title: The N terminus of Andes virus L protein suppresses mRNA and protein expression in mammalian cells.
abstract: Little is known about the structure and function of the 250-kDa L protein of hantaviruses, although it plays a central role in virus genome transcription and replication. When attempting to study Andes virus (ANDV) L protein in mammalian cells, we encountered difficulties. Even in a strong overexpression system, ANDV L protein could not be detected by immunoblotting. Deletion analysis revealed that the 534 N-terminal amino acid residues determine the low-expression phenotype. Inhibition of translation due to RNA secondary structures around the start codon, rapid proteasomal degradation, and reduced half-life time were excluded. However, ANDV L protein expression could be rescued upon mutation of the catalytic PD-E-K motif and further conserved residues of the putative endonuclease at the N terminus of the protein. In addition, wild-type ANDV L rather than expressible L mutants suppressed the level of L mRNA, as well as reporter mRNAs. Wild-type L protein also reduced the synthesis of cellular proteins in the high-molecular-weight range. Using expressible ANDV L mutants as a tool for localization studies, we show that L protein colocalizes with ANDV N and NSs but not Gc protein. A fraction of L protein also colocalized with the cellular processing (P) body component DCP1a. Overall, these data suggest that ANDV L protein possesses a highly active endonuclease at the N terminus suppressing the level of its own as well as heterologous mRNAs upon recombinant expression in mammalian cells.
authors.affiliation: Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Heinemann
authors.forename: Jonas
authors.initials: J
authors.lastname: Schmidt-Chanasit
authors.forename: Stephan
authors.initials: S
authors.lastname: Günther
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D015536:Down-Regulation; D004720:Endonucleases; D005786:Gene Expression Regulation; D006476:Orthohantavirus; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011506:Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014764:Viral Proteins; C500570:L protein, hantavirus; D012324:RNA-Dependent RNA Polymerase; D004720:Endonucleases