Human endogenous retrovirus-K (HERV-K) reverse transcriptase (RT) structure and biochemistry reveals remarkable similarities to HIV-1 RT and opportunities for HERV-K-specific inhibition.

Human endogenous retroviruses (HERVs) comprise nearly 8% of the human genome and are derived from ancient integrations of retroviruses into the germline. The biology of HERVs is poorly defined, but there is accumulating evidence supporting pathological roles in diverse diseases, such as cancer, autoimmune, and neurodegenerative diseases. Functional proteins are produced by HERV-encoded genes, including reverse transcriptases (RTs), which could be a contributor to the pathology attributed to aberrant HERV-K expression. To facilitate the discovery and development of HERV-K RT potent and selective inhibitors, we expressed active HERV-K RT and determined the crystal structure of a ternary complex of this enzyme with a double-stranded DNA substrate. We demonstrate a range of RT inhibition with antiretroviral nucleotide analogs, while classic nonnucleoside analogs do not inhibit HERV-K RT. Detailed comparisons of HERV-K RT with other known RTs demonstrate similarities to diverse RT families and a striking similarity to the HIV-1 RT asymmetric heterodimer. Our analysis further reveals opportunities for selective HERV-K RT inhibition.

Susceptibility of Human Endogenous Retrovirus Type K to Reverse Transcriptase Inhibitors.

Human endogenous retroviruses (HERVs) make up 8% of the human genome. The HERV type K (HERV-K) HML-2 (HK2) family contains proviruses that are the most recent entrants into the human germ line and are transcriptionally active. In HIV-1 infection and cancer, HK2 genes produce retroviral particles that appear to be infectious, yet the replication capacity of these viruses and potential pathogenicity has been difficult to ascertain. In this report, we screened the efficacy of commercially available reverse transcriptase inhibitors (RTIs) at inhibiting the enzymatic activity of HK2 RT and HK2 genomic replication. Interestingly, only one provirus, K103, was found to encode a functional RT among those examined. Several nucleoside analogue RTIs (NRTIs) blocked K103 RT activity and consistently inhibited the replication of HK2 genomes. The NRTIs zidovudine (AZT), stavudine (d4T), didanosine (ddI), and lamivudine (3TC), and the nucleotide RTI inhibitor tenofovir (TDF), show efficacy in blocking K103 RT. HIV-1-specific nonnucleoside RTIs (NNRTIs), protease inhibitors (PIs), and integrase inhibitors (IIs) did not affect HK2, except for the NNRTI etravirine (ETV). The inhibition of HK2 infectivity by NRTIs appears to take place at either the reverse transcription step of the viral genome prior to HK2 viral particle formation and/or in the infected cells. Inhibition of HK2 by these drugs will be useful in suppressing HK2 infectivity if these viruses prove to be pathogenic in cancer, neurological disorders, or other diseases associated with HK2. The present studies also elucidate a key aspect of the life cycle of HK2, specifically addressing how they do, and/or did, replicate.IMPORTANCE Endogenous retroviruses are relics of ancestral virus infections in the human genome. The most recent of these infections was caused by HK2. While HK2 often remains silent in the genome, this group of viruses is activated in HIV-1-infected and cancer cells. Recent evidence suggests that these viruses are infectious, and the potential exists for HK2 to contribute to disease. We show that HK2, and specifically the enzyme that mediates virus replication, can be inhibited by a panel of drugs that are commercially available. We show that several drugs block HK2 with different efficacies. The inhibition of HK2 replication by antiretroviral drugs appears to occur in the virus itself as well as after infection of cells. Therefore, these drugs might prove to be an effective treatment by suppressing HK2 infectivity in diseases where these viruses have been implicated, such as cancer and neurological syndromes.

Expression of the pol gene of human endogenous retroviruses HERV-K and -W in leukemia patients.

The human endogenous retroviruses (HERVs) are a family of endogenous retroviruses that integrated into the germ cell DNA of primates over 30 million years ago. HERV expression seems impaired in several diseases, ranging from autoimmune to neoplastic disorders. The purpose of this study was to evaluate the overall endogenous retroviral transcription profile in bone marrow (BM) samples. A total of 30 paediatric high-risk leukaemia patients (lymphoid and myeloid malignancies) were tested for HERVs virus gene expression. Our findings show that HERV-K expression was significantly higher in leukaemia patients when compared to healthy donors of a similar median age. We observed a significantly high expression of HERV-K in acute lymphoblastic leukemia (ALL) patients. In this study, we also found a relative overexpression of the endogenous retrovirus HERV-K in BM cells from the majority of leukemia samples analyzed, in particular in ALL. This overexpression might be related to lymphatic leukemogenesis and it warrants further investigations.

