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ABSTRACT: Sequence comparisons of the integrase (IN) proteins from different retroviruses have identified several highly conserved residues. We have introduced mutations at 16 of these sites into the integrase gene of human immunodeficiency virus type 1 and analyzed the phenotypes of the resulting viruses. The viruses were all normal for p24 content and reverse transcriptase activity. In addition, all of the mutants could infect T-cell lines and undergo reverse transcription, as assessed by PCR analysis. Most of the mutant viruses also had normal Western blot (immunoblot) profiles, although three of the mutations resulted in reduced signals for IN relative to the wild type on the immunoblots and mutation of residue W235 completely abolished recognition of the protein by pooled sera from human immunodeficiency virus type 1-positive patients. Mutations that have previously been shown to abolish activity in in vitro studies produced noninfectious viruses. The substitution of W235 was notable in producing a noninfectious virus, despite previous reports of this residue being nonessential for IN activity in vitro (A.D. Leavitt, L. Shiue, and H.E. Varmus, J. Biol. Chem. 268:2113-2119, 1993). In addition, we have identified four highly conserved residues that can be mutated without any affect on viral replication in T-cell lines.
Journal of Virology 09/1994; 68(8):4768-75. · 5.40 Impact Factor
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ABSTRACT: Replication of the human immunodeficiency virus (HIV-1) depends upon the viral TAT protein. TAT stimulates gene expression via a target response sequence (TAR) located within the HIV-1 LTR. As TAR is located in the transcribed region it could act as a signal in either the DNA, the RNA, or both. To test whether TAT acts on transcription and/or posttranscriptionally, we produced TAT in yeast and monitored its activity after microinjection into the nucleus or cytoplasm of Xenopus oocytes. The TAT protein stimulated TAR-dependent expression, but this activation was not inhibited by transcriptional inhibitors. Furthermore, TAR-containing RNA, produced in vitro, was "activated" by TAT after coinjection into oocytes. This activation only occurred, however, when the RNA was injected into the nucleus and not into the cytoplasm. Our data indicate, therefore, that in the Xenopus system TAT acts on presynthesized RNA and that the nucleus is involved in this action.
Cell 08/1989; 58(2):269-79. · 32.40 Impact Factor
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ABSTRACT: The pol gene of the human immunodeficiency virus (HIV-1) is expressed as a gag:pol fusion, arising from a ribosomal frameshift that brings the overlapping, out-of-phase gag and pol genes into translational phase. In this study, we show that HIV frameshifting is mediated by a very short sequence in the viral RNA. We demonstrate the importance of a homopolymeric run within this sequence and conclude that HIV frameshifting is not dependent on stem-loop structures downstream from the frameshift site. Our analysis also indicates that the sequence requirements are identical in mammalian and yeast systems.
Cell 01/1989; 55(6):1159-69. · 32.40 Impact Factor
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ABSTRACT: The complete nucleotide sequence of a mouse retro-element is presented. The cloned element is composed of 4,834 base pairs (bp) with long terminal repeats of 568 bp separated by an internal region of 3,698 bp. The element did not appear to have any open reading frames that would be capable of encoding the functional proteins that are normally produced by retro-elements. However, some regions of the genome showed some homology to retroviral gag and pol open reading frames. There was no region in VL30 corresponding to a retroviral env gene. This implies that VL30 is related to retrotransposons rather than to retroviruses. The sequence also contained regions that were homologous to known reverse transcriptase priming sites and viral packaging sites. These observations, combined with the known transcriptional capacity of the VL30 promoter, suggest that VL30 relies on protein functions of other retro-elements, such as murine leukemia virus, while maintaining highly conserved cis-active promoter, packaging, and priming sites necessary for its replication and cell-to-cell transmission.
