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      Short Nascent cDNA Fragments in the Presence of Nucleocapsid Protein during Reverse Transcription

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          Abstract

          Pausing and strand-transfers occur frequently during reverse transcription. To investigate how nucleocapsid protein (NC) affects pausing and strand-transfer of nascent cDNA fragments, we directly cloned and sequenced new reverse transcripts in the presence or absence of NC using our recently develop method that clones cDNA products without their amplification by PCR. The nascent cDNA yield was lower and their lengths were shorter in the presence of NC during reverse transcription than in the absence of NC. Our results showed that NC promoted more frequent pausing of nascent cDNA fragments during reverse transcription and resulted in short reverse transcripts.

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          DNA recombination during PCR.

          PCR co-amplification of two distinct HIV1 tat gene sequences lead to the formation of recombinant DNA molecules. The frequency of such recombinants, up to 5.4% of all amplified molecules, could be decreased 2.7 fold by a 6 fold increase in Taq DNA polymerase elongation time. Crossover sites mapped essentially to three discrete regions suggesting specific Taq DNA polymerase pause or termination sites. PCR mediated recombination may be a problem when studying heterogeneous genetic material such as RNA viruses, multigene families, or repetitive sequences. This phenomenon can be exploited to create chimeric molecules from related sequences.
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            Retroviral recombination and reverse transcription.

            Recombination occurs at a high rate in retroviral replication, and its observation requires a virion containing two different RNA molecules (heterodimeric particles). Analysis of retroviral recombinants formed after a single round of replication revealed that (i) the nonselected markers changed more frequently than expected from the rate of recombination of selected markers; (ii) the transfer of the initially synthesized minus strand strong stop DNA was either intramolecular or intermolecular; (iii) the transfer of the first synthesized plus strand strong stop DNA was always intramolecular; and (iv) there was a strong correlation between the type of transfer of the minus strand strong stop DNA and the number of template switches observed. These data suggest that retroviral recombination is ordered and occurs during the synthesis of both minus and plus strand DNA.
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              The remarkable frequency of human immunodeficiency virus type 1 genetic recombination.

              The genetic diversity of human immunodeficiency virus type 1 (HIV-1) results from a combination of point mutations and genetic recombination, and rates of both processes are unusually high. This review focuses on the mechanisms and outcomes of HIV-1 genetic recombination and on the parameters that make recombination so remarkably frequent. Experimental work has demonstrated that the process that leads to recombination--a copy choice mechanism involving the migration of reverse transcriptase between viral RNA templates--occurs several times on average during every round of HIV-1 DNA synthesis. Key biological factors that lead to high recombination rates for all retroviruses are the recombination-prone nature of their reverse transcription machinery and their pseudodiploid RNA genomes. However, HIV-1 genes recombine even more frequently than do those of many other retroviruses. This reflects the way in which HIV-1 selects genomic RNAs for coencapsidation as well as cell-to-cell transmission properties that lead to unusually frequent associations between distinct viral genotypes. HIV-1 faces strong and changeable selective conditions during replication within patients. The mode of HIV-1 persistence as integrated proviruses and strong selection for defective proviruses in vivo provide conditions for archiving alleles, which can be resuscitated years after initial provirus establishment. Recombination can facilitate drug resistance and may allow superinfecting HIV-1 strains to evade preexisting immune responses, thus adding to challenges in vaccine development. These properties converge to provide HIV-1 with the means, motive, and opportunity to recombine its genetic material at an unprecedented high rate and to allow genetic recombination to serve as one of the highest barriers to HIV-1 eradication.
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                Author and article information

                Contributors
                Journal
                Infectious Diseases and Translational Medicine
                Infect. Dis. Transl. Med.
                Infect. Dis. Transl. Med.
                International Biological and Medical Journals Publishing House Co., Limited (Room E16, 3/f, Yongda Commercial Building, No.97, Bonham Stand (Sheung Wan), HongKong )
                2411-2917
                31 October 2017
                31 October 2017
                : 3
                : 2
                : 22-24 (pp. )
                Affiliations
                From National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun 130012, China
                From National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun 130012, China
                From National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun 130012, China
                From National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun 130012, China
                From National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun 130012, China
                From National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun 130012, China
                Author notes
                Correspondence to: Feng Gao, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA; Email: fgao@duke.edu
                Article
                10.11979/idtm.201702003
                a6dc56a9-8d8a-4875-86a7-b4622694dcc5

                This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                Page count
                Figures: 3, Tables: 0, References: 23, Pages: 3
                Product
                Categories
                Brief Report

                Medicine,Infectious disease & Microbiology
                Reverse transcription,Strand transfer,Pausing,Nucleocapsid protein

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