Molecular phylogeny of the extinctcave lion Panthera leo spelaea

To reconstruct the phylogenetic position of the extinct cave lion (Panthera leo spelaea), we sequenced 1 kb of the mitochondrial cytochrome b gene from two Pleistocene cave lion DNA samples (47 and 32 ky B.P.). Phylogenetic analysis shows that the ancient sequences form a clade that is most closely related to the extant lions from Africa and Asia; at the same time, cave lions appear to be highly distinct from their living relatives. Our data show that these cave lion sequences represent lineages that were isolated from lions in Africa and Asia since their dispersal over Europe about 600 ky B.P., as they are not found among our sample of extant populations. The cave lion lineages presented here went extinct without mitochondrial descendants on other continents. The high sequence divergence in the cytochrome b gene between cave and modern lions is notable

Intracellular Localization of the Peanut Clump Virus Replication Complex in Tobacco BY-2 Protoplasts Containing Green Fluorescent Protein-Labeled Endoplasmic Reticulum or Golgi Apparatus

RNA-1 of Peanut clump virus (PCV) encodes the proteins P131 and P191, containing the signature motifs of replication proteins, and P15, which regulates viral RNA accumulation. In PCV-infected protoplasts both P131 and P191 were immunodetected in the perinuclear region. Laser scanning confocal microscopy (LSCM) showed that P131 and P191 colocalized with neosynthesized 5-bromouridine 5′-triphosphate-labeled RNA and double-stranded RNA, demonstrating that they belong to the replication complex. On the contrary, the P15 fused to the enhanced green fluorescent protein (EGFP) never colocalized with the two proteins. In endoplasmic reticulum (ER)-GFP transgenic BY-2 protoplasts, the distribution of the green fluorescent-labeled ER was strongly modified by PCV infection. LSCM showed that both P131 and P191 colocalized with ER green fluorescent bodies accumulating around the nucleus during infection. The replication process was not inhibited by cerulenin and brefeldin A, suggesting that PCV replication does not depend on de novo-synthesized membrane and does not require transport through the Golgi apparatus. Electron microscopy of ultrathin sections of infected protoplasts showed aggregates of broken ER but also visualized vesicles, some of which resembled modified peroxisomes. The results suggest that accumulation of PCV during infection is accompanied by specific association of PCV RNA-1-encoded proteins with membranes of the ER and other organelles. The concomitant extensive rearrangement of these membranous structures leads to the formation of intracellular compartments in which synthesis and accumulation of the viral RNA occur in defined areas

Functional characterization of the proteolytic activity of the tomatoblack ring nepovirus RNA-1-encoded polyprotein

AbstractTranslation of tomato black ring virus (TBRV) RNA-1 in a rabbit reticulocyte lysate leads to the synthesis of a 250Kpolyprotein which cleaves itself into smaller proteins of 50, 60, 120, and 190K. Polypeptides synthesized from synthetic transcripts corresponding to different regions of TBRV RNA-1 are processed only when they encode the 23K protein delimited earlier by sequence homology with the cowpea mosaic virus 24K protease. The proteolytic activity of this protein is completely lost by mutating residues C170 (to I) or L188 (to H), residues which align with conserved residues of the viral serine-like proteases. The 120K protein is generated by cleavage of the dipeptide K/A localized in front of the VPg but is not further cleaved in vitro at the K/S site (at the C terminus of the VPg) or between the protease and polymerase domains. However, both the protein VPgProPol (120K) and the protein ProPol (117K) produced in vitro from synthetic transcripts can cleave in trans the RNA-2-encoded 150K polyprotein, but they cannot cleave in trans polypeptides containing a cleavage site expressed from RNA-1 transcripts in which the protease cistron is absent or modified