In this study, by using a series of insertion and deletion mutants, we examined the role of the hypervariable hinge region and the flanking sequences of VSV P protein in viral RNA synthesis and recovery of infectious particles. Our results suggest that some of the sequences in this central region are required for the activity of the protein in viral RNA synthesis. Furthermore, our studies demonstrate that sequences present in this region play a critical role in the assembly and generation of infectious VSV, thus delineating a previously unidentified role for the P protein in the life cycle of this virus.
The hinge region of the P protein links two functionally well-characterized domains of the protein, namely, domains I and II, which have been shown to play pivotal roles in viral RNA transcription and replication, respectively (20
). It is maintained throughout evolution in all vesiculoviruses, though with much flexibility and considerable variability. In addition, compared to the hinge region in VSVNJ
, the region in Chandipura virus is longer and that in VSVI
is shorter (37
). One postulated function of this hypervariable region is that it acts as a spacer to maintain the correct spatial arrangement of the two active domains of the protein (4
). Experimental demonstration using in vitro transcription studies in which the function of the P protein was shown to be affected by alteration of the spatial organization of the two functional domains (I and II) through disruption of the hypervariable region (9
) is consistent with this interpretation. Analogous to the VSV P hinge region, putative spacer-like regions within other transcription-associated proteins with modular structures, e.g., yeast transcriptional activators, have been described (25
Although this hinge region of the P protein shows considerable sequence plasticity, it is not necessarily without any role in the biology of the virus. Our mutational studies provide for the first time a definitive role for this domain in the function of the protein in viral RNA synthesis. Tn-mediated insertions showed that positioning of the insertions at different regions affected the transcription and replication activity of P protein to various extents. Insertions at some positions (amino acids 136 and 180) reduced the replication activities to <20%. The lower activities might be due to the perturbation of the functional domains which might affect their contacts with various components of the replication machinery. It is unlikely that the sequence of the insertion per se is responsible for the observed reduction in transcription and replication, as some of the insertion mutants (PTn196) retained nearly full activities in viral RNA synthesis. Although the activity of the P protein was affected by the insertions, there was no apparent correlation between the level of activity and the position of the insertion in the hinge region relative to either of the functional domains. The reason(s) for this is not clear at this time. It should be noted that although the insertion of 19 amino acids at various positions within the hinge region affected P protein function to various extents, none of the insertion mutants was fully defective. On the other hand, the insertion of larger sequences (for example, enhanced green fluorescent protein) at some of these positions led to completely inactive P protein, whereas functional P proteins could be obtained by the insertion of enhanced green fluorescent protein at other positions (Das and Pattnaik, unpublished data).
We and others have previously shown that phosphorylation in domain I residues is the major determinant of transcription activity of P protein with no significant adverse effects on replication (39
), whereas phosphorylation of domain II residues is required for replication but not for transcription (20
). In the case of the insertion mutants within the hinge region, both replication and transcription functions of the P protein were affected (Fig. ). These results suggest that although domain I and II are involved in discrete functions of the protein, the spatial arrangement of the functional domains relative to each other is important for the overall function of the P protein.
Our results from studies with the deletion mutants not only further strengthened the notion that proper spacing of the domains is important for optimal activity of the P protein but also revealed that sequences within this region are required for its function. Mutants of P protein (PΔ1, PΔ2, and PΔ3) from which 20 amino acids, spanning residues 131 to 150, 151 to 170, or 171 to 190, from the amino-terminal part of the hinge were deleted possessed significant levels of activity, whereas mutants from which 20 or 10 amino acids, spanning residues 191 to 210 or 211 to 220 (as in PΔ4 and PΔ5), from the carboxy-terminal part of the hinge region were deleted resulted in fully defective P protein (Fig. ). From further deletion mapping studies, it became clear that the region between amino acids 140 and 201 is able to tolerate deletions without significant adverse effects on viral RNA synthesis. It is possible that the deletion of residues in the hinge region could bring the functional domains I and II closer to each other and potentially influence the activity of the protein by steric hindrance of the functional domains as has been seen for PΔ1, PΔ2, PΔ3, PΔ6, and PΔ7 mutants. Alternatively, since the activity of a polypeptide is determined by its properly folded structure (46
), the removal of the hinge region may have an adverse effect on the three-dimensional structure of the P protein, resulting in reduced activity. We conclude that the length of the hinge region is critical for optimal functioning of P protein. This observation is consistent with previous results obtained from various in vitro transcription studies in which disruption of the linkage of the P protein functional domains negatively affected the viral polymerase activity (9
The observations that the removal of 20 (residues 191 through 210) or 10 (residues 211 through 220) amino acids from the hinge region led to completely nonfunctional P proteins (PΔ4 and PΔ5), whereas the removal of 60 (residues 141 through 200) amino acids resulted in a functional protein (PΔ7), suggesting that residues 201 through 220 play an important role in the function of the protein. The exact role that this region plays in the function of the P protein is currently a matter of speculation.
