Variation in boldness and novelty response between rural and urban predatory birds: The Chimango Caracara, Milvago chimango as study case.

The study of traits that enable species to thrive in urban habitats is critical to a better understanding the evolution of urban ecosystems. Here, we examined variation in boldness, neophobia, and exploratory behavior in a generalist raptor, the Chimango Caracara, Milvago chimango, occurring in areas with different urbanization level. We also focused on the relationship between object exploration and neophobia in rural and urban population of this species. The results showed that birds from more urbanized habitats showed more tolerance to humans than rural raptors, without any effect of age and sex in this trait. Rural birds were also more neophobic and were slower to explore than urban raptors, though they dedicated a similar amount of time to exploring novel objects, indicating a non-correspondence between the speed to approach and contact these objects and the amount of exploration performed. Finally, we found a correlation between exploration speed and neophobia in rural birds, whereas for urban raptors this correlation was not observed. Our results show that urbanization not only influences the expression of risk-taking behaviors and novelty responses in the chimango, but can also modify the relationship between exploration and neophobia.

Processing of an HIV replication intermediate by the human DNA replication enzyme FEN1.

The role of human FEN1 (flap endonuclease-1), an RTH1 (RAD two homolog-1) class nuclease, in the replication of human immunodeficiency virus (HIV) type 1 has been examined using model substrates. FEN1 is able to endonucleolytically cleave a primer annealed to a template, but with a 5'-unannealed tail. The HIV (+)-strand is synthesized as two discontinuous segments, with the upstream segment displacing the downstream segment to form a central (+)-strand overlap. Given a substrate with the exact HIV nucleotide sequence, FEN1 was able to remove the overlap. After extension of the upstream primer with DNA polymerase epsilon, human DNA ligase I was able to complete the continuous double strand as would occur for an integrated provirus. FEN1 may represent a target for new therapeutic interventions.

Strand displacement synthesis in the central polypurine tract region of HIV-1 promotes DNA to DNA strand transfer recombination.

Two distinct plus strand initiation sites have been identified in human immunodeficiency virus (HIV), the central polypurine tract (cPPT) and the polypurine tract located just upstream of the U3 region (U3-PPT). When synthesis from the U3-PPT reaches the cPPT, the elongating primer causes limited strand displacement of the product created from the cPPT. We examined whether reverse transcriptase (RT) catalyzed strand transfer recombination is promoted by this process. Using a substrate having the viral sequence of the displaced region, we measured transfer of an elongating DNA primer from a donor DNA to an acceptor DNA. Strand transfer synthesis was only efficient when RT was performing strand displacement synthesis. Transfer efficiency was directly related to acceptor concentration but independent of the reaction time. Transfer could occur to acceptors containing 80, 40, or 20 nucleotides of homology with the template DNA. Using different acceptors, we found that DNA to DNA transfer occurred at positions throughout the donor template, except near the 5' end. This shows that a number of the sequences downstream of the cPPT region can promote transfer, but once synthesis has progressed to the point where the downstream segment is completely displaced transfer is not allowed. When the DNA to DNA transfer reactions were performed using a template containing nonviral sequences, the transfer efficiency dropped significantly. This indicates that transfer efficiency is determined by the sequences of the templates used. HIV-RT RNase H-dependent strand transfer between RNA templates is well documented. We propose a quite different mechanism for DNA to DNA transfer, consistent with the ability of RNase H minus RT to perform this reaction. If these DNA to DNA transfer events occur in vivo, they will result in plus strand recombination.

Relationship between plus strand DNA synthesis removal of downstream segments of RNA by human immunodeficiency virus, murine leukemia virus and avian myeloblastoma virus reverse transcriptases.

During retroviral reverse transcription the genomic RNA is degraded by the RNase H activity of reverse transcriptase (RT). Previous results suggest that after RNA-directed DNA synthesis, fragments of RNA remain annealed to the newly synthesized DNA [DeStefano et al.(1991) J. Biol.Chem. 266, 7423-7431]. These must be removed to allow synthesis of the second DNA strand. We measured the ability of HIV-, AMV- and MuLV-RT to coordinate DNA-dependent DNA synthesis and removal of downstream segments of RNA. The substrates employed were DNA templates having upstream DNA and downstream RNA primers. We found that none of the wild type RTs elongated the upstream DNA without simultaneous degradation of the RNA. Consistent with these results, HIV-, AMV- and MuLV-RT showed relatively higher affinity for RNA than for DNA oligonucleotides bound to a DNA template. Differences were observed in the RNA degradation and DNA extension patterns generated by the different RTs. AMV-RT degraded the RNA to segments 11-12 nt long, and readily elongated the upstream DNA to the end of the template. MuLV- and HIV-RT degraded the RNA primarily to segments 15-16 nt long. At low concentrations of the latter two RTs, the DNA primer stalled when it encountered the 5'-end of the RNA. In sufficient excess, all of the RTs elongated the upstream primer without stalling. Even though we were unable to detect displacement of the downstream RNA by the wild type RTs, MuLV- and HIV-RT lacking RNase H, were able to elongate the upstream DNA to the end of the template without degradation of the RNA. This suggests that degradation of downstream pieces of RNA is not absolutely required before synthesis of the plus strand DNA. The implications of these findings for viral replication are discussed.

Strand displacement synthesis of the long terminal repeats by HIV reverse transcriptase.

