary structures of wild-type, loop and bulge mutated RNAs and their minimum free energy (ΔG) were predicted using Mfold [21]. All data sets are presented as mean ± SD from at least 3 independent experiments and * indicates p < 0.05
Mentions: 5’UTR of HIV-1 genomic RNA contains several well-defined RNA elements with important roles in HIV-1 replication. These elements include TAR, PolyA loop, TLE, PBS, and the stem-loops 1 to 3 (SL1 to SL3) (Fig. 2a) [15, 18]. To better define the eEF1A binding region in the 5’UTR RNA, two truncations of the 5’ UTR RNAs were synthesized. The first truncation was made by removing the stem-loops SL1-SL3 (designated 5’UTR-ΔSL1-3). The second RNA contained only TAR and polyA regions (designated TAR + polyA). The interaction of the truncated 5’UTR RNAs, 5’UTR-ΔSL1-3 and TAR + polyA with eEF1A were examined using BLI assay by immobilizing biotinylated RNAs onto biosensors as described earlier. The 5’UTR-ΔSL1-3 RNA showed a similar binding profile with eEF1A as the intact 5’UTR RNA, and the maximum responses (nm) of the interaction with 90 nM eEF1A is 0.32 nm (no significant difference compared to the intact 5’UTR), whilst the interaction of TAR + polyA with eEF1A was significantly reduced (p < 0.05) (Fig. 2b). The results suggest that RNA sequences after polyA and before SL1-3, which includes the TLE and PBS (nt 106 to 224), are important for optimal interaction with eEF1A.Fig. 2

Bottom Line: Truncation and substitution mutations in the 5'UTR RNA demonstrated that a stem-loop formed by nucleotides 142 to 170, which encompass a reported tRNA anticodon-like-element, binds to eEF1A.Mutations that altered the stem-loop structure by changing two highly conserved sequence clusters in the stem-loop region result in reduction of the interaction with eEF1A in vitro.HIV-1 virus harbouring the same 5'UTR mutations significantly reduced the interaction of eEF1A with HIV-1 reverse transcription complex (RTC), reverse transcription and replication. eEF1A interacts with 5'UTR of HIV-1 genomic RNA and the interaction is important for late DNA synthesis in reverse transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Herston, QLD, 4029, Australia.

ABSTRACT

Background: The cellular protein eukaryotic translation elongation factor 1A (eEF1A) binds to aminoacylated transfer RNAs and delivers them to the ribosome during translation. eEF1A also binds to RNA secondary structures present in genomes of several RNA viruses and plays important roles in their replication. As a RNA binding protein, whether eEF1A can bind with HIV-1 genomic RNA has not been investigated and was the aim of the study.

Methods: RNA-protein interaction was determined by reversible crosslink co-immunoprecipitation (RC-Co-IP) and biolayer Interferometry assay (BLI). eEF1A binding region within RNA was mapped by deletion and mutation analysis. Virus with genomic RNA mutations were examined for eEF1A-RT interaction by proximity ligation assay, for reverse transcription by qPCR and for replication by CAp24 ELISA in cells.

Results: The interaction of eEF1A with 5'UTR of HIV-1 genomic RNA was detected in cells and in vitro. Truncation and substitution mutations in the 5'UTR RNA demonstrated that a stem-loop formed by nucleotides 142 to 170, which encompass a reported tRNA anticodon-like-element, binds to eEF1A. Mutations that altered the stem-loop structure by changing two highly conserved sequence clusters in the stem-loop region result in reduction of the interaction with eEF1A in vitro. HIV-1 virus harbouring the same 5'UTR mutations significantly reduced the interaction of eEF1A with HIV-1 reverse transcription complex (RTC), reverse transcription and replication.

Conclusion: eEF1A interacts with 5'UTR of HIV-1 genomic RNA and the interaction is important for late DNA synthesis in reverse transcription.

No MeSH data available.


Related in: MedlinePlus