Daniel Elleder

Retroviruses in foreign species and the problem of provirus silencing.

Retroviruses are known to integrate in the host cell genome as proviruses, and therefore they are prone to cell-mediated control at the transcriptional and posttranscriptional levels. This plays an important role especially after retrovirus heterotransmission to foreign species, but also to differentiated cells. In addition to host cell-mediated blocks in provirus expression, also so far undefined host specificities, deciding upon the pathogenic manifestation of retrovirus heterotransmission, are in play. In this respect, we discuss especially the occurrence of wasting disease and immunodeficiency syndrome, which we established also in avian species using avian leukosis virus subgroup C (ALV-C) inoculated in mid-embryogenesis in duck or chicken embryos. The problem of provirus downregulation in foreign species or in differentiated cells has been in the recent years approached experimentally. From a series of observations it became apparent that provirus downregulation is mediated by its methylation, especially in the region of proviral enhancer-promoter located in long terminal repeats (LTR). Several strategies have been devised in order to protect the provirus from methylation using LTR modification and/or introducing in the LTR sequence motifs acting as antimethylation tags. In such a way the expression of retroviruses and vectors in foreign species, as well as in differentiated cells, has been significantly improved. The complexity of the mechanisms involved in provirus downregulation and further possibilities to modulate it are discussed.

Preferential integration of human immunodeficiency virus type 1 into genes, cytogenetic R bands and GC-rich DNA regions: insight from the human genome sequence

After entering a cell during infection, the human immunode¢ciency virus type 1 (HIV-1) undergoes a series of steps including reverse transcription of its genome and culminating in integration of proviral DNA into the host chromosomes. The further fate of the individual provirus is to a great extent in£uenced by the e⁄ciency of provirus transcription, dependent upon the site of its integration [1]. How HIV-1 and other retroviruses choose their integration sites is still far from completely understood. It seems that the integration is not strictly speci¢c, because most or all genomic regions are potential targets, but neither is it a random event. Locally, there are up to several hundred-fold di¡erences in the usage of target sites due to the local DNA structure, bending, distortion and wrapping around nucleosomes (reviewed in [2]). The non-randomness on the scale of genomic regions has been much less addressed [3]. Methods employed to study in vivo retrovirus integration sites include restriction enzyme digestions and blotting, £uorescence in situ hybridization, PCRbased assays, and most importantly cloning and sequencing the virus^host integration junctions. Most studies analyze only a small number of integration sites, or focus on selected genomic regions. To date, the most representative study is provided by Carteau et al. [4]. It lists a set of 61 HIV-1 integration site sequences obtained after short experimental infection of the human T-cell line SupT1. Of these, 59 sequences are available in GenBank, together with 104 control genomic sequences for comparison. The authors analyzed the sequences using the nr, dbEST and MONTH databases as of November 1997. They concluded that there is no signi¢cant di¡erence between integration sites and controls, except that centromeric alphoid repeats are selectively absent at integration sites. The availability of the human genome sequence [5] creates a great opportunity for a new genome-wide analysis of these data. By mapping the exact positions of the integration sites we can analyze large DNA regions £anking the proviruses and describe the genomic features present. We used the BLAT program to map the genomic positions of integration sites in the most recent GoldenPath assembly of 6 August 2001 (http://genome.ucsc.edu). Of 59 sequences available in GenBank, we succeeded in mapping 48, where the level of homology was satisfactory (Table 1 in supplementary material on the web; http://www.elsevier.com/PII/ S0014579302026121). For each mapped integration we collected several genomic features available in the GoldenPath assembly. The ¢rst was the presence of transcribed sequences, either as the ‘known protein coding genes’ category (from the RefSeq project) or as the ‘human mRNAs from GenBank’ category. In addition, 800 cytogenetic band resolution is available, light or dark according to Giemsa staining. Next we calculated the GC level of 100 kb regions surrounding the integration sites symmetrically and the gene densities along these £anking regions. We compared these data with the whole genome summary statistics that we calculated for the GoldenPath assembly and looked for any di¡erences indicating possible integration preference. In our analysis, 54.2% (26 of 48) of the mapped integration sites fall in genes, which is signi¢cantly higher compared to the genome average calculated as 22.2% (P6 0.00001, M2 test). For the broader category of mRNAs this comparison is 68.7% to 30.7% (P6 0.00001, M2 test). This implies that potentially transcribed regions represent strongly preferred targets for HIV-1 integration. Out of the 33 integrations in transcription units, 18 and 15 are in sense and antisense orientation, respectively, 28 map to introns, two to exons, two to 5P untranslated regions (UTR) and one to a 3P UTR. Giemsa light (R) and dark (G) bands were targeted in 68.7% and 31.2%, compared to genome averages of 44.0% and 48.5%, respectively, estimated from the GoldenPath assembly (P6 0.003, M2 test). The average GC content of 100 kb regions £anking the integration sites was calculated to be 44.4%, higher than the whole genome average of 41.0%. The distribution of integrations is clearly biased, with more hits belonging to the GCricher genomic regions (Fig. 1).

