Characterization of Human Endogenous Retrovirus Type K Virus-like Particles Generated from Recombinant Baculoviruses

AbstractThe family of human endogenous retrovirus type K (HERV-K) comprises members with long open reading frames (ORF) for retroviral proteins. The existence of a biologically active provirus with replicative capacities has not yet been demonstrated. To confirm the assumption that HERV-K codes for the previously observed retrovirus-like particles (human teratocarcinoma-derived virus, HTDV) in human teratocarcinoma cells, we have constructed recombinant full-length HERV-K cDNA-based baculoviruses withgag, pro, pol,andenvORFs. Two viral constructs were used for infections of insect cells, one bearing 67 bp of the 5′ untranslated region upstream of the 5′ splice donor (SD) site and of the retroviral genes, the second omitting the SD sequence. For both recombinant viruses, indirect immunofluorescence and laser scan analyses revealed expression of HERV-K Gag protein. Electron microscopy studies demonstrated efficient production of virus-like particles (VLPs) at the cytoplasmic cell membranes. These VLPs are morphologically identical with the HTDV phenotype. In immunoelectron microscopy of ultrathin frozen sections, anti-HERV-K Gag antibodies specifically reacted with HERV-K VLPs. In Western blots, in addition to the 76-kDa precursor protein, the putative major core protein with an apparent molecular mass of 32 kDa exhibited predominant immunoreactivity with anti-Gag antiserum. In contrast, neither HERV-K Env nor cORF proteins could be detected due to inefficient mRNA splicing. Purified particles from insect cell culture supernatants tested in an ultrasensitive reverse transcriptase assay revealed weak polymerase activity. The data demonstrate that HERV-K codes for retroviral particles of the HTDV phenotype

Perturbation Analysis of the Kuramoto Phase Diffusion Equation Subject to Quenched Frequency Disorder

The Kuramoto phase diffusion equation is a nonlinear partial differential equation which describes the spatio-temporal evolution of a phase variable in an oscillatory reaction diffusion system. Synchronization manifests itself in a stationary phase gradient where all phases throughout a system evolve with the same velocity, the synchronization frequency. The formation of concentric waves can be explained by local impurities of higher frequency which can entrain their surroundings. Concentric waves in synchronization also occur in heterogeneous systems, where the local frequencies are distributed randomly. We present a perturbation analysis of the synchronization frequency where the perturbation is given by the heterogeneity of natural frequencies in the system. The nonlinearity in form of dispersion, leads to an overall acceleration of the oscillation for which the expected value can be calculated from the second order perturbation terms. We apply the theory to simple topologies, like a line or the sphere, and deduce the dependence of the synchronization frequency on the size and the dimension of the oscillatory medium. We show that our theory can be extended to include rotating waves in a medium with periodic boundary conditions. By changing a system parameter the synchronized state may become quasi degenerate. We demonstrate how perturbation theory fails at such a critical point.Comment: 22 pages, 5 figure

Zipf law in the popularity distribution of chess openings

We perform a quantitative analysis of extensive chess databases and show that the frequencies of opening moves are distributed according to a power-law with an exponent that increases linearly with the game depth, whereas the pooled distribution of all opening weights follows Zipf's law with universal exponent. We propose a simple stochastic process that is able to capture the observed playing statistics and show that the Zipf law arises from the self-similar nature of the game tree of chess. Thus, in the case of hierarchical fragmentation the scaling is truly universal and independent of a particular generating mechanism. Our findings are of relevance in general processes with composite decisions.Comment: 5 pages, 4 figure

Third WHO Global Consultation on regulatory requirements for xenotransplantation clinical trials, Changsha, Hunan, China December 12-14, 2018: "The 2018 Changsha Communiqué" The 10-Year Anniversary of The International Consultation on Xenotransplantation

After feedback from the working parties, the final session focused on drafting proposed revisions of the WHO documents, and resulted in the formulation of the draft “Third WHO Global Consultation on Regulatory Requirements for Xenotransplantation Clinical Trials, The 2018 Changsha Communiqué.” This draft was submitted to WHO in February 2019 for WHO and World Health Assembly consideration. If approved, the 2018 Changsha Communiqué will then be posted on the websites of WHO, IXA, and TTS, and published in Xenotransplantation. This report includes summaries of the various sessions, followed by the abstracts of invited speakers from the update sessions

Relative Age of Proviral Porcine Endogenous Retrovirus Sequences in Sus scrofa Based on the Molecular Clock Hypothesis

Porcine endogenous retroviruses (PERV) are discussed as putative infectious agents in xenotransplantation. PERV classes A, B, and C harbor different envelope proteins. Two different types of long terminal repeat (LTR) structures exist, of which both are present only in PERV-A. One type of LTR contains a distinct repeat structure in U3, while the other is repeatless, conferring a lower level of transcriptional activity. Since the different LTR structures are distributed unequally among the proviruses and, apparently, PERV is the only virus harboring two different LTR structures, we were interested in determining which LTR is the ancestor. Replication-competent viruses can still be found today, suggesting an evolutionary recent origin. Our studies revealed that the age of PERV is at most 7.6 × 10(6) years, whereas the repeatless LTR type evolved approximately 3.4 × 10(6) years ago, being the phylogenetically younger structure. The age determined for PERV correlates with the time of separation between pigs (Suidae, Sus scrofa) and their closest relatives, American-born peccaries (Tayassuidae, Pecari tajacu), 7.4 × 10(6) years ago

