In vitro higher-order oligomeric assembly of the respiratory syncytial virus M2-1 protein with longer RNAs

Abstract

The respiratory syncytial virus (RSV) M2-1 protein is a transcriptional antitermination factor crucial for efficiently synthesizing multiple full-length viral mRNAs. During RSV infection, M2-1 exists in a complex with mRNA within cytoplasmic compartments called inclusion body-associated granules (IBAGs). Prior studies showed that M2-1 can bind along the entire length of viral mRNAs instead of just gene-end (GE) sequences, suggesting that M2-1 has more sophisticated RNA recognition and binding characteristics. Here, we analyzed the higher oligomeric complexes formed by M2-1 and RNAs in vitro using size exclusion chromatography (SEC), electrophoretic mobility shift assays (EMSA), negative stain electron microscopy (EM), and mutagenesis. We observed that the minimal RNA length for such higher oligomeric assembly is about 14 nucleotides for polyadenine sequences, and longer RNAs exhibit distinct RNA-induced binding modality to M2-1, leading to enhanced particle formation frequency and particle homogeneity as the local RNA concentration increases. We showed that particular cysteine residues of the M2-1 cysteine-cysteine-cystine-histidine (CCCH) zinc-binding motif are essential for higher oligomeric assembly. Furthermore, complexes assembled with long polyadenine sequences remain unaffected when co-incubated with ribonucleases or a zinc chelation agent. Our study provided new insights into the higher oligomeric assembly of M2-1 with longer RNA.IMPORTANCERespiratory syncytial virus (RSV) causes significant respiratory infections in infants, the elderly, and immunocompromised individuals. The virus forms specialized compartments to produce genetic material, with the M2-1 protein playing a pivotal role. M2-1 acts as an anti-terminator in viral transcription, ensuring the creation of complete viral mRNA and associating with both viral and cellular mRNA. Our research focuses on understanding M2-1's function in viral mRNA synthesis by modeling interactions in a controlled environment. This approach is crucial due to the challenges of studying these compartments in vivo. Reconstructing the system in vitro uncovers structural and biochemical aspects and reveals the potential functions of M2-1 and its homologs in related viruses. Our work may contribute to identifying targets for antiviral inhibitors and advancing RSV infection treatment.

Keywords: M2-1 protein; RNA; electrophoretic mobility shift assays (EMSA); higher-order oligomer; mutagenesis; negative stain electron microscopy (EM); respiratory syncytial virus (RSV); size exclusion chromatography (SEC).

Fig 1

Characterization of M2-1:RNA poly24A Complex through SEC and EM Imaging. (A) Gel filtration elution profile of apo M2-1 (top) and the M2-1 and poly24A complex (bottom) using Superdex 200. The blue and orange lines represent the 280 and 260 nm absorption, respectively. The elution volume of the apo M2-1 is 13.83, and the elution volume of the complex consisting of M2-1 with RNA poly24A is 9.07. (B) SDS-PAGE gel run to analyze the eluate of M2-1 with RNA poly24A complex. Additionally, (C) examines the relative size of the M2-1:RNA complex. Well 1 is the NativeMark Unstained Protein Standard. Well 2 is the M2-1 and RNA poly24A complex; (D) representative negative stain EM image of the apo M2-1 and the M2-1 with RNA poly24A complex.

Fig 2

Gel filtration profiles of M2-1 with different RNA oligos. The elution profiles show the major peaks of the M2-1:RNA complex monitored at an absorbance of 280 nm and 260 nm. The region where the complex is expected to elute has been highlighted with a dotted black box. (A) Elution profiles of M2-1 and different lengths of RNA SH gene end complexes. (B) Elution profiles for M2-1 and the same length of RNA as 16 nt complexes. (C) Elution profiles for M2-1:RNA PolyA complexes of differing lengths smaller than Poly24A. (D) Elution profiles for M2-1:RNA complexes of polyA, polyU, and polyAU (mixed) sequences.

