{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T12:11:57Z","timestamp":1790770317242,"version":"4.1.0"},"reference-count":38,"publisher":"American Society for Microbiology","issue":"1","license":[{"start":{"date-parts":[[2017,1,1]],"date-time":"2017-01-01T00:00:00Z","timestamp":1483228800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000060","name":"HHS | NIH | National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R37 AI059371"],"award-info":[{"award-number":["R37 AI059371"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Virol"],"published-print":{"date-parts":[[2017,1]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            We report an\n            <jats:italic>in vitro<\/jats:italic>\n            RNA synthesis assay for the RNA-dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in\n            <jats:named-content content-type=\"genus-species\">Escherichia coli<\/jats:named-content>\n            and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19 nucleotides (nt) of the rabies virus genome, we demonstrate that L alone initiates synthesis on naked RNA and that P serves to enhance the initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11 to 50 of P and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with those of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P protein in which the oligomerization domain (OD) and the CTD were replaced by those of VSV P stimulated RABV RdRP activity on naked RNA but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis, extending our knowledge of ribonucleoprotein interactions that are critical for gene expression.\n          <\/jats:p>\n          <jats:p>\n            <jats:bold>IMPORTANCE<\/jats:bold>\n            The current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P, information on both the structure and function of the L protein is lacking. This study reports the expression and purification of the full-length L protein of RABV and the characterization of its RdRP activity\n            <jats:italic>in vitro<\/jats:italic>\n            . The study provides a new assay that has utility for screening inhibitors and understanding their mechanisms of action, as well as defining new interactions that are required for RdRP activity.\n          <\/jats:p>","DOI":"10.1128\/jvi.01508-16","type":"journal-article","created":{"date-parts":[[2016,10,20]],"date-time":"2016-10-20T03:24:21Z","timestamp":1476933861000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":40,"title":["An\n            <i>In Vitro<\/i>\n            RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity"],"prefix":"10.1128","volume":"91","author":[{"given":"Benjamin","family":"Morin","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bo","family":"Liang","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erica","family":"Gardner","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robin A.","family":"Ross","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA"},{"name":"Brigham and Women's Hospital, Department of Medicine, Division of Infectious Disease, Boston, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sean P. J.","family":"Whelan","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","first-page":"360","article-title":"Re-evaluating the burden of rabies in Africa and Asia","volume":"83","author":"Knobel DL","year":"2005","unstructured":"Knobel DL Cleaveland S Coleman PG Fevre EM Meltzer MI Miranda ME Shaw A Zinsstag J Meslin FX . 2005. Re-evaluating the burden of rabies in Africa and Asia. Bull World Health Organ 83:360\u2013368.","journal-title":"Bull World Health Organ"},{"key":"e_1_3_2_3_2","volume-title":"Rabies","author":"WHO","year":"2014","unstructured":"WHO. 2014. Rabies. World Health Organization, Geneva, Switzerland. http:\/\/www.who.int\/rabies\/en\/ . 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