In vivo evolution of an emerging zoonotic bacterial pathogen in an immunocompromised human host.

Zoonotic transfer of animal pathogens to human hosts can generate novel agents, but the genetic events following such host jumps are not well studied. Here we characterize the mechanisms driving adaptive evolution of the emerging zoonotic pathogen Bordetella hinzii in a patient with interleukin-12 receptor ß1 deficiency. Genomic sequencing of 24 B. hinzii isolates cultured from blood and stool over 45 months revealed a clonal lineage that had undergone extensive within-host genetic and phenotypic diversification. Twenty of 24 isolates shared an E9G substitution in the DNA polymerase III ε-subunit active site, resulting in a proofreading deficiency. Within this proofreading-deficient clade, multiple lineages with mutations in DNA repair genes and altered mutational spectra emerged and dominated clinical cultures for more than 12 months. Multiple enzymes of the tricarboxylic acid cycle and gluconeogenesis pathways were repeatedly mutated, suggesting rapid metabolic adaptation to the human environment. Furthermore, an excess of G:C > T:A transversions suggested that oxidative stress shaped genetic diversification during adaptation. We propose that inactivation of DNA proofreading activity in combination with prolonged, but sub-lethal, oxidative attack resulting from the underlying host immunodeficiency facilitated rapid genomic adaptation. These findings suggest a fundamental role for host immune phenotype in shaping pathogen evolution following zoonotic infection.

Differences in HERV-K LTR insertions in orthologous loci of humans and great apes.

The classification of the long terminal repeats (LTRs) of the human endogenous retrovirus HERV-K (HML-2) family was refined according to diagnostic differences between the LTR sequences. The mutation rate was estimated to be approximately equal for LTRs belonging to different families and branches of human endogenous retroviruses (HERVs). An average mutation rate value was calculated based on differences between LTRs of the same HERV and was found to be 0.13% per million years (Myr). Using this value, the ages of different LTR groups belonging to the LTR HML-2 subfamily were found to vary from 3 to 50Myr. Orthologous potential LTR-containing loci from different primate species were PCR amplified using primers corresponding to the genomic sequences flanking LTR integration sites. This allowed us to calculate the phylogenetic times of LTR integrations in primate lineages in the course of the evolution and to demonstrate that they are in good agreement with the LTR ages calculated from the mutation rates. Human-specific integrations for some very young LTRs were demonstrated. The possibility of LTRs and HERVs involvement in the evolution of primates is discussed.

Long terminal repeats of human endogenous retrovirus K family (HERV-K) specifically bind host cell nuclear proteins.

Solitary long terminal repeats (LTRs) of the human endogenous retroviruses, scattered in several thousand copies throughout the human genome, are potentially capable of affecting the expression of closely located genes. To assess their regulatory potential, the LTR sequences of one of the most abundant HERV families (HERV-K) were screened for the presence of binding sites for the host cell nuclear factors using mobility shift and UV-crosslinking assays. It was shown that the LTR sequences of two subfamilies harbor a specific binding site for a complex consisting of at least three proteins, ERF1, ERF2 and ERF3 of 98, 91 and 88 kDa apparent molecular mass, respectively. This binding site is located in the 5' region of the LTR U3 element. The preservation of the specific protein binding site in different HERV-K LTR sequences suggests their possible role in regulation of nearby located genes.

[Long terminal repeat of the human endogenous retrovirus HERV-K in the intron of the ZNF91 gene]. / Dlinnyi kontsevoi povtor éndogennogo retrovirusa cheloveka HERV-K v introne gena ZNF91.

The relative positions of the ZNF91 gene (exon-intron organization) and the HERV-K human endogenous virus long terminal repeat (LTR), which was earlier found to be located in the ZNF91 gene locus, on the EcoRI restriction metric map of the human chromosome 19 were determined with a high resolution. The direction of the ZNF91 gene transcription relative to the chromosome 19 telomeres was determined. The HERV-K LTR was localized to the ZNF91 gene intron 19. The role of retroviral sequences in the evolution of the ZNF91 gene family is discussed.