Molecular and Cellular Biology 09/1988; 8(8):2989-98. · 5.53 Impact Factor
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ABSTRACT: The genetic organization of the yeast transposon Ty resembles that of higher eukaryotic retroviruses and other elements such as the copia-like sequences of Drosophila. The Ty genome is 5.9 kb (10(3) bases) long. It has 340 bp (base pairs) terminal repeats known as delta sequences and it produces a terminally redundant 5.7 kb RNA that starts in the 5' delta and ends in the 3' delta. Ty transcription is directed by signals upstream and downstream of the major RNA start site and is regulated by the mating-type configuration of the cell. The 5.7 kb transcriptional unit is divided into two overlapping open reading frames, TYA and TYB. TYA occupies approximately the first quarter of the transcriptional unit while TYB occupies the rest. TYB overlaps TYA by either 38 or 44 nucleotides, depending on the element, and is in the plus one reading frame with respect to TYA. TYA is expressed to produce protein p1 (50 x 10(3) Mr) and TYB is expressed as a TYA:TYB fusion protein, p3 (190 x 10(3) Mr). Both of these proteins are subsequently cleaved to produce proteins p2, p4, p5, p6, reverse transcriptase and a protease that is responsible for some of these cleavage events. These proteins are assembled into virus-like particles (Ty-VLPs) that contain Ty RNA and reverse transcriptase activity. It is likely that the Ty-VLPs are units of transposition as Ty transposes via an RNA intermediate.
Journal of cell science. Supplement 02/1987; 7:155-67.
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ABSTRACT: The Ty element of yeast is a member of a class of eukaryotic transposons which bear a striking resemblance to retroviral proviruses in their structure and expression strategies (1,2). A direct comparison can be drawn between the production of a fusion protein encoded by Ty, resulting from a frameshift event which fuses two out-of-phase open reading frames TYA and TYB, and the production of Pr180gag-pol in a retrovirus such as Rous Sarcoma Virus (RSV) (3,4). We present data which shows, definitively, that RNA splicing is not responsible for the frameshift in Ty. By in vitro mutation of a class I element, Ty1-15, we demonstrate that 31 nucleotides contained within the region where the TYA and TYB open reading frames overlap direct the frameshift. Within this short sequence there is a region of homology with a class II element which we show is also able to frameshift.
Nucleic Acids Research 10/1986; 14(17):7001-16. · 8.03 Impact Factor
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ABSTRACT: Two new Ty determined proteins have been identified by placing a Ty transcriptional unit under the control of a high efficiency yeast expression vector. One of these proteins is the product of a post-translational processing event and it binds nucleic acids. A previously identified protein, pl (Tyl-15), has also been shown to bind nucleic acids and to be modified by phosphorylation.
Nucleic Acids Research 10/1985; 13(17):6249-63. · 8.03 Impact Factor
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ABSTRACT: The Ty transposable elements of Saccharomyces cerevisiae form a heterogeneous family within which two broad structural classes (I and II) exist. The two classes differ by two large substitutions and many restriction sites. We show that, like class I elements a class II element, Tyl-17, also appears to contain at least two major protein coding regions, designated TYA and TYB, and the organisational relationship of these regions has been conserved. The TYA genes of both classes encode proteins, designated p1 proteins, with an approximate molecular weight of 50 Kd and, despite considerable variation between the TYA regions at the DNA level, the structures of these proteins are remarkably similar. These observations strongly suggest that the p1 proteins of Ty elements are functionally significant and that they have been subject to selection.
Nucleic Acids Research 07/1985; 13(11):4097-112. · 8.03 Impact Factor
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Nature 05/1985; 315:691-692. · 36.28 Impact Factor
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ABSTRACT: Eukaryotic transposons such as the Ty element of yeast or the copia-like sequences of Drosophila show structural and functional similarities to both prokaryotic transposons and retroviral proviruses, but the prokaryotic transposons and retroviral proviruses use markedly different expression strategies which yield products having entirely different functions. To determine the phylogenetic relationship between eukaryotic transposons, prokaryotic transposons and retroviruses, we have sought to identify and characterize the proteins encoded by the yeast Ty element and to describe the strategies used to express these proteins. We show here that the yeast transposon produces a fusion protein by a specific frameshifting event that fuses two out-of-phase open reading frames (ORFs). The process is remarkably similar to that used by retroviruses such as Rous sarcoma virus (RSV) to produce Pr180gag-pol.
Nature 313(5999):243-6. · 36.28 Impact Factor
