Abstract APOBEC3G (A3G), a host protein that inhibits HIV-1 reverse transcription and replication in the absence of Vif, displays cytidine deaminase single-stranded (ss) nucleic acid binding activities. nucleocapsid (NC) also binds acids has unique property, chaperone activity, which is crucial for efficient transcription. Here we report interplay between A3G, NC transcriptase (RT) effect highly purified A3G on individual reactions occur during We find did not affect kinetics NC-mediated...

APOBEC3G (APO3G), a cytidine deaminase with two zinc finger domains, inhibits human immunodeficiency virus type 1 replication in the absence of Vif. Here, we provide comprehensive molecular analysis and nucleic acid binding activities APO3G using pure system containing only one protein component, i.e., highly purified, catalytically active enzyme expressed baculovirus system. We demonstrate that deaminates cytosines single-stranded DNA (ssDNA) only, whereas it binds efficiently to ssDNA...

During the first strand transfer in reverse transcription, minus-strand strong-stop DNA [(-) SSDNA] is annealed to 3' end of acceptor RNA a reaction mediated by base-pairing between terminal repeat sequences and their complement DNA. The large stem-loop structure region known as TAR could interfere with this annealing reaction. We have developed an vitro human immunodeficiency virus type 1 (HIV-1) system investigate effect on transfer. Mutational analysis demonstrates that presence donor...

Many properties of the RNA code which were discussed at 1963 Cold Spring Harbor meeting based on information obtained with randomly ordered synthetic polynucleotides. Most questions concerning raised that time related to its fine structure, is, order bases within codons. After meetings a relatively simple means determining nucleotide sequences codons was devised depends upon ability trinucleotides known sequence stimulate AA-sRNA binding ribosomes (Nirenberg and Leder, 1964). In this paper,...

Production of particles with the ultrastructural appearance C-type virions persisted for at least 6 h in actinomycin D-treated cells infected murine leukemia virus. This phenomenon occurred despite severe inhibition viral RNA synthesis. Virus present a 6-h harvest sedimented sucrose gradients buoyant density characteristic tumor viruses (1.16 g/cm(3)) and exhibited high levels reverse transcriptase activity response to exogenous template polyriboadenylic acid.oligo deoxythymidylic acid range...

Human APOBEC3A is a single-stranded DNA cytidine deaminase that restricts viral pathogens and endogenous retrotransposons, has role in the innate immune response. Furthermore, its potential to act as genomic mutator implications for carcinogenesis. A deeper understanding of APOBEC3A's nucleic acid-binding properties, which central biological activities, been limited by lack structural information. Here we report nuclear magnetic resonance solution structure show critical interface...

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ABSTRACT The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) has two zinc fingers, each containing the invariant metal ion binding residues CCHC. Recent reports indicate that mutations in CCHC motifs are deleterious for reverse transcription vivo. To identify transcriptase (RT) reactions affected by such changes, we have probed finger functions NC-dependent RT-catalyzed HIV-1 minus- and plus-strand transfer model systems. Our approach was to examine activities...

After a retrovirus particle is released from the cell, dimeric genomic RNA undergoes change in conformation. We have previously proposed that this change, termed maturation of dimer, due to action nucleocapsid (NC) protein on within virus particle. now report treatment 345-base synthetic fragment Harvey sarcoma with recombinant or HIV-1 NC converts less stable form more form. This phenomenon thus appears reproduce retroviral completely defined system vitro. To our knowledge, first example an...

A group of conserved hydrophobic residues faces the interior coiled-coil-like structure within N-terminal domain human immunodeficiency virus type 1 (HIV-1) capsid protein (CA). It has been suggested that these are important for maintaining stable and functional activity. To investigate this possibility, we constructed two HIV-1 clones, in which Trp23 or Phe40 was changed to Ala. We also a third mutant, D51A, mutation destroys salt bridge between Pro1 Asp51. All three mutants replication...

