Comparison of the amino acid sequences of the N proteins of vesiculoviruses revealed the existence of several conserved regions. The studies presented in this communication focused on a region spanning residues 282 to 291 (GLSSKSPYSS) of the VSV N protein, which is highly conserved among rhabdoviruses and is located in the central region of the molecule. By mutational analysis, we show here that this region plays critical roles in N-P interaction as well as RNA encapsidation functions of the protein. Our results further show that single- amino-acid alterations in this region have dramatic effects on transcription and replication functions of the N-RNA template, suggesting that the N protein, in part, regulates N-RNA template functions.
The crystal structure of the N-RNA complex suggests that this particular domain is located in close proximity to the central cavity of the N molecule that is formed by the amino- and carboxy-terminal lobes of the protein (15
). The central cavity sequesters the viral RNA in the N-RNA complex (15
). Our observation that Y289A mutant N protein does not encapsidate the viral RNA (Fig. ) suggests that this particular residue is possibly involved in maintaining the integrity of the cavity for encapsidating the RNA. Additionally, because this mutation also resulted in the loss of binding of the P protein, it further suggests that the binding site for the P protein is in close proximity to or on the opposite side of the RNA binding cavity. Alternatively, the Y289A mutation may cause misfolding of the protein, resulting in the loss of the P binding site as well as the RNA binding. Thus, a single mutation in this highly conserved region affects the overall structure of the protein in such a manner that at least two major functions of the N protein are impaired. The structural role of the highly conserved region is also consistent with its location in the center of the neck region of the molecule. Alteration of the Y289 residue to alanine might result in exposure of nearby buried residues in the groove, thereby altering the overall conformation, leading to loss of the RNA encapsidation function of the protein. The suggestion that Y289 may be critical for holding up the RNA cavity is further strengthened by the observation that mutation of Y289 to phenylalanine or tryptophan, the two hydrophobic residues with aromatic rings, has no negative effect on RNA encapsidation or the replication or transcription functions of the protein, although replacement with the bulkier tryptophan residue resulted in a reduced or conditional (ts
) phenotype for transcription. Mutants with K, E, or N substituted at this position are most likely too dramatic to fold the protein correctly and therefore do not interact with the P protein or encapsidate the RNA.
Structure of the N-RNA complex of VSV suggests involvement of at least six positively charged residues from the two lobes of the protein whose side chains directly bind the phosphate backbone of RNA (15
). The comparison of the N-RNA structure of VSV with that of rabies virus supports this notion (22
). Our data show that mutation of K286 to alanine resulted in a protein which could still encapsidate the RNA, albeit less efficiently (Fig. ). These results suggest that mutation of K286 to alanine might have resulted in recruitment of additional positively charged residues located in close proximity for RNA encapsidation or that the RNA encapsidation function of the protein may not require the involvement of all the positively charged residues identified in the crystal structure (15
). It would be interesting to examine how many of these positively charged residues located in the RNA binding cavity can be altered without adversely affecting the encapsidation or replication functions of the protein.
Although the K286A mutation in N resulted in less-efficient RNA encapsidation, it was interesting to see that the mutant N protein supported replication consistently at a higher level than the wt N protein. Furthermore, mutation of several residues (S285, S287, P288, and S291) in close proximity to K286 individually to alanine also supported higher levels of replication than the wt N protein (Fig. ). The reason(s) for the hyperactivity of these mutant RNP templates in replication is not known at this time, but it is possible that mutations in this stretch of residues reduced their grips on the RNA in the RNP template such that these template RNAs are more accessible to the viral replicase machinery. However, the same mutations had varied effects on the transcription functions of the RNP template (Fig. ). In this case, it may be possible that the viral transcriptase machinery associates with the RNA template less efficiently or not at all. The differential activity of the same RNP template for replication and transcription suggests that the viral transcription and replication machinery may recognize the RNP template distinctly and further supports the data showing that transcriptase and replicase of VSV are distinct molecular complexes (16
). Furthermore, our results suggest that the structures of the N protein and the resulting N-RNA template, in part, seem to play a significant role in regulating the template functions in transcription and replication. Previous studies using the polR1 mutant of VSV with a single-amino-acid substitution in the N protein (R179H) have suggested that the N-RNA template regulates the viral transcription and replication functions (4
). Our results presented here, and the interpretation that the structure of the N protein regulates the N-RNA template functions, are consistent with those for previous studies. It will be interesting to examine the crystal structures of the N-RNA complexes with these mutations to provide further insights into how the transcriptase and replicase recognize the same template distinctly and execute the transcription and replication activities with such varied efficiencies.
Previous biochemical and mutational studies have shown that the P binding domain in the N protein lies at the carboxy-terminal end of N, including the last 5 residues (11
). Recent structural studies also suggest that residues located in the extended loop of the carboxy-terminal lobe play a role in N-P interaction since deletion of six residues (NΔ347-352) resulted in an N protein that interacted weakly with the P protein (34
). Since we were unable to detect N-P interaction using Y289A mutant in co-IP assays (Fig. ), it is possible that the Y289A mutant does not interact with the P protein at all or interacts very weakly. The Y289A mutant may have misfolded in such a manner that the P binding site and the RNA binding cavity may have collapsed, resulting in loss of P binding and RNA encapsidation. Mutation of the Y289 residue to F or W resulted in N proteins that interacted well with the P protein, suggesting that the hydrophobic and aromatic nature of this residue is critical for N-P interaction and the N protein functions. In this context, it is noteworthy that the serine residue (S290) located adjacent to Y289 in this highly conserved region, when altered to tryptophan (S290W), resulted in an N protein that maintained its interaction with the P protein but lost its RNA encapsidation function (34
). It is possible that this highly conserved region not only forms part of the RNA binding cavity of the N protein but also is critical for the overall structure of the protein such that mutations in this region have negative effects on N-P interactions as well.
Several of the N mutants that generated defective or less competent RNP templates in transcription exhibited the ts
phenotype. At 33°C, the temperature used as permissive in this study, the N-RNA template with the L283A mutant showed transcription activity at a level similar to that for the wt N protein and significantly higher activity than the mutant template at 37°C, indicating that the mutant protein had recovered full activity in transcription at the lower temperature. Other mutants (K286A, Y289W, and S290A) also exhibited the ts
phenotype for transcription, although the level of transcription at the permissive temperature was only about 10 to 15% of that for the wt N protein at the same temperature. These data suggest that structural alterations at the lower temperature result in RNP templates that can be readily recognized by the transcriptase complex to transcribe. It has been shown that the stability of the RNA polymerase-template RNA complex is sensitive to temperature (5
). The low temperature could have restored the stability of the mutant N protein-RNA template-transcriptase complex. However, this was not observed for the Y289A mutant tested, indicating that the specific structural changes of some mutant N-RNA templates due to temperature shiftdown are more favorably recognized by the viral transcriptase machinery for transcription. It should be noted that using the VSV infectious clone (20
), we have inserted the L283A and K286A mutant N protein-coding regions and have recovered these two mutant VSVs at the lower permissive temperature. Initial studies have shown that these viruses exhibit the ts
phenotype for virus growth and overall RNA synthesis. More-detailed studies are being carried out to characterize these viruses and will be reported elsewhere.
Thus, from the results presented in this communication, we conclude that the highly conserved region of the N protein located near the cavity between the two lobes of the protein plays an important role in the overall structure of the protein and that the N-RNA template functions are partly regulated by the structure of the N-RNA complex. The study has identified several key residues whose alterations affect RNA encapsidation, interaction with the P protein, and the N-RNA template functions in transcription and replication.