Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Retroviral integrase (IN) functions to insert retroviral DNA into the host cell chromosome in a highly coordinated manner. IN catalyzes two biochemically separable reactions: processing of the viral DNA ends and joining of these ends to the host DNA. Previous studies suggested that these two reactions are chemically similar and are carried out by a single active site that is characterized by a highly conserved constellation of carboxylate residues, the D,D(35)E motif. We report here the crystal structure of the isolated catalytic domain of avian sarcoma virus (ASV) IN, solved using multiwavelength anomalous diffraction data for a selenomethionine derivative and refined at 1.7 A resolution. The protein is a crystallographic dimer with each monomer featuring a five-stranded mixed beta-sheet region surrounded by five alpha-helices. Based on the general fold and the arrangement of catalytic carboxylate residues, it is apparent that ASV IN is a member of a superfamily of proteins that also includes two types of nucleases, RuvC and RNase H. The general fold and the dimer interface are similar to those of the analogous domain of HIV-1 IN, whose crystal structure has been determined at 2.5 A resolution. However, the ASV IN structure is more complete in that all three critical carboxylic acids, Asp64, Asp121 and Glu157, are ordered. The ordered active site and the considerably higher resolution of the present structure are all important to an understanding of the mechanism of retroviral DNA integration, as well as for designing antiviral agents that may be effective against HIV.
J Mol Biol 1995 Oct 20
PMID:High-resolution structure of the catalytic domain of avian sarcoma virus integrase. 756 93

The genome of avian sarcoma virus CT10 encodes a fusion protein in which viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Transformation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specific cellular proteins with molecular weights of 130,000, 110,000, and 70,000 (p130, p110, and p70, respectively), an event which has been correlated with cell transformation. In this study, we have identified the 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10-CEF) as paxillin, a cytoskeletal protein suggested to be important for organizing the focal adhesion. Tyrosine-phosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunoprecipitation studies. Moreover, a bacterially expressed recombinant glutathione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is sufficient for binding. Mapping of the sequence specificity of a GST-CrkSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipitation studies, an acetylated A(p)YDAPA hexapeptide was able to quantitatively inhibit the binding of GST-CrkSH2 to paxillin and p130, suggesting that it meets the minimal structural requirements necessary for the interaction of CrkSH2 with physiological targets. To investigate the mechanism by which v-Crk elevates the tyrosine phosphorylation of paxillin in vivo, we have treated normal CEF and CT10-CEF with sodium vanadate to inhibit protein tyrosine phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and that v-Crk binding may interrupt this balance to increase the steady-state level of tyrosine phosphorylation. By contrast, the 130-kDa protein was not tyrosine phosphorylated upon vanadate treatment of normal CEF and only weakly affected in the CT10-CEF, suggesting that a different mechanism may be involved in its phosphorylation.
Mol Cell Biol 1993 Aug
PMID:Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts. 768 42

Incomplete splicing is essential for retroviral replication; and in simple retroviruses, splicing regulation appears to occur entirely in cis. Our previous studies, using avian sarcoma virus, indicated that weak splicing signals allow transcripts to escape the splicing pathway. We also isolated a series of avian sarcoma virus mutants in which env mRNA splicing was regulated by mechanisms distinct from those of the wild-type virus. In vitro splicing experiments with one such mutant (insertion suppressor 1 [IS1]) revealed that exon 1 and lariat-exon 2 intermediates were produced (step 1) but the exons were not efficiently ligated (step 2). In this work, we have studied the mechanism of this second-step block as well as its biological relevance. Our results show that the second-step block can be overcome by extending the polypyrimidine tract, and this causes an oversplicing defect in vivo. The requirement for regulated splicing was exploited to isolate new suppressor mutations that restored viral growth by down-regulating splicing. One suppressor consisted of a single U-to-C transition in the polypyrimidine tract; a second included this same change as well as an additional U-to-C transition within a uridine stretch in the polypyrimidine tract. These suppressor mutations affected primarily the second step of splicing in vitro. These results support a specific role for the polypyrimidine tract in the second step of splicing and confirm that, in a biological system, uridines and cytosines are not functionally equivalent within the polypyrimidine tract. Unlike the wild-type virus, the second-step mutants displayed significant levels of lariat-exon 2 in vivo, suggesting a role for splicing intermediates in regulation. Our results indicate that splicing regulation can involve wither the first or second step.
Mol Cell Biol 1995 May
PMID:Genetic selection for balanced retroviral splicing: novel regulation involving the second step can be mediated by transitions in the polypyrimidine tract. 773 46