[New putative gene from human retrovirus-containing locus on chromosome 19]. / Novyi potentsial'nyi gen is retrovirusosoderzhashchego lokusa 19-i khromosomy cheloveka.

By means of cDNA selection, a cDNA clone corresponding to a putative gene was isolated. The identified gene was mapped to human chromosome 19 (region 19q12) to the locus containing the human endogenous retrovirus HERV-K LTR. The distance between the LTR and the cDNA fragment is about 20 kb. RT-PCR analysis of the total RNA revealed that the corresponding gene is preferentially expressed in the brain.

[Structural characteristics of four long terminal repeats (LTR) of human endogenous retroviruses and features of their integration sites]. / Strukturnye kharakteristiki chetyrekh dlinnykh kontsevykh povtorov (LTR) endogennykh chelovecheskikh virusov i osobennosti uchastkov ikh integratsii.

Four LTR-containing regions of human chromosome 19 were sequenced by the primer walking technique using strings of short oligonucleotides tightly bound to the template. A comparative and evolutionary analysis of sequences homologous to human endogenous retroviruses (HERV) was performed, and the prototypes of the LTRs were determined. Analysis of the chromosome 19 sequences adjacent to LTR revealed that LTRs of HERV-K share a common location with other retroposons.

Variation in patterns of human meiotic recombination.

In the last 30 years it has become evident that patterns of meiotic recombination can be highly variable among individuals. The evidence comes from both low and high resolution analyses of hotspots of recombination in human and other species. In addition, a comparison of the recombination profiles in closely related species such as human and chimpanzee reveals essentially no correlation in the position of hotspots. Although the variation in hotspots of meiotic recombination is clearly documented, the mechanisms responsible for such variation are far from being understood. Here we will review the available evidence of natural variation in meiotic recombination and will discuss potential implications of this variation on the functional mechanisms of crossover formation and control.

A human endogenous retrovirus-like (HERV) LTR formed more than 10 million years ago due to an insertion of HERV-H LTR into the 5' LTR of HERV-K is situated on human chromosomes 10, 19 and Y.

A chimeric long terminal repeat (LTR) containing the whole LTR of a human endogenous retrovirus-like element of the H family (HERV-H) inserted downstream of the core enhancer region of the 5' LTR of a HERV-K retroelement was detected and sequenced in the human 19p12 locus, known to be enriched with genes encoding zinc finger proteins. Similar chimeras were also detected in human chromosomes 10 and Y in human-hamster hybrid cells containing individual human chromosomes. This finding was interpreted as evidence of transpositions of the chimera in the genome. PCR analyses detected the chimera in the genomes of chimpanzee and gorilla, but not in that of orangutan. These data demonstrate that the chimera appeared in the primate germ cells more than 10 million years ago, before divergence of the human/chimpanzee and the gorilla lineages. The combination of the two LTRs forms a new regulatory system that can be involved in nearby gene expression.

High resolution mapping DNAs by R-loop atomic force microscopy.

R-loops formed by short RNA transcripts have been imaged by atomic force microscopy (AFM) at a constant force in the height mode. The technique was applied to mapping the human endogenous retrovirus K10 family (HERV-K10) long terminal repeats (LTR) within individual plasmids and cosmids. RNA probes specific for the U3 (384 nt) and U5 (375 nt) LTR regions separated by a span of 200 bp were used for R-loop formation with LTRs located within plasmid (3.8 kb) or cosmid ( approximately 40 kb) DNAs. R-loops stabilized by glyoxal treatment and adsorbed onto the mica surface in the presence of magnesium ions looked like looped out segments of RNA:DNA hybrids. The total yield of R-loops was usually approximately 95%. The RNA:DNA hybrids were found to be 12-15% shorter than the corresponding DNA:DNA duplex. The two regions of the LTR could be easily discerned in the AFM images as clearly separated loops. R-loop positions determined on cosmids by AFM were accurate to approximately 0.5% of the cosmid length. This technique might be easily adapted for mapping various sequences such as gene exons or regulatory regions and for detecting insertions, deletions and rearrangements that cause human genetic diseases.