The structural basis of recognition and removal of cellular mRNA 7-methyl G 'caps' by a viral capsid protein: a unique viral response to host defense

The single segment, double-stranded RNA genome of the L-A virus (L-A) of yeast encodes two proteins: the major coat protein Gag (76 kDa) and the Gag-Pol fusion protein (180 kDa). The icosahedral L-A capsid is formed by 120 copies of Gag and has architecture similar to that seen in the reovirus, blue tongue virus and rice dwarf virus inner protein shells. Gag chemically removes the m7GMP caps from host cellular mRNAs. Previously we identified a trench on the outer surface of Gag that included His154, to which caps are covalently attached. Here we report the refined L-A coordinates at 3.4 angstroms resolution with additional structural features and the structure of L-A with bound m7GDP at 6.5 angstroms resolution, which shows the conformational change of the virus upon ligand binding. Based on site-directed mutations, residues in or adjacent to the trench that are essential (or dispensable) for the decapping reaction are described here. Along with His154, the reaction requires a cluster of positive charge adjoining the trench and residues Tyr 452, Tyr150 and either Tyr or Phe at position 538. A tentative mechanism for decapping is proposed.     

Zinc- and sequence-dependent binding to nucleic acids by the N-terminal zinc finger of the HIV-1 nucleocapsid protein: NMR structure of the complex with the Psi-site analog, dACGCC.

The nucleic acid interactive properties of a synthetic peptide with sequence of the N-terminal CCHC zinc finger (CCHC = Cys-X2-Cys-X4-His-X4-Cys; X = variable amino acid) of the human immunodeficiency virus (HIV) nucleocapsid protein, Zn(HIV1-F1), have been studied by 1H NMR spectroscopy. Titration of Zn(HIV1-F1) with oligodeoxyribonucleic acids containing different nucleotide sequences reveals, for the first time, sequence-dependent binding that requires the presence of at least one guanosine residue for tight complex formation. The dynamics of complex formation are sensitive to the nature of the residues adjacent to guanosine, with residues on the 3' side of guanosine having the largest influence. An oligodeoxyribonucleotide with sequence corresponding to a portion of the HIV-1 psi-packaging signal, d(ACGCC), forms a relatively tight complex with Zn(HIV1-F1) (Kd = 5 x 10(-6) M). Two-dimensional nuclear Overhauser effect (NOESY) data indicate that the bound nucleic acid exists predominantly in a single-stranded, A-helical conformation, and the presence of more than a dozen intermolecular NOE cross peaks enabled three-dimensional modeling of the complex. The nucleic acid binds within a hydrophobic cleft on the peptide surface. This hydrophobic cleft is defined by the side chains of residues Val1, Phe4, Ile12, and Ala13. Backbone amide protons of Phe4 and Ala13 and the backbone carbonyl oxygen of Lys2 that lie within this cleft appear to form hydrogen bonds with the guanosine O6 and N1H atoms, respectively. In addition, the positively charged side chain of Arg14 is ideally positioned for electrostatic interactions with the phosphodiester backbone of the nucleic acid. The structural findings provide a rationalization for the general conservation of these hydrophobic and basic residues in CCHC zinc fingers, and are consistent with site-directed mutagenesis results that implicate these residues as direct participants in viral genome recognition.     

The L1 family of long interspersed repetitive DNA in rabbits: Sequence,copy number,conserved open reading frames,and similarity to keratin

Summary The L1 family of long interspersed repetitive DNA in the rabbit genome (L1Oc) has been studied by determining the sequence of the five L1 repeats in the rabbit -like globin gene cluster and by hybridization analysis of other L1 repeats in the genome. L1Oc repeats have a common 3 end that terminates in a poly A addition signal and an A-rich tract, but individual repeats have different 5 ends, indicating a polar truncation from the 5 end during their synthesis or propagation. As a result of the polar truncations, the 5 end of L1Oc is present in about 11,000 copies per haploid genome, whereas the 3 end is present in at least 66,000 copies per haploid genome. One type of L1Oc repeat has internal direct repeats of 78 bp in the 3 untranslated region, whereas other L1Oc repeats have only one copy of this sequence. The longest repeat sequenced, L1Oc5, is 6.5 kb long, and genomic blot-hybridization data using probes from the 5 end of L1Oc5 indicate that a full length L1Oc repeat is about 7.5 kb long, extending about 1 kb 5 to the sequenced region. The L1Oc5 sequence has long open reading frames (ORFs) that correspond to ORF-1 and ORF-2 described in the mouse L1 sequence. In contrast to the overlapping reading frames seen for mouse L1, ORF-1 and ORF-2 are in the same reading frame in rabbit and human L1s, resulting in a discistronic structure. The region between the likely stop codon for ORF-1 and the proposed start codon for ORF-2 is not conserved in interspecies comparisons, which is further evidence that this short region does not encode part of a protein. ORF-1 appears to be a hybrid of sequences, of which the 3 half is unique to and conserved in mammalian L1 repeats. The 5 half of ORF-1 is not conserved between mammalian L1 repeats, but this segment of L1Oc is related significantly to type II cytoskeletal keratin.