We previously reported the isolation of PR2257, a novel replication defective avian sarcoma virus which transduced the entire c-src coding region together with about 900 bp of c-src 3' non coding sequences. This virus originated from a chicken sarcoma induced by inoculation of a transformation-defective Rous sarcoma virus carrying only replicative genes. The 5' end of PR2257 was formed by a splice junction between viral leader sequences and the first exon of c-src. To understand the mechanism of 3' recombination between viral and cellular sequences, we analyzed the genomic organization of the 3' region in chicken and quail src DNA. We report that this sequence is colinear with that of a chicken src cDNA. Therefore, exon 12, which encodes the last 68 amino acids of c-src, also contains all 3' non coding sequences present in c-src mRNA and consequently, appears to be the last and largest (about 2 kbp) exon of c-src. We also show that the 3' regions of chicken and quail c-src genes does not contain the additional open reading frame (orf) which was previously reported (Dorai et al. (1991) Mol. Cell. Biol. 11, 4165-4176), and that no other significant conserved open reading frames could be found in this region for both species. Therefore, this region of src does not code for another protein. Taken together, our results suggest that PR2257 was generated by recombination at the RNA level. However, because of the absence of introns in this region of c-src, we cannot formally rule out the possibility that this recombination took place at the DNA level.
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PMID:The 3' region of c-src gene mRNA is entirely included in exon 12 and does not encode another protein. 804 78

A portion of the avian sarcoma virus (ASV) primary RNA transcripts is alternatively spliced in chicken embryo fibroblast cells to two different messages, the src and env mRNAs. Frameshift mutations of the viral genome causing premature translation termination within the src gene result in a decreased steady-state level of the src mRNA. In marked contrast, frameshift mutations at various positions of the env gene do not decrease the level of the env mRNA. We show that the src gene product is not required in trans for splicing and accumulation of src mRNA. Conversely, the truncated Src proteins do not act negatively in trans to decrease specifically the levels of src mRNA. Taken together, these results indicate that the frameshift mutations act in cis to reduce src mRNA levels. A double mutant with a lesion in the src initiator AUG and a frameshift within the src gene demonstrated wild-type RNA levels, indicating that the src mRNA must be recognized as a translatable mRNA for the effect on src mRNA levels to occur. Our results indicate that the reduced levels do not result from decreased cytoplasmic stability of the mature src mRNA. We also show that the src gene frameshift mutations affect src mRNA levels when expressed from intronless src cDNA clones. We conclude that the reduction of src mRNA levels triggered by the presence of frameshift mutations within the src gene occurs while it is associated with the nucleus. Our data also strongly suggest that this occurs at a step of RNA processing or transport independent of RNA splicing.
Mol Cell Biol 1994 Mar
PMID:Frameshift mutations in the v-src gene of avian sarcoma virus act in cis to specifically reduce v-src mRNA levels. 811 16

Subgroup C avian sarcoma viruses efficiently infect and transform but poorly replicate in duck cells. Nucleotide sequence analysis of Prague strain of Rous sarcoma virus adapted by numerous passages on duck embryonic fibroblasts (daPr-RSV-C) showed that adaptation of originally chicken virus to duck cells correlated with changes in viral genome, first of all in gp85-coding domain of env-gene. Besides, changes in LTR and src-gene sequences could play a role in widening of host range for this virus. The major changes of daPr-RSV-C in comparison with original Pr-RSV-C appeared to be the result of homologous recombinations with corresponding regions of chicken endogenous retroviruses.
Mol Biol (Mosk)
PMID:[Complete nucleotide sequence of Rous sarcoma virus variants adapted to duck cells]. 838 33

We solved the structure and traced the complete active site of the catalytic domain of the human immunodeficiency virus type 1 integrase (HIV-1 IN) with the F185H mutation. The only previously available crystal structure, the F185K mutant of this domain, lacks one of the catalytically important residues, E152, located in a stretch of 12 disordered residues [Dyda et al. (1994) Science 266, 1981-1986]. It is clear, however, that the active site of HIV-1 IN observed in either structure cannot correspond to that of the functional enzyme, since the cluster of three conserved carboxylic acids does not create a proper metal-binding site. The conformation of the loop was compared with two different conformations found in the catalytic domain of the related avian sarcoma virus integrase [Bujacz et al. (1995) J. Mol. Biol. 253, 333-346]. Flexibility of the active site region of integrases may be required in order for the enzyme to assume a functional conformation in the presence of substrate and/or cofactors.
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PMID:The catalytic domain of human immunodeficiency virus integrase: ordered active site in the F185H mutant. 897 1

