Limits...
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

Bulge and loop mutations in 5’UTR of HIV-1 genomic RNA resulted in reduced association of eEF1A and RT in virus infected cells. a TZM-bl cells were infected with WT, Loop-M, Bulge-M virus as indicated equal to 100 ng CAp24. Association of eEF1A with HIV-1 RTC in virus infected TZM-bl cells was determined by proximity ligation assay using anti-eEF1A and anti-RT antibodies at 2 h of post-infection. A red foci represents a positive signal that was visualized using a DeltaVision Core imaging system and analysis was performed from more than 200 cells. b The levels of genomic RNA at 2 h of post-infection were determined by RT-PCR and possible DNA contamination was examined by PCR without RT
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4525723&req=5

Fig3: Bulge and loop mutations in 5’UTR of HIV-1 genomic RNA resulted in reduced association of eEF1A and RT in virus infected cells. a TZM-bl cells were infected with WT, Loop-M, Bulge-M virus as indicated equal to 100 ng CAp24. Association of eEF1A with HIV-1 RTC in virus infected TZM-bl cells was determined by proximity ligation assay using anti-eEF1A and anti-RT antibodies at 2 h of post-infection. A red foci represents a positive signal that was visualized using a DeltaVision Core imaging system and analysis was performed from more than 200 cells. b The levels of genomic RNA at 2 h of post-infection were determined by RT-PCR and possible DNA contamination was examined by PCR without RT

Mentions: The Bulge-M and Loop-M mutations were introduced into a HIV-1 proviral plasmid DNA and the virus was made by plasmid transfection into HEK293T cells. An equivalent amount of each mutant and wild-type virus, normalised to CAp24 levels, was used to infect TZM-bl cells. Association of eEF1A with HIV-1 RT, a surrogate marker of HIV-1 reverse transcription complex (RTC), was determined by PLA at 2 h post-infection. PLA is a modified fluorescent in situ hybridization method to detect protein-protein interactions that produces fluorescent foci if the two PLA antibodies are in proximity. Heat-inactivated virus, which is defective for viral entry, was used to measure non-specific foci made by the PLA antibodies. There was a significant reduction in the number of foci present in cells infected with the mutant compared to wild type virus (P < 0.01), while inactivated wild type, which are defective for viral entry, virus produced very few foci (Fig. 3a). The levels of virus entry into cells, as determined by measurement of genomic RNA in the cytoplasm at 2 h of post-infection, were similar indicating that reduced levels of eEF1A-RTC association in mutated virus infection were not due to differences in viral entry (Fig. 3b). As expected, heat-inactivated virus sharply reduced levels of viral RNA in cells. The results suggest that the RNA stem-loop structure altered by the mutations is important for interaction between eEF1A and RT.Fig. 3


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)

Bulge and loop mutations in 5’UTR of HIV-1 genomic RNA resulted in reduced association of eEF1A and RT in virus infected cells. a TZM-bl cells were infected with WT, Loop-M, Bulge-M virus as indicated equal to 100 ng CAp24. Association of eEF1A with HIV-1 RTC in virus infected TZM-bl cells was determined by proximity ligation assay using anti-eEF1A and anti-RT antibodies at 2 h of post-infection. A red foci represents a positive signal that was visualized using a DeltaVision Core imaging system and analysis was performed from more than 200 cells. b The levels of genomic RNA at 2 h of post-infection were determined by RT-PCR and possible DNA contamination was examined by PCR without RT
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4525723&req=5

Fig3: Bulge and loop mutations in 5’UTR of HIV-1 genomic RNA resulted in reduced association of eEF1A and RT in virus infected cells. a TZM-bl cells were infected with WT, Loop-M, Bulge-M virus as indicated equal to 100 ng CAp24. Association of eEF1A with HIV-1 RTC in virus infected TZM-bl cells was determined by proximity ligation assay using anti-eEF1A and anti-RT antibodies at 2 h of post-infection. A red foci represents a positive signal that was visualized using a DeltaVision Core imaging system and analysis was performed from more than 200 cells. b The levels of genomic RNA at 2 h of post-infection were determined by RT-PCR and possible DNA contamination was examined by PCR without RT
Mentions: The Bulge-M and Loop-M mutations were introduced into a HIV-1 proviral plasmid DNA and the virus was made by plasmid transfection into HEK293T cells. An equivalent amount of each mutant and wild-type virus, normalised to CAp24 levels, was used to infect TZM-bl cells. Association of eEF1A with HIV-1 RT, a surrogate marker of HIV-1 reverse transcription complex (RTC), was determined by PLA at 2 h post-infection. PLA is a modified fluorescent in situ hybridization method to detect protein-protein interactions that produces fluorescent foci if the two PLA antibodies are in proximity. Heat-inactivated virus, which is defective for viral entry, was used to measure non-specific foci made by the PLA antibodies. There was a significant reduction in the number of foci present in cells infected with the mutant compared to wild type virus (P < 0.01), while inactivated wild type, which are defective for viral entry, virus produced very few foci (Fig. 3a). The levels of virus entry into cells, as determined by measurement of genomic RNA in the cytoplasm at 2 h of post-infection, were similar indicating that reduced levels of eEF1A-RTC association in mutated virus infection were not due to differences in viral entry (Fig. 3b). As expected, heat-inactivated virus sharply reduced levels of viral RNA in cells. The results suggest that the RNA stem-loop structure altered by the mutations is important for interaction between eEF1A and RT.Fig. 3

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