{"doi":"10.1128/jvi.75.17.7791-7802.2001","title":"Genetic Analysis of the Pestivirus Nonstructural Coding Region: Defects in the NS5A Unit Can Be Complemented in<i>trans</i>","abstract":"<jats:title>ABSTRACT</jats:title><jats:p>The functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5′ and 3′ untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act in<jats:italic>cis</jats:italic>or in<jats:italic>trans</jats:italic>, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued in<jats:italic>trans</jats:italic>, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to be<jats:italic>cis</jats:italic>dominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of the pestivirus replication machinery.</jats:p>","journal":"Journal of Virology","year":2001,"id":35004,"datarank":3.273466498745562,"base_score":4.248495242049359,"endowment":4.248495242049359,"self_citation_contribution":0.637274286307404,"citation_network_contribution":2.636192212438158,"self_endowment_contribution":0.637274286307404,"citer_contribution":2.636192212438158,"corpus_percentile":null,"corpus_rank":null,"citation_count":69,"citer_count":56,"citers_with_citation_signal":52,"citers_with_endowment":52,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":150716,"name":"Olaf Isken","orcid":null,"position":1,"is_corresponding":false},{"id":180211,"name":"Norbert Tautz","orcid":null,"position":2,"is_corresponding":false},{"id":150717,"name":"Sven-Erik Behrens","orcid":null,"position":3,"is_corresponding":false},{"id":150715,"name":"Claus W. Grassmann","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"base_score":4.248495242049359,"endowment":4.248495242049359,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"11483722","pmcid":null,"openalex_id":"https://openalex.org/W2118283523","authors":[],"funders":[],"total_grants":0,"fwci":10.7173,"citation_percentile":0.98695385,"influential_citations":2,"citation_trend":[{"year":2012,"count":2},{"year":2013,"count":3},{"year":2014,"count":3},{"year":2015,"count":3},{"year":2016,"count":2},{"year":2017,"count":1},{"year":2018,"count":2},{"year":2021,"count":2},{"year":2023,"count":2},{"year":2025,"count":2}],"oa_status":"closed","license":"https://journals.asm.org/non-commercial-tdm-license","oa_locations":[{"url":"https://europepmc.org/articles/pmc115021?pdf=render","host_type":"GREEN"},{"url":"https://journals.asm.org/doi/pdf/10.1128/JVI.75.17.7791-7802.2001","host_type":"publisher"},{"url":"https://doi.org/10.1128/jvi.75.17.7791-7802.2001","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/11483722","host_type":"repository"},{"url":"https://www.ncbi.nlm.nih.gov/pmc/articles/115021","host_type":"repository"}],"fields_of_study":["Animal Disease Management and Epidemiology","Viral Infections and Immunology Research","Animal Virus Infections Studies","Biology","Medicine","Animals","Bovine Virus Diarrhea-Mucosal Disease","Cattle","Cell Line","Diarrhea Viruses, Bovine Viral","Genetic Complementation Test","Mutation","Open Reading Frames","Protein Biosynthesis","Replicon","Reverse Transcriptase Polymerase Chain Reaction","Transfection","Viral Nonstructural Proteins","Virus Replication"],"mesh_terms":["Animals","Diarrhea Viruses, Bovine Viral","Bovine Virus Diarrhea-Mucosal Disease","Cattle","Cell Line","Genetic Complementation Test","Mutation","Replicon","Transfection","Protein Biosynthesis","Virus Replication","Open Reading Frames","Viral Nonstructural Proteins","Reverse Transcriptase Polymerase Chain Reaction"],"keywords":["Biology","Replicon","NS5B","NS3","Subgenomic mRNA","Open reading frame","Viral replication","Pestivirus","Virology","Genetics","NS5A","RNA","NS2-3 protease","RNA-dependent RNA polymerase","Virus","Gene","Genome","Hepatitis C virus","Peptide sequence","Flaviviridae","Hepacivirus"],"sdg_mappings":[],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-06-09T23:09:02.538138Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}