Structural remodeling of the HIV-1 genomic RNA by EMCV IRES insertion impairs viral maturation and fitness

Description

The HIV-1 genomic RNA contains highly structured cis-acting elements that regulate multiple stages of the viral replication cycle, including translation, genome packaging, and particle maturation. Disruption of these RNA structural networks may therefore compromise viral fitness and infectivity. To investigate the impact of large-scale RNA structural remodeling on HIV-1 replication, we analyzed an HIV-1 infectious clone containing the EMCV Internal Ribosome Entry Site (IRES) followed by the gfp reporter gene inserted downstream of nef (NL4-3-IRES). In silico RNA folding analyses predicted extensive global and local conformational changes throughout the viral genomic RNA induced by EMCV IRES insertion. Functionally, NL4-3-IRES exhibited a ~2.5-fold reduction in viral infectivity associated with impaired PR-mediated Gag processing and reduced viral maturation. IRES-containing viral particles displayed decreased incorporation of mature p24-Capsid, Reverse Transcriptase, and Integrase proteins, consistent with defects in viral particle composition. Polysome profiling further revealed altered ribosome association patterns among distinct HIV-1 RNA species. Importantly, similar replication defects were also observed in an independent HIV-1 background. Together, these findings demonstrate that structural remodeling of the HIV-1 genomic RNA induced by EMCV IRES insertion disrupts viral maturation and reduces HIV-1 infectivity, reinforcing the critical role of RNA structural organization in the HIV-1 replication cycle.

Steps to reproduce

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.

Institutions

• Universidade Federal do Rio de Janeiro

  RJ, Rio de Janeiro

Categories

Licence