Hidden genes in birds

AbstractWe report that a subset of avian genes is characterized by very high GC content and long G/C stretches. These sequence characteristics correlate with the frequent absence of these genes from genomic databases. We provide several examples where genes in this subset are mistakenly reported as missing in birds. www.dx.doi.org/10.1186/s13059-015-0725-y

Two Different Molecular Defects in the Tva Receptor Gene Explain the Resistance of Two tvar Lines of Chickens to Infection by Subgroup A Avian Sarcoma and Leukosis Viruses

ABSTRACT The subgroup A to E avian sarcoma and leukosis viruses (ASLVs) are highly related and are thought to have evolved from a common ancestor. These viruses use distinct cell surface proteins as receptors to gain entry into avian cells. Chickens have evolved resistance to infection by the ASLVs. We have identified the mutations responsible for the block to virus entry in chicken lines resistant to infection by subgroup A ASLVs [ASLV(A)]. The tva genetic locus determines the susceptibility of chicken cells to ASLV(A) viruses. In quail, the ASLV(A) susceptibility allele tvas encodes two forms of the Tva receptor; these proteins are translated from alternatively spliced mRNAs. The normal cellular function of the Tva receptor is unknown; however, the extracellular domain contains a 40-amino-acid, cysteine-rich region that is homologous to the ligand binding region of the low-density lipoprotein receptor (LDLR) proteins. The chicken tvas cDNAs had not yet been fully characterized; we cloned the chicken tva cDNAs from two lines of subgroup A-susceptible chickens, line H6 and line 0. Two types of chicken tvas cDNAs were obtained. These cDNAs encode a longer and shorter form of the Tva receptor homologous to the Tva forms in quail. Two different defects were identified in cDNAs cloned from two different ASLV(A)-resistant inbred chickens, line C and line 72. Line C tvar contains a single base pair substitution, resulting in a cysteine-to-tryptophan change in the LDLR-like region of Tva. This mutation drastically reduces the binding affinity of TvaR for the ASLV(A) envelope glycoproteins. Line 72tvar2 contains a 4-bp insertion in exon 1 that causes a change in the reading frame, which blocks expression of the Tva receptor.

Polymorphic integrations of an endogenous gammaretrovirus in the mule deer genome

ABSTRACT Endogenous retroviruses constitute a significant genomic fraction in all mammalian species. Typically they are evolutionarily old and fixed in the host species population. Here we report on a novel endogenous gammaretrovirus (CrERVγ; for cervid endogenous gammaretrovirus) in the mule deer (Odocoileus hemionus) that is insertionally polymorphic among individuals from the same geographical location, suggesting that it has a more recent evolutionary origin. Using PCR-based methods, we identified seven CrERVγ proviruses and demonstrated that they show various levels of insertional polymorphism in mule deer individuals. One CrERVγ provirus was detected in all mule deer sampled but was absent from white-tailed deer, indicating that this virus originally integrated after the split of the two species, which occurred approximately one million years ago. There are, on average, 100 CrERVγ copies in the mule deer genome based on quantitative PCR analysis. A CrERVγ provirus was sequenced and contained intact open reading frames (ORFs) for three virus genes. Transcripts were identified covering the entire provirus. CrERVγ forms a distinct branch of the gammaretrovirus phylogeny, with the closest relatives of CrERVγ being endogenous gammaretroviruses from sheep and pig. We demonstrated that white-tailed deer (Odocoileus virginianus) and elk (Cervus canadensis) DNA contain proviruses that are closely related to mule deer CrERVγ in a conserved region of pol; more distantly related sequences can be identified in the genome of another member of the Cervidae, the muntjac (Muntiacus muntjak). The discovery of a novel transcriptionally active and insertionally polymorphic retrovirus in mammals could provide a useful model system to study the dynamic interaction between the host genome and an invading retrovirus.

Identification of a 3-aminoimidazo(1,2-a)pyridine inhibitor of HIV-1 reverse transcriptase

BackgroundDespite the effectiveness of highly active antiretroviral therapy (HAART), there remains an urgent need to develop new human immunodeficiency virus type 1 (HIV-1) inhibitors with better pharmacokinetic properties that are well tolerated, and that block common drug resistant virus strains.MethodsHere we screened an in-house small molecule library for novel inhibitors of HIV-1 replication.ResultsAn active compound containing a 3-aminoimidazo[1,2-a]pyridine scaffold was identified and quantitatively characterized as a non-nucleoside reverse transcriptase inhibitor (NNRTI).ConclusionsThe potency of this compound coupled with its inexpensive chemical synthesis and tractability for downstream SAR analysis make this inhibitor a suitable lead candidate for further development as an antiviral drug.