In an earlier study on minus-strand DNA synthesis catalyzed by murine leukemia virus reverse transcriptase, we described a prominent pause site near the polypurine tract (J. Guo, W. Wu, Z. Y. Yuan, K. Post, R. J. Crouch, and G . Levin, Biochemistry 34:5018-5029, 1995). We now report that pausing at this is due to stem-loop structure in RNA template, formed interaction of number bases tract, including six G's, 3' sequence which includes four C's. Addition human immunodeficiency type 1 (HIV-1)...

Murine leukemia virus particles assembled in actinomycin D-treated cells were detected by determination of reverse transcriptase [RNA-dependent DNA polymerase (nucleotidyltransferase)] activity and radioimmunoassay the major virion protein, p30. The levels enzyme p30 protein both 30-40% relative to control over an 8 hr period, whereas after 3 or 4 infectivity was reduced 95%. Thus, virions produced absence RNA synthesis represent a fairly homogeneous population defective particles. Although...

Cytoplasmic extracts of chicken embryo fibroblast cells infected with Semliki Forest virus were subjected to isopycnic centrifugation in discontinuous sucrose gradients. Seven distinct bands usually formed. The four upper contained predominantly smooth membranes and the lowest band was enriched rough endoplasmic reticulum. One fraction (fraction 5), banding at a density 1.16 g/cm 3 , found be heavily pulse-labeled ribonucleic acid (RNA), viral RNA polymerase, forms associated replication....

INTRACYTOPLASMIC MEMBRANOUS STRUCTURES OF A UNIQUE TYPE WERE ASSOCIATED WITH THE REPLICATION THREE GROUP ARBOVIRUSES: Semliki Forest virus (SFV), Sindbis virus, or Western equine encephalomyelitis virus. The structures, referred to as type 1 cytopathic vacuoles (CPV-1), were membrane-limited and characteristically lined by regular membranous spherules measuring 50 nm in diameter. typically contained a fine central density, but neither cores nor virions. Detection of CPV-1 electron microscopy...

Viral ribonucleic acid (RNA) from Semliki Forest virus- and Sindbis virus-infected cells was analyzed by electrophoresis on polyacrylamide gels. In contrast to earlier results obtained sucrose density gradient centrifugation, all of the known viral RNA forms (i.e., 42 S , 26 replicative form, intermediate) were very clearly separated. The high resolution electrophoretic method permitted identification two new single-stranded species. addition, form shown be heterogeneous consist at least...

The pol gene of murine leukemia virus and other mammalian type C retroviruses is expressed by read-through suppression an in-frame UAG codon which separates the gag coding regions. In this study, we have analyzed sequence requirements for placing different portions wild-type mutant viral sequences from gag-pol junction between reporter genes testing transcripts these constructs in reticulocyte lysates. We find that signal contained within first 57 nucleotides on 3' side codon. Our results...

ABSTRACT APOBEC3G (APO3G) is a cytidine deaminase that restricts replication of vif -defective human immunodeficiency virus type 1 (HIV-1). Like other members the cellular family, APO3G has propensity to form homo-multimers. In current study, we investigated functional determinants for multimerization and studied role catalytic activity, encapsidation, antiviral activity. We found capable forming multimeric complexes in transfected HeLa cells. Interestingly, was exquisitely sensitive RNase...

Human APOBEC3A (A3A) is a single-domain cytidine deaminase that converts deoxycytidine residues to deoxyuridine in single-stranded DNA (ssDNA). It inhibits wide range of viruses and endogenous retroelements such as LINE-1, but it can also edit genomic DNA, which may play role carcinogenesis. Here, we extend our recent findings on the NMR structure A3A report structural, biochemical cell-based mutagenesis studies further characterize A3A's nucleic acid binding activities. We find binds ssRNA,...

Human APOBEC3B (A3B) is a member of the APOBEC3 (A3) family cytidine deaminases, which function as DNA mutators and restrict viral pathogens endogenous retrotransposons. Recently, A3B was identified major source genetic heterogeneity in several human cancers. Here, we determined solution nuclear magnetic resonance structure catalytically active C-terminal domain (CTD) performed detailed analyses its deaminase activity. The core comprises central five-stranded β-sheet with six surrounding...