Solution Conformations of Prototype Foamy Virus Integrase and Its Stable Synaptic Complex with U5 Viral DNA

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Highlights? Missing domains in PFV intasome revealed by SAXS/SANS ? PFV IN undergoes dramatic changes in conformation and oligomerization upon binding DNA ? Strand transfer inhibitors do not alter quaternary structure of PFV intasome     

Blockade of NGF and trk receptors inhibits increased peripheral mechanical sensitivity accompanying cystitis in rats

Visceral inflammation, including that arising from bladder inflammation, reduces the threshold to sensation of innocuous or noxious stimuli applied to peripheral structures (referred hyperalgesia). Cystitis may induce transient or persistent plastic changes mediated by neurotrophins, particularly nerve growth factor (NGF), which contribute to increased nociceptive input. In this study, acute or subacute cystitis was induced in female rats by one or three (at 72-h intervals) 400-microl intravesical instillations of 1 mM acrolein. Sensitivity of the hindpaws to mechanical and thermal stimuli was determined before and 4, 24, 48, 72, and 96 h after treatment. Other groups of rats were treated with intravesical or intrathecal k252a [a nonspecific antagonist of tyrosine kinase (trk) receptors, including trkA, the high-affinity receptor for NGF] before the first or third acrolein instillation. Some rats were intraperitoneally injected with specific NGF-neutralizing antiserum or normal serum before acrolein instillation. Acute and subacute cystitis induced mechanical, but not thermal, referred hyperalgesia that was attenuated by intravesical pretreatment with k252a. Systemic treatment with NGF-neutralizing antiserum before instillation of acrolein suppressed subsequent mechanical referred hyperalgesia. Expression of NGF was increased within the bladder by acute or subacute cystitis and in L6/S1 dorsal root ganglia by subacute cystitis. These results suggest that the bladder-derived NGF acting via trk receptors at least partially mediates peripheral sensitization to mechanical stimuli associated with acute and subacute acrolein-induced cystitis.     

Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution

The ITS sequences of Acropora spp. are the shortest so far identified in any metazoan and are among the shortest seen in eukaryotes; ITS1 was 70-80 bases, and ITS2 was 100-112 bases. The ITS sequences were also highly variable, but base composition and secondary structure prediction indicate that divergent sequence variants are unlikely to be pseudogenes. The pattern of variation was unusual in several other respects: (1) two distinct ITS2 types were detected in both A. hyacinthus and A. cytherea, species known to hybridize in vitro with high success rates, and a putative intermediate ITS2 form was also detected in A. cytherea; (2) A. valida was found to contain highly (29%) diverged ITS1 variants; and (3) A. longicyathus contained two distinct 5.8S rDNA types. These data are consistent with a reticulate evolutionary history for the genus Acropora.      

Molluscum Contagiosum Virus Topoisomerase: Purification, Activities, and Response to Inhibitors

Molluscum contagiosum virus (MCV), the only member of the Molluscipoxvirus genus, causes benign papules in healthy people but disfiguring lesions in immunocompromised patients. The sequence of MCV has been completed, revealing that MCV encodes a probable type I topoisomerase enzyme. All poxviruses sequenced to date also encode type I topoisomerases, and in the case of vaccinia virus the topoisomerase has been shown to be essential for replication. Thus, inhibitors of the MCV topoisomerase might be useful as antiviral agents. We have cloned the gene for MCV topoisomerase, overexpressed and purified the protein, and begun to characterize its activities in vitro. Like other eukaryotic type I topoisomerases, MCV topoisomerase can relax both positive and negative supercoils. An analysis of the cleavage of plasmid and oligonucleotide substrates indicates that cleavage by MCV topoisomerase is favored just 3′ of the sequence 5′ (T/C)CCTT 3′, resulting in formation of a covalent bond to the 3′ T residue, as with other poxvirus topoisomerases. We identified solution conditions favorable for activity and measured the rate of formation and decay of the covalent intermediate. MCV topoisomerase is sensitive to inhibition by coumermycin A1 (50% inhibitory concentration, 32 μM) but insensitive to five other previously reported topoisomerase inhibitors. This work provides the point of departure for studies of the mechanism of function of MCV topoisomerase and the development of medically useful inhibitors.     

The mobility of an HIV-1 integrase active site loop is correlated with catalytic activity.

Replication of HIV-1 requires the covalent integration of the viral cDNA into the host chromosomal DNA directed by the virus-encoded integrase protein. Here we explore the importance of a protein surface loop near the integrase active site using protein engineering and X-ray crystallography. We have redetermined the structure of the integrase catalytic domain (residues 50-212) using an independent phase set at 1.7 A resolution. The structure extends helix alpha4 on its N-terminal side (residues 149-154), thus defining the position of the three conserved active site residues. Evident in this and in previous structures is a conformationally flexible loop composed of residues 141-148. To probe the role of flexibility in this loop, we replaced Gly 140 and Gly 149, residues that appear to act as conformational hinges, with Ala residues. X-ray structures of the catalytic domain mutants G149A and G140A/G149A show further rigidity of alpha4 and the adjoining loop. Activity assays in vitro revealed that these mutants are impaired in catalysis. The DNA binding affinity, however, is minimally affected by these mutants as assayed by UV cross-linking. We propose that the conformational flexibility of this active site loop is important for a postbinding catalytic step.     

Role of the PWWP domain of lens epithelium-derived growth factor (LEDGF)/p75 cofactor in lentiviral integration targeting

LEDGF/p75 is a chromatin-interacting, cellular cofactor of HIV integrase that dictates lentiviral integration site preference. In this study we determined the role of the PWWP domain of LEDGF/p75 in tethering and targeting of the lentiviral pre-integration complex, employing potent knockdown cell lines allowing analysis in the absence of endogenous LEDGF/p75. Deletion of the PWWP domain resulted in a diffuse subnuclear distribution pattern, loss of interaction with condensed chromatin, and failure to rescue proviral integration, integration site distribution, and productive virus replication. Substitution of the PWWP domain of LEDGF/p75 with that of hepatoma-derived growth factor or HDGF-related protein-2 rescued viral replication and lentiviral integration site distribution in LEDGF/p75-depleted cells. Replacing all chromatin binding elements of LEDGF/p75 with full-length hepatoma-derived growth factor resulted in more integration in genes combined with a preference for CpG islands. In addition, we showed that any PWWP domain targets SMYD1-like sequences. Analysis of integration preferences of lentiviral vectors for epigenetic marks indicates that the PWWP domain is critical for interactions specifying the relationship of integration sites to regions enriched in specific histone post-translational modifications.