Tubulin acts as a positive transcription factor for in vitro RNA synthesis by two different negative-strand viruses: Sendai virus, a paramyxovirus; vesicular stomatitis virus (VSV), a rhabdovirus. A monoclonal antibody directed against f3-tubulin completely inhibited not only mRNA synthesis and RNA replication catalyzed in vitro by extracts of cells infected with either virus but also mRNA synthesis by detergent-disrupted purified vrions. The synthesis of both a leaderlike RNA and the NP mRNA directed by detergent-disrupted purified Sendai virions was shown to be totally dependent on the addition of purified tubulin. The addition of purified tubulin, although not required, also stimulated mRNA synthesis directed by detergent-disrupted VSV vrions 2-to 7-fold.Finally, there appears to be an association between tubulin and the L protein of VSV, since both monoclonal and polyclonal anti-tubulin antisera specifically immunoprecipitated not only tubulin but also the L protein of two different VSV serotypes from the soluble protein fraction of infected cells.Two different groups of negative-strand RNA viruses, the rhabdoviruses [vesicular stomatitis virus (VSV)] and the paramyxoviruses (Sendai virus) share similar strategies for their reproduction (1, 2). Each virus carries within the virion an RNA-dependent RNA polymerase, composed ofthe L and NS subunits for VSV or the L and P subunits for Sendai virus (3,4). When purified virus is treated with detergent, the disrupted virions transcribe first a leader RNA and then the mRNAs sequentially from the nucleocapsid RNA template (5-7). After viral mRNA and protein synthesis in the infected cell, the negative-strand genome RNA of either virus is replicated through a positive-strand complementary RNA intermediate (1,2). Both the positive-and negative-genome length RNAs are encapsidated by the N (VSV) or NP (Sendai virus) proteins to form nucleocapsid structures concomitant with the synthesis of their respective genome RNAs. We have developed cell-free systems for VSV (8) and for Sendai virus (9) that support both the faithful transcription and replication of the genome RNAs of the wild-type viruses and the replication ofthe corresponding defective-interfering (DI) viruses under conditions of mixed infection.In this communication, we have exploited these in vitro systems to identify any host cell proteins that may be required in the process of viral RNA synthesis. It is known that the addition of extracts of uninfected cells will stimulate transcription directed by VSV or Sendai virus (5, 10, 11). We report here that tubulin appears to be one host protein that can act as a positive transcription factor for both of these viruses. We show that the addition of a monoclonal antibody directed against A-tubulin completely inhibited all viral RNA synthesis in extracts of cells infected with either VSV or Sendai virus. The addition of purified tubulin, moreover, was required for the in vitro synthesis of Sendai virus leader-like RNA and NP mRNA from detergent-treated virions...

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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.