Endogenous lentivirus in Malayan colugo (Galeopterus variegatus), a close relative of primates

BackgroundA significant fraction of mammalian genomes is composed of endogenous retroviral (ERV) sequences that are formed by germline infiltration of various retroviruses. In contrast to other retroviral genera, lentiviruses only rarely form ERV copies. We performed a computational search aimed at identification of novel endogenous lentiviruses in vertebrate genomes.FindingsUsing the in silico strategy, we have screened 104 publicly available vertebrate genomes for the presence of endogenous lentivirus sequences. In addition to the previously described cases, the search revealed the presence of endogenous lentivirus in the genome of Malayan colugo (Galeopterus variegatus). At least three complete copies of this virus, denoted ELVgv, were detected in the colugo genome, and approximately one hundred solo LTR sequences. The assembled consensus sequence of ELVgv had typical lentivirus genome organization including three predicted accessory genes. Phylogenetic analysis placed this virus as a distinct subgroup within the lentivirus genus. The time of insertion into the dermopteran lineage was estimated to be more than thirteen million years ago.ConclusionsWe report the discovery of the first endogenous lentivirus in the mammalian order Dermoptera, which is a taxon close to the Primates. Lentiviruses have infiltrated the mammalian germline several times across millions of years. The colugo virus described here represents possibly the oldest documented endogenization event and its discovery can lead to new insights into lentivirus evolution. This is also the first report of an endogenous lentivirus in an Asian mammal, indicating a long-term presence of this retrovirus family in Asian continent.

Discovery of an endogenous Deltaretrovirus in the genome of long-fingered bats (Chiroptera: Miniopteridae)

Significance Retroviruses copy their RNA genome into complementary DNA, which is then inserted into the host chromosomal DNA as an obligatory part of their life cycle. Such integrated viral sequences, called proviruses, are passed to the infected cell progeny on cellular division. If germline cells are targeted, the proviruses become vertically inherited as other host genes and are called endogenous retroviruses. Deltaretroviruses, which include important human and veterinary pathogens (HTLV-1 and BLV), are the last retroviral genus for which endogenous forms were not known. We have identified a case of endogenous Deltaretrovirus, which entered the genome of long-fingered bat ancestors more than 20 million years ago. This finding opens the way for elucidating the deep evolutionary history of deltaretroviruses. Retroviruses can create endogenous forms on infiltration into the germline cells of their hosts. These forms are then vertically transmitted and can be considered as genetic fossils of ancient viruses. All retrovirus genera, with the exception of deltaretroviruses, have had their representation identified in the host genome as a virus fossil record. Here we describe an endogenous Deltaretrovirus, identified in the germline of long-fingered bats (Miniopteridae). A single, heavily deleted copy of this retrovirus has been found in the genome of miniopterid species, but not in the genomes of the phylogenetically closest bat families, Vespertilionidae and Cistugonidae. Therefore, the endogenization occurred in a time interval between 20 and 45 million years ago. This discovery closes the last major gap in the retroviral fossil record and provides important insights into the history of deltaretroviruses in mammals.

Characterization of Two Historic Smallpox Specimens from a Czech Museum

Although smallpox has been known for centuries, the oldest available variola virus strains were isolated in the early 1940s. At that time, large regions of the world were already smallpox-free. Therefore, genetic information of these strains can represent only the very last fraction of a long evolutionary process. Based on the genomes of 48 strains, two clades are differentiated: Clade 1 includes variants of variola major, and clade 2 includes West African and variola minor (Alastrim) strains. Recently, the genome of an almost 400-year-old Lithuanian mummy was determined, which fell basal to all currently sequenced strains of variola virus on phylogenetic trees. Here, we determined two complete variola virus genomes from human tissues kept in a museum in Prague dating back 60 and 160 years, respectively. Moreover, mass spectrometry-based proteomic, chemical, and microscopic examinations were performed. The 60-year-old specimen was most likely an importation from India, a country with endemic smallpox at that time. The genome of the 160-year-old specimen is related to clade 2 West African and variola minor strains. This sequence likely represents a new endemic European variant of variola virus circulating in the midst of the 19th century in Europe.

Induction and characterization of a replication competent cervid endogenous gammaretrovirus (CrERV) from mule deer cells.

Endogenous retroviruses (ERVs) were acquired during evolution of their host organisms after infection and mendelian inheritance in the germline by their exogenous counterparts. The ERVs can spread in the host genome and in some cases they affect the host phenotype. The cervid endogenous gammaretrovirus (CrERV) is one of only a few well-defined examples of evolutionarily recent invasion of mammalian genome by retroviruses. Thousands of insertionally polymorphic CrERV integration sites have been detected in wild ranging mule deer (Odocoileus hemionus) host populations. Here, we describe for the first time induction of replication competent CrERV by cocultivation of deer and human cells. We characterize the physical properties and tropism of the induced virus. The genomic sequence of the induced virus is phylogenetically related to the evolutionarily young endogenous CrERVs described so far. We also describe the level of replication block of CrERV on deer cells and its capacity to establish superinfection interference.