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DNA shuffling facilitated the evolution of a human immunodeficiency virus type 1 (HIV-1) variant with enhanced replication in pig-tailed macaque peripheral blood mononuclear cells (pt mPBMC). This variant consists exclusively of HIV-1-derived sequences with the exception of simian immunodeficiency virus (SIV) nef . Sequences spanning the gag -protease-reverse transcriptase ( gag - pro -RT) region from several HIV-1 isolates were shuffled and cloned into a parental HIV-1 backbone containing SIV nef . Neither this full-length parent nor any of the unshuffled HIV-1 isolates replicated appreciably or sustainably in pt mPBMC. Upon selection of the shuffled viral libraries by serial passaging in pt mPBMC, a species emerged which replicated at substantially higher levels (50 to 100 ng/ml p24) than any of the HIV-1 parents and most importantly, could be continuously passaged in pt mPBMC. The parental HIV-1 isolates, when selected similarly, became extinct. Analyses of full-length improved proviral clones indicate that multiple recombination events in the shuffled region and adaptive changes in the rest of the genome contributed synergistically to the improved phenotype. This improved variant may prove useful in establishing a pig-tailed macaque model of HIV-1 infection.

Manufacturing of retroviral vectors for gene therapy is complicated by the sensitivity of these viruses to stress forces during purification and concentration. To isolate viruses that are resistant to these manufacturing processes, we performed breeding of six ecotropic murine leukemia virus (MLV) strains by DNA shuffling. The envelope regions were shuffled to generate a recombinant library of 5 x 106 replication-competent retroviruses. This library was subjected to the concentration process three consecutive times, with amplification of the surviving viruses after each cycle. Several viral clones with greatly improved stabilities were isolated, with the best clone exhibiting no loss in titer under conditions that reduced the titers of the parental viruses by 30- to 100-fold. The envelopes of these resistant viruses differed in DNA and protein sequence, and all were complex chimeras derived from multiple parents. These studies demonstrate the utility of DNA shuffling in breeding viral strains with improved characteristics for gene therapy.

DNA shuffling and screening technologies recombine and evolve genes in vitro to rapidly obtain molecules with improved biological activity and fitness. In this way, genes from related strains are bred like plants or livestock and their successive progeny are selected. These technologies have also been called molecular breeding-directed molecular evolution. Recent developments in bioinformatics-assisted computer programs have facilitated the design, synthesis and analysis of DNA shuffled libraries of chimeric molecules. New applications in vaccine development are among the key features of DNA shuffling and screening technologies because genes from several strains or antigenic variants of pathogens can be recombined to create novel molecules capable of inducing immune responses that protect against infections by multiple strains of pathogens. In addition, molecules such as co-stimulatory molecules and cytokines have been evolved to have improved T-cell proliferation and cytokine production compared with the wild-type human molecules. These molecules can be used to immunomodulate vaccine responsiveness and have multiple applications in infectious diseases, cancer, allergy and autoimmunity. Moreover, DNA shuffling and screening technologies can facilitate process development of vaccine manufacturing through increased expression of recombinant polypeptides and viruses. Therefore, DNA shuffling and screening technologies can overcome some of the challenges that vaccine development currently faces.

Murine cells do not support efficient assembly and release of human immunodeficiency virus type 1 (HIV-1) virions. HIV-1-infected mouse cells that express transfected human cyclin T1 synthesize abundant Gag precursor polyprotein, but inefficiently assemble and release virions. This assembly defect may result from a failure of the Gag polyprotein precursor to target to the cell membrane. Plasma membrane targeting of the precursor is mediated by the amino-terminal region of polyprotein. To compensate for the assembly block, we substituted the murine leukemia virus matrix coding sequences into an infectious HIV-1 clone. Transfection of murine fibroblasts expressing cyclin T1 with the chimeric proviruses resulted in viruses that were efficiently assembled and released. Chimeric viruses, in which the cytoplasmic tail of the transmembrane subunit, gp41, was truncated to prevent potential interference between the envelope glycoprotein and the heterologous matrix, could infect human and murine cells. They failed to further replicate in the murine cells, but replicated with delayed kinetics in human MT-4 cells. These findings may be useful for establishing a murine model for HIV-1 replication.