Infection Barriers to Successful Xenotransplantation Focusing on Porcine Endogenous Retroviruses

Xenotransplantation may be a solution to overcome the shortage of organs for the treatment of patients with organ failure, but it may be associated with the transmission of porcine microorganisms and the development of xenozoonoses. Whereas most microorganisms may be eliminated by pathogen-free breeding of the donor animals, porcine endogenous retroviruses (PERVs) cannot be eliminated, since these are integrated into the genomes of all pigs. Human-tropic PERV-A and -B are present in all pigs and are able to infect human cells. Infection of ecotropic PERV-C is limited to pig cells. PERVs may adapt to host cells by varying the number of LTR-binding transcription factor binding sites. Like all retroviruses, they may induce tumors and/or immunodeficiencies. To date, all experimental, preclinical, and clinical xenotransplantations using pig cells, tissues, and organs have not shown transmission of PERV. Highly sensitive and specific methods have been developed to analyze the PERV status of donor pigs and to monitor recipients for PERV infection. Strategies have been developed to prevent PERV transmission, including selection of PERV-C-negative, low-producer pigs, generation of an effective vaccine, selection of effective antiretrovirals, and generation of animals transgenic for a PERV-specific short hairpin RNA inhibiting PERV expression by RNA interference

Evolutionary Spread and Recombination of Porcine Endogenous Retroviruses in the Suiformes

Different Suiformes with increasing phylogenetic distance to the common pig (Sus scrofa) were assayed for the presence of porcine endogenous retroviruses (PERV) in general (pol gene), while the distribution of long terminal repeat (LTR) types (with or without repeats in U3) and env genes (classes A, B, and C) were determined in detail. PERV was not detectable in the most distantly related species, while classes PERV-A and PERV-B are present in Suiformes originating in the Pliocene epoch, and class PERV-C was detectable only in S. scrofa and in closely related species originating in the Holocene epoch. This distribution pattern of PERV classes is in line with our previous study on the age of PERV (45) and suggests an African origin of about 7.5 million years ago (MYA) and a gradual spread of PERV through the Suiformes. It seems likely that PERV-C originated more recently (1.5 to 3.5 MYA) by recombination with a homologue of unknown descent, while the origin of the repeatless LTR was a separate event approximately 3.5 MYA

Analyses of prevalence and polymorphisms of six replication-competent and chromosomally assigned porcine endogenous retroviruses in individual pigs and pig subspecies

AbstractAs porcine endogenous retroviruses (PERV) productively infect human cells in vitro, they pose a serious risk in xenotransplantation and xenogeneic cell therapies. We have analyzed the prevalence of six well-characterized full-length PERV, five of them being replication-competent and four of them being chromosomally assigned (J. Virol. 75 (2001) 5465; J. Virol. 76 (2002) 2714). These analyses revealed a heterogeneous distribution of PERV among individuals and, as no PERV is present in every pig, it seems feasible to generate pigs free of functional PERV by conventional breeding. Conversely, as PERV are polymorphic, single proviruses may have escaped detection and this kind of assay must be performed for every herd used in xenotransplantation or xenogeneic cell therapies. In addition, specific proviruses show internal point mutations which significantly affect their replicational capacities. As there are two different types of PERV LTR structures showing varying levels of transcriptional capacity (J. Virol. 75 (2001) 6933), an analysis of 21 distinct chromosomal locations revealed that PERV which harbor highly active LTRs with repeat elements in U3 are dominant

The transcription factor Sp1 regulates the myeloid-specific expression of the human hematopoietic cell kinase (HCK) gene through binding to two adjacent GC boxes within the HCK promoter-proximal region

The human hemopoietic cell kinase (HCK) is a member of the src family of protein tyrosine kinases specifically expressed in myeloid cells and to a minor extent in B-lymphoid cells. HCK expression is up-regulated at the transcriptional level during myeloid differentiation of hematopoietic cells. To elucidate the molecular basis of the differential HCK gene expression, the genomic region containing the HCK promoter was isolated and functionally characterized. A DNA fragment containing 101 base pairs of the 5′-flanking sequence showed strong promoter activity in the macrophage cell line RAW264 but was inactive in the non-monocytic cell lines HUT-78 and NIH-3T3. Site-directed mutagenesis of the proximal promoter region showed that two GC-rich sequence elements are essential for transcriptional activity in myeloid cells. Electrophoretic mobility shift analysis using nuclear extracts obtained from RAW264 cells and from the promonocytic cell line U-937 revealed the formation of at least three distinct protein-DNA complexes at each of these sites, one of which was found to contain the transcription factor Sp1. Expression of a reporter gene linked to the −101HCK promoter region was up-regulated by Sp1, but not by other members of the Sp1 family of transcription factors, in Drosophila Schneider cells. A synergistic effect onHCK promoter activity was observed at high concentrations of Sp1. Our results show that Sp1 plays an essential role in the regulation of the differential gene expression of the HCKgene