Impact of IRES sequence insertrion in the HIV-1 genome
Description
The HIV-1 genome contains several RNA structures involved with viral genomic RNA packaging; which is directly related with viral maturation . Thus, mutations leading to structural alterations in the viral genomic RNA have the potential to impact these processes and consequently impair viral infectivity. The insertion of a highly structured RNA element at the 3’-end of the HIV-1 genome may cause deleterious alterations or hide signalling sequences important for viral replication. To characterise this further, we took advantage of an HIV-1 infectious clone harbouring a ECMV Internal Ribosome Entry Site (IRES) sequence followed by the gfp reporter gene introduced downstream of the nef gene (NL4-3-IRES). In cells, NL4-3-IRES was 2.5-fold less infectious than the wild type NL4-3 and had a 2-fold reduction in Gag processing. Mature particles showed less incorporation of p24-Capsid, Reverse Transcriptase, and Integrase proteins within NL4-3-IRES viral particles. In silico molecular modeling analysis revealed that the genomic RNA of NL4-3-IRES had several conformational changes compared with the wild type NL4-3 sequence, especially in regions related to viral maturation, demonstrating that the presence of an IRES element within the 3’ end of the HIV-1 genome has profound impacts on virus assembly and maturation and consequently reduced viral infectivity. This dataset consists in Western blot, RT-qPCR, ELISA and infectivity assay results. Also, results from a iodixanol gradiente, in which each fraction was measured by optical density, can be found. The data can be interpreted by comparing the values of the measuring units from each assay or experiment. The secondary structure of the genome of wild type HIV-1, depleted of not fo Nef gene and with or without IRES sequence, was solved. It can be interpreted by comparing the differences, both local and global, in their structures.
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Plasmids Plasmids used: pNL4-3 and pNL4-3-ΔNef, pBR-NL4-3-IRES and pBR-NL4-3-ΔNef-IRES-eGFP, pNLHX-PLAP_N+ and pNLHX-PLAP_N-. Viral quantification The amount of p24 protein in the viral supernatant was quantified by ELISA assay, using the HIV-1 p-24 Antigen ELISA kit. TZM-bl cells were plated in 96-well plates with 2,0 x 106. Infected TZM-bl cells were incubated at 37°C and 5% CO2 for 48 hours. After that, cells were fixed with 1% formaldehyde and incubated with X-Gal substrate at 37°C and 5% CO2 for 24h hours. The viral titer was measured by blue spots counting. Iodixanol Gradient With a 1,2% difference in the iodixanol concentration, 8 fractions (from 9,6% up to 18%), with 1mL each, were carefully placed in crescent order in an SW41Ti rotor tube. The viral supernatant, in a total of 2,0 mL, was added to the top of the gradient and then, centrifugated by 1 hour at 30,000 rpm. After that, 9 fractions of 1,0 mL were collected and submitted to protein precipitation with trichloroacetic acid (TCA). 750µL of TCA was added to 200µL of supernatant (ratio of 1:4) and then, incubated for 10 minutes at 4°C followed by centrifugation at 14,000 x g for 5 minutes. The resulted pellet was washed three times with 200 µL of acetone 100% at 4°C and centrifuged at 14,000 x g at 4°C for 5 minutes. The final pellet was heated at 95°C and stoked at -80°C. Polysome Gradient Five different sucrose concentration, from 7% up to 47%, was diluted in polysome buffer. The gradient was incubated at 4ºC for 24 hours. The cell was lysate with 400 µL of polysome lysing buffer and incubated at 4ºC for 5 minutes. The cell lysate was homogenized with a syringe coupled to an 8 x 0,3 mm needle, followed by a 5-minute incubation at 4ºC. Next, the sample was centrifugated at 300 xg for 5 minutes at 4°C, and the supernatant was added to the sucrose gradient. The gradient was centrifuged for 3 hours at 35000 rpm at 4°C, using the SW41Ti rotor. After centrifugation, 1,0 mL of samples were collected and stoked at -80°C. Western Blot The primary antibodies α-p24, α-Nef, α-Env, α-RT, α-IN, were used to detected HIV-1 proteins. α-GAPDH and α-β-actin were used as a loading control. Polymerase chain reaction The cDNA synthesis was conducted using the High-Capacity Reverse Transcription kit with 2,0 µL of total RNA. The real-time polymerase chain reaction was conducted to amplify and measure the full-length viral mRNA using Power SYBR Green Master Kit. RNA structure Modeling The secondary RNA structure of the entire HIV-1 genome was solved using the CLC Main Workbench 8 (QIAGEN). For settings parameters, the non-paring base permanence was allowed. Statistical Analyzes Statistical analyzes were performed using GraphPad Prism 8® software, with One-way Anova tests for multiple comparisons and a T-test for double comparisons. The differences were considered statistically significant when p <0.05.