Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity

Figure 1. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reactivity-constrained lowest energy secondary structure of the 636-nt Rous sarcoma virus (RSV) 5′-leader RNA. The secondary structure model was generated by applying averaged normalized SHAPE reactivity from three independent trials as pseudo free-energy constraints in RNAstructure [53]. Nucleotides are colored in accordance to SHAPE reactivity as indicated in the key. Nucleotides that could not be analyzed due to primer annealing (3′ end) or the lack of resolution (5′ end) are shown in grey. MΨ is indicated by a black dashed box. Stem loops in µΨ and loops that were tested in the dimerization assays (DIS-0, DIS-1/L3, DIS-2/SL-A, SL-B and SL-C) are labeled. The AUG start codon is indicated by a purple box.

Figure 2. (A) RNA constructs used in the dimerization studies. Sequences in the regions containing DIS-2/SL-A, DIS-1/L3 and DIS-0 are shown. Nucleotides in the loop regions are underlined. Tetraloop mutations are highlighted in red. (B) Native agarose gel showing the results of dimerization assays of the wild-type (WT) RSV 5′-leader and dimerization initiation signal (DIS) loop mutants. RNAs (0.5 μM) were folded under low salt (L) or under high salt (H), as described in the Materials and Methods. Locations for monomeric (M) and dimeric RNA bands (D) are indicated. This gel shown is representative of three independent replicates. (C) Native agarose gel showing the results of dimerization assays of the WT RSV 5′-leader and more extensive stem loop deletion mutants. The conditions are the same as panel (B). This gel shown is representative of two independent replicates.

Figure 3. (A) Schematic of the constructs used in the infectivity studies. (B) Immunoblots of Gag in 50 µg of lysates collected from QT6 cells transfected with RC.pAT.V8 WT, RC.pAT.V8 ∆DIS-2, RC.pAT.V8 ∆DIS-1, RC.pAT.V8 DIS-DM or RC.V8 Bal31 mCherry. Lysates were collected in RIPA at 3 and 6 dpt. (C) Immunoblots of virions collected from QT6 cells transfected with RC.pAT.V8 WT, RC.pAT.V8 ∆SL-A, RC.pAT.V8 ∆L3, RC.pAT.V8 ∆L3+SL-A and RC.V8 Bal31 mCherry (top panel, 2 dpt), and immunoblots of Gag in 50 µg of lysates collected from QT6 cells infected with viruses produced from the transfected cells (bottom panel, 3, 6, 14 and 17 dpi).

Figure 4. Crosslinking (XL)-SHAPE analysis of RSV Gag∆PR binding to the RSV 5′-leader RNA. (A) Schematic showing individual domains of RSV Gag and RSV Gag∆PR. (B) XL-SHAPE results mapped to the secondary structure of the RSV 5′-leader RNA. Sites with decreased and increased SHAPE reactivity upon protein binding are indicated by green and red arrows, respectively. Identified crosslinking sites are labeled with stars. All identified sites have reactivity changes of ≥0.3 and p < 0.05 based on unpaired, two-tailed student t-tests, compared with the no protein control. The MΨ region (dashed box) and PBS are indicated. Results are based on the average of at least 3 independent experiments.

Figure 5. (A) Secondary structure of RSV MΨ. Six mutant MΨ constructs tested are indicated by red boxes and black arrows. Additional nucleotides that were added to facilitate in vitro transcription are shown in grey. (B,C) Bar graphs of Kd,1M (B) and Zeff (C) values determined by fluorescence anisotropy (FA) salt titration binding assays using RSV Gag∆PR and RSV 167, MΨ-WT and six MΨ mutants. Values of three to four trials are indicated by black dots and the average is indicated by the height of the bar. p values were determined in comparison with MΨ-WT using unpaired, two-tailed student t-tests. (***) p < 0.001; (**) p < 0.01; (*) p < 0.05; (n.s.) not significant.