We present evidence that the formation of NP-P and P-L protein complexes is essential for replication of the genome of Sendai defective interfering (DI-H) virus in vitro, using extracts of cells expressing these viral proteins from plasmids. Optimal replication of DI-H nucleocapsid RNA required extracts of cells transfected with critical amounts and ratios of each of the plasmids and was threeto fivefold better than replication with a control extract prepared from a natural virus infection. Extracts in which NP and P proteins were coexpressed supported replication of the genome of purified DI-H virus which contained endogenous polymerase proteins, but extracts in which NP and P were expressed separately and then mixed were inactive. Similarly, the P and L proteins must be coexpressed for biological activity. The replication data thus suggest that two protein complexes, NP-P and P-L, are required for nucleocapsid RNA replication and that these complexes must form during or soon after synthesis of the proteins. Biochemical evidence in support of the formation of each complex includes coimmunoprecipitation of both proteins of each complex with an antibody specific for one component and cosedimentation of the subunits of each complex. We propose that the P-L complex serves as the RNA polymerase and NP-P is required for encapsidation of newly synthesized RNA.

We have developed a cell-free system derived from measles virus-infected cells that supported the transcription and replication of measles virus RNA in vitro. The data suggest that tubulin may be required for these reactions, since an anti-fl-tubulin monoclonal antibody inhibited viral RNA synthesis and the addition of purified tubulin stimulated measles virus RNA synthesis in vitro. Tubulin may be a subunit of the viral RNA polymerase, since two different anti-tubulin antibodies, one specific for the t-and another specific for the ~-subunit of tubulin, coimmunoprecipitated the measles virus L protein as well as tubulin from extracts of measles virus-infected cells. Other experiments further implicated actin in the budding process during virus maturation, as there appeared to be a specific association of actin in vitro only with nucleocapsids that have terminated RNA synthesis, which is presumably a prerequisite to budding.

The nucleocapsid protein (N, 525 amino acids) of measles virus plays a central role in the replication of the viral genomic RNA. Its functions require interactions with itself and with other viral components. The N protein encapsidates genomic RNA, a function reflected in its ability to self-assemble into nucleocapsid-like particles in the absence of other viral proteins. The substrate for the packaging of nascent RNA during RNA replication is a complex between the N and phosphoprotein (P). The domains on the N protein that promote binding to P protein and self-assembly have been identified utilizing a series of N protein deletions. Two noncontiguous regions, amino acids 4-188 and 304-373 of N protein, are required for the formation of the soluble N-P complex, while deletion of amino acids 189-239 did not affect N-P binding. Amino acids 240-303 appear to be necessary for the stability of the protein. The N-terminal 398 amino acids are all required for the formation of organized nucleocapsid-like particles, since deletion of the central region from amino acids 189-373 completely abolished N-N interaction, and deletion of amino acids 4-188 and 374-492 caused the formation of unstructured aggregates.

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The Sendai virus nested set of C proteins which are expressed in an alternative open reading frame from the P mRNA has been shown to downregulate viral RNA synthesis. Utilizing a glutathione S-transferase (gst) C fusion protein (gstC), we have shown that C protein forms a complex with the L, but not the P, subunit of the viral RNA polymerase. When P, L, and gstC are coexpressed, an oligomer of P, through its interaction with L, is also bound to beads. Since binding of C to L in the P-L complex does not disrupt P binding, the C and P binding sites appear to be different. GstC binding to L occurs only when the proteins are coexpressed in the same cell. The gstC, but not gst, protein inhibits viral transcription in vitro, showing that the fusion protein retains biological function. Pulse-chase experiments of the various complexes show that L protein synthesized alone has a half-life of 1. 2 hr, which is increased 12.5-fold by binding P, but is not significantly increased by binding gstC. Analyses of complex formation with truncations of L protein show that the C-terminal 1333 amino acids of L are not required for binding C. The dose-response curves show that replication of the genomic DI-H RNA is more sensitive to inhibition by C protein than is the synthesis of DI leader RNA, suggesting that the downregulation of RNA synthesis may be more complex than just the inhibition of the initiation of RNA synthesis.

The interactions of Sendai virus proteins required for viral RNA synthesis have been characterized both by the yeast two-hybrid system and through the use of glutathione S-transferase (gst)-viral fusion proteins synthesized in mammalian cells. Using the two-hybrid system we have confirmed the previously identified P-L (RNA polymerase), NPo-P (encapsidation substrate), and P-P complexes and now demonstrate NP-NP and NPo-V protein interactions. Expression of gstP and P proteins and binding to glutathione-Sepharose beads as a measure of complex formation confirmed the P-P interaction. The P-gstP binding occurred only on expression of the proteins in the same cell and was mapped to amino acids 345-411. We also show that full-length and deletion gstV and gstW proteins bound NPo protein when these sets of proteins were coexpressed and have identified one required region from amino acids 78-316. Neither gstV nor gstW bound NP assembled into nucleocapsids. Furthermore, both V and W proteins lacking the N-terminal 77 amino acids inhibited DI-H genome replication in vitro, showing the biological relevance of the remaining region. We propose that the specific inhibition of genome replication by V and W proteins occurs through interference with either the formation or the use of the NPo-P encapsidation substrate.

To begin to map functional domains of the Sendai P-L RNA polymerase complex we wanted to characterize the P binding site on the Sendai L protein. Analysis of in vitro and in vivo P-L polymerase complex formation with carboxyl-truncations of the L protein showed that the N-terminal half of the protein was required. Site-directed mutagenesis of the Sendai virus L gene was employed to change amino acids within a highly conserved region of the N-terminal domain I from amino acids (aa) 348-379 singly or in pairs from the Sendai to the corresponding measles L sequence or to alanine. The mutant L proteins coexpressed with the viral P and NP proteins in mammalian cells were assayed for their ability to form the P-L complex and to synthesize RNA in vitro and showed a variety of defective phenotypes. While most of the mutant L proteins still formed the P-L polymerase complex, a change from serine to arginine at aa 368 and a three-amino-acid insertion at aa 379 virtually abolished both complex formation and RNA synthesis. Changes of aas 370 and 376-377 in the L protein gave only small decreases in viral RNA synthesis. Substitutions at either aas 349-350 or aas 354-355 and a three-amino-acid insertion at aa 348 in the L protein yielded enzymes that catalyzed significant transcription, but were defective in DI RNA replication, thus differentially affecting the two processes. Since DI leader RNA, but not genome RNA, was still synthesized by this class of mutants, the defect in replication appears to be in the ability of the mutant enzyme to package newly synthesized nascent RNA. Single changes at aas 362, 363, and 366 in the L protein gave enzymes with severely decreased overall RNA synthesis, although some leader RNA was synthesized, suggesting that they cannot transcribe or replicate past the leader gene. These studies have identified a region in conserved domain I critical for multiple functions of the Sendai virus L protein.

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