title: Silencing and complementation of reovirus core protein mu2: functional correlations with mu2-microtubule association and differences between virus- and plasmid-derived mu2.
abstract: A low-copy component of mammalian reovirus particles is mu2, an 83-kDa protein encoded by the M1 viral genome segment and packaged within the viral core. Previous studies have identified mu2 as a nucleoside triphosphate phosphohydrolase (NTPase) as well as an RNA 5'-triphosphate phosphohydrolase (RTPase), putatively involved in reovirus RNA synthesis and/or 5'-capping. Other studies have identified mu2 as a microtubule-binding protein, which also associates with the viral factory matrix protein muNS and thereby anchors the factories to cellular microtubules during infections by most reovirus strains. To extend studies of mu2 functions during infection, we tested a small interfering RNA (siRNA) directed against the M1 plus-strand RNAs of reovirus strains Type 1 Lang (T1L) and Type 3 Dearing (T3D). The siRNA strongly suppressed mu2 expression by either strain and reduced infectious yields in a strain-dependent manner. This first strain difference was genetically mapped to the M1 genome segment and tentatively assigned to a single mu2 sequence polymorphism, Pro/Ser208, which also determines a T1L-T3D strain difference in microtubule association. The siRNA-based defect in mu2 expression was rescued by plasmids, containing silent mutations in the siRNA-targeted sequence, which encoded either T1L or T3D mu2, but the growth defect was rescued only by T1L mu2. This second strain difference was also mapped to Pro/Ser208, in that swapping this one residue between T1L and T3D mu2 reversed the rescue phenotypes. Thus, the T1L-T3D strain difference in mu2-microtubule association was correlated not only with the extent of reduction in infectious yields by the siRNA but also with the extent of rescue by plasmid-derived mu2. In addition, the rescue capacity of T1L mu2 was abrogated by nocodazole treatment, providing independent evidence for the importance of mu2-microtubule association in plasmid-based rescue. In two separate cases, the results revealed functional differences between virus- and plasmid-derived mu2. Ala substitutions within the NTP-binding motif of T1L mu2 also abrogated its rescue capacity, suggesting that the NTPase or RTPase activity of mu2 is additionally required for effective viral growth.
authors: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors: John
authors: J
authors: Carvalho
authors: Michelle M
authors: MM
authors: Arnold
authors: Max L
authors: ML
authors: Nibert
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D020868:Gene Silencing; D005814:Genes, Viral; D005816:Genetic Complementation Test; D012089:Mammalian orthoreovirus 3 / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D051379:Mice; D008870:Microtubules / Q000378:metabolism / Q000821:virology; D008969:Molecular Sequence Data; D043583:Nucleoside-Triphosphatase / Q000378:metabolism; D030102:Orthoreovirus, Mammalian / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014162:Transfection; D014758:Viral Core Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D034741:RNA, Small Interfering; D014758:Viral Core Proteins; D014764:Viral Proteins; C410663:mu2 protein, Reovirus; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase; D043583:Nucleoside-Triphosphatase