ena-STUDY-Institute of Molecular Genetics of the ASCR, v. v. i.-05-11-2018-19:26:06:546-934
New insight into RNA composition of the HIV-1 virion
The techniques for studies of chemical structure of RNA such as LC/MS or methylation profilingemploying deep sequencing have been discovered only recently and they have not been applied toviral RNA. We focused on HIV-1 viral particles. Here, we present the first employment of liquidchromatography- mass spectrometry (LC/MS) analysis of RNA from viral particle. This directanalytical method allowed us to reveal the real amount of host tRNAs co-packed in HIV-1 particle.After isolation of viral particles and isolation of RNA from viral packageome, the RNA wasenzymatically digested into form of nucleosides and analysed. Surprisingly, high amount of 1-methyladenosine (m 1 A) in RNA digest led us to preparation of four deep sequencing libraries withparticular focus on m 1 A profiling. The known amount of co-packed tRNA species (containing one m 1 Aper molecule) did not correspond to the detected level of m 1 A in LC/MS analysis. Deep sequencingdata confirmed the presence of m 1 A in tRNA but did not bring any evidence of m 1 A in genomic HIV-1RNA neither in the most abundant guiding 7SL RNA. The employment of new technique (LC/MS) forstudies of RNA allow us new insight into viral particle and here we bring evidence that per 2molecules of genomic RNA, 14 copies of 7SL RNA the HIV-1 particle contains approximately 322tRNA copies (4.6 fold more than expected).
The techniques for studies of chemical structure of RNA such as LC/MS or methylation profilingemploying deep sequencing have been discovered only recently and they have not been applied toviral RNA. We focused on HIV-1 viral particles. Here, we present the first employment of liquidchromatography- mass spectrometry (LC/MS) analysis of RNA from viral particle. This directanalytical method allowed us to reveal the real amount of host tRNAs co-packed in HIV-1 particle.After isolation of viral particles and isolation of RNA from viral packageome, the RNA wasenzymatically digested into form of nucleosides and analysed. Surprisingly, high amount of 1-methyladenosine (m 1 A) in RNA digest led us to preparation of four deep sequencing libraries withparticular focus on m 1 A profiling. The known amount of co-packed tRNA species (containing one m 1 Aper molecule) did not correspond to the detected level of m 1 A in LC/MS analysis. Deep sequencingdata confirmed the presence of m 1 A in tRNA but did not bring any evidence of m 1 A in genomic HIV-1RNA neither in the most abundant guiding 7SL RNA. The employment of new technique (LC/MS) forstudies of RNA allow us new insight into viral particle and here we bring evidence that per 2molecules of genomic RNA, 14 copies of 7SL RNA the HIV-1 particle contains approximately 322tRNA copies (4.6 fold more than expected).