Genetic recombination is a major force driving the evolution of many viruses. Recombination between two copackaged retroviral genomes may occur at rates as high as 40% per replication cycle1. This enables genetic information to be shuffled rapidly, leading to recombinants with new patterns of mutations and phenotypes. The in vitro process of DNA shuffling2,3 (molecular breeding) mimics this mechanism on a vastly parallel and accelerated scale. Multiple homologous parental sequences are recombined in parallel, leading to a diverse library of complex recombinants from which desired improvements can be selected. Different proteins and enzymes have been improved using DNA shuffling4,5,6. We report here the first application of molecular breeding to viruses. A single round of shuffling envelope sequences from six murine leukaemia viruses (MLV) followed by selection yielded a chimaeric clone with a completely new tropism for Chinese Hamster Ovary (CHOK1) cells. The composition and properties of the selected clone indicated that this particular permutation of parental sequences cannot be readily attained by natural retroviral recombination. This example demonstrates that molecular breeding can enhance the inherently high evolutionary potential of retroviruses to obtain desired phenotypes. It can be an effective tool, when information is limited, to optimize viruses for gene therapy and vaccine applications when multiple complex functions must be simultaneously balanced.

We measured the levels of a somatic, 4977 bp deletion of mitochondrial DNA (mtDNA4977) in paired neural retinal and optic nerve tissues from 14 adults and 1 infant using a quantitative PCR assay. MtDNA is prone to free radical damage, and areas in the brain that are exposed to high levels of free radicals are observed to accumulate higher levels of the mtDNA4977 deletion. The levels of mtDNA deletions also increase with age in many tissues. Despite the presence of a free radical rich environment, mtDNA from the neural retina possessed extremely low mtDNA4977 levels (0.0001-0.001%). Deletion levels were always lower than those in the optic nerve from the same eye and do not appear to increase with age. Our results suggest that antioxidant defenses in the neural retina are effective in protecting mtDNA against oxidative damage.

Publisher Summary This chapter describes polymerase chain reaction (PCR) techniques to measure the proportion of mitochondrial DNA (mtDNA 4977 ) to wild-type mtDNA. The quantitative method has been used to measure the levels of the deletion in different regions of the brains of aged individuals. The method can be used for the quantitation of other rare mtDNA deletions in brain or other tissues of humans or other species. The parameters for PCR need to be individually optimized and exponential ranges of amplification determined for each PCR target. Once these conditions are determined, the technique is quite reproducible. In diseases such as chronic progressive external ophthalmopelgia, Kearn-Sayre's syndrome, and Pearson's syndrome, a significant proportion (20-80%) of the mitochondrial DNA (mtDNA) carry large deletions of up to 10 kb. The chapter discusses PCR-based techniques used in the laboratory for the detection and semiquantitative and quantitative measurement of the levels of the mtDNA deletion, the so-called common deletion that removes 4977 bp of mtDNA sequence between two 13-bp repeats.

Publisher Summary This chapter discusses the use of polymerase chain reaction (PCR) in quantitative applications. Although the sensitivity of PCR has rendered it the method of choice in the detection of low amounts of nucleic acid target, its utility as a quantitative tool has been slower in gaining credibility. The ability of PCR to make rare sequences detectable also makes it extremely challenging to extrapolate the amounts of initial input template molecules from the measured amounts of product molecules after many cycles of amplification. In the past few years, a number of PCR strategies based on different methodological and technical approaches have been adopted for the semiquantitative or quantitative analysis of nucleic acids. These strategies deal with the basic problem of making the amplification process as predictable as possible. Because of the exponential nature of PCR, even minor variations in efficiency can lead to large deviations at the end of the reaction that will confound the quantitative relationship between the product and initial template. An understanding of the theoretical and technical aspects of the kinetics of PCR product accumulation is imperative to designing quantitative PCR experiments.

The levels of a specific mitochondrial DNA deletion (mtDNA4977) measured in 12 brain regions of 6 normal adults 39 to 82 years old exhibited striking variation among anatomical locations. Comparisons of the same region among individuals showed an increase of mtDNA4977 with age. The three regions with the highest levels, caudate, putamen and substantia nigra, are characterized by a high dopamine metabolism. The breakdown of dopamine by mitochondrial MAO produces H2O2 which can lead to oxygen radical formation. We suggest that mtDNA4977 may be the “tip of the iceberg” of the spectrum of somatic mutations produced by oxidative damage.

An assay that selectively amplifies a specific deletion of the mitochondrial genome has been used to study the extent of the deletion's accumulation in a variety of human tissues. The deletion occurs at much higher levels in nervous and muscle tissues than in all other tissues studied. The variation in deletion level between the same tissues in different persons of similar age appears to be less than the variation among tissues within an individual. Tests for artifactual explanations of the level differences were each negative. Three cellular parameters that are correlated with the level of the deletion are identified. The preferential accumulation of deleterious mitochondrial mutations in a restricted subset of aging human tissues may compound deficiencies of function in those tissues that accrue with age.