Fig 3

EMSA experiments. (A–D) Presents the results of EMSA experiments, investigating the binding interactions between protein M2-1 and different RNA oligos (Poly24A, Poly19A, Poly14A, and Poly9A) at with gradient changed molecular ratio of M2-1 with RNA. Each panel displays the EMSA results for a specific RNA oligo. The molecular ratio between M2-1 and RNA is indicated above the corresponding lanes, and the final RNA concentration used in the binding reactions is 0.5 µM. Corresponding binding curves with the highest goodness of fit are shown below the gel images. (B–D) The presented fitted curves were determined from one-site specific binding with Hill slope saturation binding analyses. (A) The presented fitted curve was determined from the one-site total and nonspecific saturation binding analysis.

Fig 4

EMSA experiments of M2-1 mutant with RNA. The results of EMSA experiments focus on the binding reactions of various M2-1 mutants RNA poly24A. These experiments were conducted using two specific protein and RNA ratios: 7.4 and 1.8. In figures A–D, the first three wells function as references: well 1 is a negative control, and wells 2 and 3 are positive controls. (A) EMSA results for M2-1 mutants R4A, C7A, and F9A. (B) EMSA results for M2-1 mutants F9Y, H22A, and F23W. (C) EMSA results for M2-1 mutants T91D, K92A, and K150A. (D) EMSA results for M2-1 mutant R151D. (E) Specific cysteine residues of the zinc-binding motif of RSV M2-1 are highlighted (C7, C15, and C21).

Fig 5

Characterization of M2-1:RNA complex by EM imaging and 2D classification. Representative field of M2-1 with RNA poly 19A (A) and poly 24A (B) complex from a micrograph acquired at 92,000 nominal magnifications. The concentration of protein M2-1 was fixed at 10.8 µM, and the protein and RNA complex was made based on the following M2-1:RNA ratios: 15.1, 1.8, and 0.4, respectively. 2D class-averaging images for the M2-1 with RNA poly24A and M2-1 with RNA poly19A complexes are included beside the negative stain images wherein substantial particle formation was observed, as was the case with the 1.8 and 0.4 M2-1:RNA ratios for both the poly19A and poly24A RNA sequences.

Fig 6

The stability analysis of the M2-1:RNA poly24A complex. (A) RNase A digestion was employed to investigate its stability, with negative stain EM imaging performed at 0, 1, and 3 hours at room temperature (RT). (B) examines the RNA digestion for up to 5 hours. The wells labeled 1–13 are further described as follows: 1. M2-1 and RNA poly24A complex; 2. RNA poly24A; 3. NativeMark Unstained Protein Standard; 4. M2-1 and RNA poly24A complex after RNaseA digestion at RT for 1 hour; 5. RNA extraction from the M2-1 and RNA poly24A complex from well 4; 6. M2-1 and RNA poly24A complex after RNaseA digestion at RT for 2 hours; 7. RNA extraction from the M2-1 and RNA poly24A complex from well 6; 8. M2-1 and RNA poly24A complex after RNaseA digestion at RT for 3 hours; 9. RNA extraction from the M2-1 and RNA poly24A complex from well 8; 10. M2-1 and RNA poly24A complex after RNaseA digestion at RT for 4 hours; 11. RNA extraction from the M2-1 and RNA poly24A complex from well 10; 12. M2-1 and RNA poly24A complex after RNaseA digestion at RT for 5 hours; 13. RNA extraction from the M2-1 and RNA poly24A complex from well 12. (C) Examine the complex's stability under varying conditions through EDTA chelation with increasing concentrations of EDTA. The concentration of EDTA is labeled above.

Fig 7

Potential binding modality of M2-1 with short RNA versus longer RNA. The diagram illustrates the potential binding modalities of M2-1 with short and long RNA. (A) When M2-1 binds with short RNA, the entire length of the RNA sequence is bound by a single M2-1, leading to a binding stoichiometry of 1:1. (B) A longer RNA sequence allows for more space and thus allows the binding of multiple M2-1 proteins to a single RNA strand.