The insertion and deletion mutants that are nonfunctional did not show any obvious loss of interactions of P protein with the other known components of the viral replication machinery, namely, the N and the L proteins. This observation is in agreement with previous data, obtained with a yeast two-hybrid system, in which it was shown that the acidic N-terminal domain I and the basic C-terminal domain III of P protein are involved in the interaction with the N protein, whereas the hinge region is not required for the interaction (50
). The L protein binding region on P protein has also been located at the N-terminal acidic domain (17
). It must be emphasized here that the insertion and deletion mutants that maintained their interactions with N and L proteins still lost their transcription activities. It is possible that this region of P protein spanning residues 201 through 220 may be involved in interactions with other unknown components of the viral replication machinery or with other cellular components required for viral RNA synthesis.
The observation that PTn220 acts as a dominant-negative mutant and inhibits the function of the wt P protein is quite interesting. Since PTn220 is not defective in its interaction with the N or L protein, it could therefore compete with Pwt for binding to the replication machinery resulting in the inhibition of viral RNA synthesis. Alternatively, since P protein exists in oligomeric form in infected cells (20
), PTn220 may interact with the wt P protein, forming P oligomers that are inactive in polymerase functions. On the other hand, it is intriguing that PΔ4 and PΔ5 do not function as dominant-negative mutants. Both of these proteins interacted with the N and L proteins and were somewhat expected to function as dominant-negative inhibitors of P protein function. It is possible that these deletion mutants had lost their interactions with the cellular proteins involved in viral RNA synthesis, thereby not affecting the formation of functional viral polymerase complexes. Further studies using these mutants may lead to an understanding of this phenotype.
The adverse effect of the deletion in the hinge region on virus growth is remarkable. The viruses with 20 and 40 amino acids removed from the hinge region (VSV-PΔ2 and VSV-PΔ6) grew to titers that were approximately 10- and 100-fold less, respectively, than that of the wild-type VSV (Fig. ), even though the RNA synthetic functions of the proteins were not adversely affected (Fig. ). What is more interesting is that both mutant viruses exhibited small-plaque morphology. The sizes of the plaques generated with the mutant viruses were significantly smaller than those obtained with wt VSV. The small-plaque phenotype does not appear to depend on the extent on viral macromolecular synthesis, since approximately similar levels of viral proteins and mRNAs were synthesized in infected cells. These results suggest that the extents of viral protein and RNA synthesis are not the sole determinants of viral growth kinetics and plaque size. Other factors, such as localization of viral nucleocapsids at appropriate sites for assembly and interactions of nucleocapsids with other viral proteins involved in viral assembly, in particular, with the M protein may have been affected in VSV-PΔ2 and VSV-PΔ6 viruses, resulting in retarded growth and small-plaque phenotype.
Perhaps one of the most interesting aspects of the present study is the observation that VSV-PΔ7, which has 60 amino acids (residues 141 through 200) deleted from the P protein that was still functional in viral RNA synthesis, was highly debilitated in growth and could not be amplified with multiple passages. Passaging of the virus in cell culture resulted in loss of the virus, even though the virus was initially recovered from the transfected-cell supernatants. We suspected that the mutant P protein may have lost the ability to generate infectious VSV that is competent for multiple rounds of passaging. Initial recovery of VSV-PΔ7 in transfected-cell supernatant may have been due to the presence of wt P protein synthesized from the transfected P plasmid provided in the support plasmid mix. Since the P protein possesses redistributive properties (24
), passaging of the virus may have resulted in dilution of the P protein, eventually leading to the complete loss of wt P protein and therefore, loss of VSV-PΔ7. This contention is supported by data from two experiments. First, VSV-PΔ7 could be amplified to high titers by passaging the virus in cells expressing the wt P protein (Fig. ). Second, when the plasmid encoding PΔ7 was used in the support plasmid mix for recovery of virus, VSV-PΔ7 virus could not be detected in supernatants of transfected cells as examined by immunofluorescent staining of cells infected with the supernatant (data not shown). We conclude from these studies that, in addition to having a role in viral RNA transcription and replication, the P protein also plays a role in viral assembly. These two functions are separable, since the deletion of certain regions led to a protein that is defective in viral assembly while maintaining significantly high levels of RNA synthetic activity. From examination of the deletion mutants used in this study, it appears that the region of P protein spanning amino acids 191 through 200 may be responsible for this new function. The role of P protein in VSV assembly is not known at this time. It may mediate the interaction of the viral nucleocapsid with the M protein either directly or indirectly for the assembly of particles. Alternatively, it may be involved in the transport and proper localization of the viral nucleocapsids to the sites of viral assembly. Further detailed studies will be necessary to provide an understanding of the mechanism of action of the P protein in viral assembly.
In summary, using Tn-mediated insertion mutagenesis and deletion mutagenesis, we have shown that the hinge region of the P protein plays a role in viral RNA synthesis. Furthermore, our studies demonstrate a new function for the viral P protein: it is involved in assembly of infectious VSV particles.