According to the current model for retroviral replication, strand displacement of the long terminal repeat (LTR) is a necessary step during plus strand DNA synthesis in vivo. We have investigated the ability of human immunodeficiency virus reverse transcriptase (HIV-RT) to synthesize in vitro over a 634-nucleotide HIV LTR DNA template, having or lacking a single full-length DNA downstream primer. The presence of the downstream primer resulted in an approximately 12-fold reduction in the rate of upstream primer elongation. Addition of Escherichia coli single-stranded binding protein (SSB) or human replication protein A (RP-A) enhanced strand displacement synthesis; however, addition of HIV nucleocapsid protein (NC) did not. The presence of excess single-stranded DNA complementary to the downstream primer did not stimulate displacement synthesis. Interestingly, we observed that the elongating upstream primer could readily transfer to this DNA. This observation suggests that recombination is favored during strand displacement synthesis in vivo.

Helix structure and ends of RNA/DNA hybrids direct the cleavage specificity of HIV-1 reverse transcriptase RNase H.

RNA/DNA hybrids in human immunodeficiency virus (HIV) replication are cleaved by HIV-1 reverse transcriptase (RT) H in locations determined by hybrid structure. Minus strand DNA synthesis is accompanied by cleavage of template viral RNA directed by RT positioned at the growing 3' DNA end. Some RNA remains as oligomers annealed to the new DNA strand and is cut by RTs positioned at the 5' RNA ends. We constructed substrates to the test the hypothesis that internal helix structure, rather than strand end structure, drives the RT to position at 3' DNA and 5' RNA ends. On substrates with an RNA primer recessed on a DNA template, the 5' end of the RNA had a dominant role in the determination of RNase H cleavage positions. If the 5' end region of the RNA could not anneal, cleavage would not occur. Nevertheless, we obtained evidence that helix structure promotes the binding of RT to the end of the helical region closest to the 5' RNA/3' DNA end. When a DNA primer recessed on an RNA template had a 3' unannealed region, cleavage occurred, with RT positioned solely by helical structure at the 5' RNA/3' DNA end of the annealed region of the hybrid. Using substrates having RNA primers annealed to circular DNA templates, we showed that cleavage can be independent of the presence of a DNA 3'end and is directed by the 5' RNA end. Overall, the results suggest that the RT initially binds an internal region of the hybrid and then is driven in the direction to encounter a 3' DNA or 5' RNA end, where it is positioned for catalysts by the strand end. The requirement for two modes of RNA cleavage in viral replication and the unexpected requirement for the 5' RNA end structure are discussed.

Use of an oligoribonucleotide containing the polypurine tract sequence as a primer by HIV reverse transcriptase.

A primary site for initiation of plus strand DNA synthesis in human immunodeficiency virus (HIV) corresponds to a 19-nucleotide-long purine rich sequence located just upstream of the U3 region, designated the polypurine tract (PPT). The HIV reverse transcriptase (RT) uses its RNase H activity to cut the genomic RNA after minus strand DNA synthesis. A plus strand PPT primer is formed, extended, and then removed. In vitro, the HIV-RT recognizes this primer specifically, using it much more efficiently than other RNA primers. However, the PPT still primes significantly less efficiently than DNA primers. The 19-nucleotide PPT primer is partially resistant to degradation when compared with other oligoribonucleotides. Prior to initiation of DNA synthesis, several nucleotides are removed by the RT from the 3' ends of some of the PPT primers. Cleavage is enhanced in the absence of dNTPs. We suggest that DNA synthesis suppresses primer degradation, so that primer extension and cleavage occur in proper sequence. As a result of 3' end degradation, PPT elongation products contain 5'-RNA segments from 16 to 19 nucleotides in length. These shorter segments are also generated from a longer transcript containing the PPT sequence, indicating that they are not created as a result of binding of the RT to the 5' end of the PPT oligoribonucleotide. Full-length and shorter versions of the PPT primers are cleaved from the extended DNA by RT. These experiments show that HIV-RT has a specificity to generate a primer in the region of the PPT but that the ends of the primer are not well defined.

Influence of human immunodeficiency virus nucleocapsid protein on synthesis and strand transfer by the reverse transcriptase in vitro.

Human immunodeficiency virus (HIV) nucleocapsid protein (NC) influences HIV reverse transcriptase (RT) catalyzed strand transfer synthesis from internal regions of natural sequence RNA. In the strand transfer assay reaction in vitro, primer synthesis initiated on a donor template can transfer and be completed on an acceptor template. NC was added at concentrations up to twice that needed for 100% template coating. As the concentration of NC was increased, primer extension was stimulated until NC coated approximately 50% of the template. Stimulation was caused in part by an increase in the number of primers that sustained synthesis. Subsequent increments of NC decreased synthesis. The presence of NC also increased the efficiency of the strand transfer reaction, allowing a greater proportion of extended primers to transfer from donor to acceptor templates. Processivity of the RT on the donor template was measured using both challenged and enzyme dilution assays. NC did not alter the proportion of synthesis products that reached the end of the template, indicating little effect on processivity. This result suggests that the increase in full-length product synthesis, observed in reactions where the RT repeatedly bound the primer-template, resulted from promotion of RT reassociation by NC. Consequently, since the RT could not reassociate with the template in the processivity assay, NC could not stimulate the amount of full-length synthesis. No strand transfer was observed in dilution processivity assays, suggesting that the RT must dissociate and rebind during the transfer reaction. Stimulation of synthesis, e.g. by increased dNTP concentration, normally inhibits strand transfer. Stimulation of both synthesis and transfer by NC indicates that properties of NC that improve the transfer event prevail over the negative effects of rapid synthesis on transfer efficiency.