Propagation of long terminal repeat (LTR)-bearing retrotransposons and retroviruses requires integrase (IN, EC 2.7.7.-), encoded by the retroelements themselves, which mediates the insertion of cDNA copies back into the genome. An active retrotransposon family, BARE-1, comprises approximately 7% of the barley (Hordeum vulgare subsp. vulgare) genome. We have generated models for the secondary and tertiary structure of BARE-1 IN and demonstrate their similarity to structures for human immunodeficiency virus 1 and avian sarcoma virus INs. The IN core domains were compared for 80 clones from 28 Hordeum accessions representative of the diversity of the genus. Based on the structural model, variations in the predicted, aligned translations from these clones would have minimal structural and functional effects on the encoded enzymes. This indicates that Hordeum retrotransposon IN has been under purifying selection to maintain a structure typical of retroviral INs. These represent the first such analyses for plant INs.
Mol Biol Evol 1998 Sep
PMID:The core domain of retrotransposon integrase in Hordeum: predicted structure and evolution. 972 78

The crystal structure of simian immunodeficiency virus (SIV) integrase that contains in a single polypeptide the core and the C-terminal deoxyoligonucleotide binding domain has been determined at 3 A resolution with an R-value of 0.203 in the space group P2(1)2(1)2(1). Four integrase core domains and one C-terminal domain are found to be well defined in the asymmetric unit. The segment extending from residues 114 to 121 assumes the same position as seen in the integrase core domain of avian sarcoma virus as well as human immunodeficiency virus type-1 (HIV-1) crystallized in the absence of sodium cacodylate. The flexible loop in the active site, composed of residues 141-151, remains incompletely defined, but the location of the essential Glu152 residue is unambiguous. The residues from 210-218 that link the core and C-terminal domains can be traced as an extension from the core with a short gap at residues 214-215. The C(alpha) folding of the C-terminal domain is similar to the solution structure of this domain from HIV-1 integrase. However, the dimeric form seen in the NMR structure cannot exist as related by the non-crystallographic symmetry in the SIV integrase crystal. The two flexible loops of the C-terminal domain, residues 228-236 and residues 244-249, are much better fixed in the crystal structure than in the NMR structure with the former in the immediate vicinity of the flexible loop of the core domain. The interface between the two domains encompasses a solvent-exclusion area of 1500 A(2). Residues from both domains purportedly involved in DNA binding are narrowly distributed on the same face of the molecule. They include Asp64, Asp116, Glu152 and Lys159 from the core and Arg231, Leu234, Arg262, Arg263 and Lys264 from the C-terminal domain. A model for DNA binding is proposed to bridge the two domains by tethering the 228-236 loop of the C-terminal domain and the flexible loop of the core.
J Mol Biol 2000 Feb 18
PMID:X-ray structure of simian immunodeficiency virus integrase containing the core and C-terminal domain (residues 50-293)--an initial glance of the viral DNA binding platform. 1066 6

Retroviral genomes consist of two identical RNA molecules joined non-covalently near their 5'-ends. Recently, we showed that an imperfect autocomplementary sequence, located in the L3 domain, plays an essential role in avian sarcoma-leukosis virus (ASLV) RNA dimerization in vitro. This sequence can adopt a stem-loop structure and is involved in ASLV replication. Here, we found that in the absence of nucleocapsid protein, RNA transcripts of avian leukosis virus (ALV) were able to form two types of dimers in vitro that differ in their stability: a loose dimer, formed at a physiological temperature, and a tight dimer, formed at a high temperature. A mutational analysis was performed to define the features of these dimers. The results of this analysis unambiguously confirm that the two L3 stem-loops interact directly in both types of dimers. A loop-loop interaction is the main linkage in the loose dimer. In contrast, in the tight dimer, the stem and the loop of the L3 hairpin form an extended duplex. Surprisingly, we also found that the dimerization properties defined for our ALV strain (type SR-A) differ from those found in other ASLV strains.
J Mol Biol 2000 Jun 30
PMID:Characterization of loose and tight dimer forms of avian leukosis virus RNA. 1086 97


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