Porcine endogenous retrovirus-A/C: biochemical properties of its integrase and susceptibility to raltegravir.

Porcine endogenous retroviruses (PERVs) are present in the genomes of pig cells. The PERV-A/C recombinant virus can infect human cells and is a major risk of zoonotic disease in the case of xenotransplantation of pig organs to humans. Raltegravir (RAL) is a viral integrase (IN) inhibitor used in highly active antiretroviral treatment. In the present study, we explored the potential use of RAL against PERV-A/C. We report (i) a three-dimensional model of the PERV-A/C intasome complexed with RAL, (ii) the sensitivity of PERV-A/C IN to RAL in vitro and (iii) the sensitivity of a PERV-A/C-IRES-GFP recombinant virus to RAL in cellulo. We demonstrated that RAL is a potent inhibitor against PERV-A/C IN and PERV-A/C replication with IC50s in the nanomolar range. To date, the use of retroviral inhibitors remains the only way to control the risk of zoonotic PERV infection during pig-to-human xenotransplantation.

The Roles of Syncytin-Like Proteins in Ruminant Placentation.

Recent developments in genome sequencing techniques have led to the identification of huge numbers of endogenous retroviruses (ERV) in various mammals. ERVs, which occupy 8%-13% of mammalian genomes, are believed to affect mammalian evolution and biological diversity. Although the functional significance of most ERVs remains to be elucidated, several ERVs are thought to have pivotal roles in host physiology. We and other groups recently identified ERV envelope proteins (e.g., Fematrin-1, Syncytin-Rum1, endogenous Jaagsiekte sheep retrovirus Env) that may determine the morphogenesis of the unique fused trophoblast cells, termed trinucleate cells and syncytial plaques, found in ruminant placentas; however, there are still a number of outstanding issues with regard to the role of ERVs that remain to be resolved. Here, we review what is known about how these ERVs have contributed to the development of ruminant-specific trophoblast cells.

ERVK polyprotein processing and reverse transcriptase expression in human cell line models of neurological disease.

Enhanced expression of the reverse transcriptase (RT) protein encoded by human endogenous retrovirus-K (ERVK) is a promising biomarker for several inflammatory and neurological diseases. However, unlike RT enzymes encoded by exogenous retroviruses, little work has been done to identify ERVK RT isoforms, their expression patterns, and cellular localization. Using Western blot, we showcase the ERVK gag-pro-pol polyprotein processing leading to the production of several ERVK RT isoforms in human neuronal (ReNcell CX) and astrocytic (SVGA) models of neuroinflammatory disease. Since the pro-inflammatory cytokine IFNγ plays a key role in the pathology of several ERVK-associated neurological diseases, we sought to determine if IFNγ can drive ERVK RT expression. IFNγ signalling markedly enhanced ERVK polyprotein and RT expression in both human astrocytes and neurons. RT isoforms were expressed in a cell-type specific pattern and the RT-RNase H form was significantly increased with IFNγ treatment. Fluorescent imaging revealed distinct cytoplasmic, perinuclear and nuclear ERVK RT staining patterns upon IFNγ stimulation of astrocytes and neurons. These findings indicate that ERVK expression is inducible under inflammatory conditions such as IFNγ exposure-and thus, these newly established in vitro models may be useful in exploring ERVK biology in the context of neuroinflammatory disease.

Elevated HERV-K mRNA expression in PBMC is associated with a prostate cancer diagnosis particularly in older men and smokers.

Aberrant expression of subgroup k human endogenous retroviruses (HERV-K) has been observed in prostate cancer. This subgroup is unique because it encodes sequences in the human genome containing open reading frames for near intact retroviruses. We hypothesized that HERV-K reactivation could serve as a non-invasive early disease detection marker for prostate cancer. We evaluated HERV-K gag messenger RNA (mRNA) expression in blood samples of African-American and European-American men using a case-control design via quantitative real-time PCR. Additionally, we examined HERV-K envelope protein expression in prostate tumors by immunohistochemistry. HERV-K envelope protein was commonly upregulated in prostate tumors, but more so in tumors of African-American than European-American patients (61% versus 40%, P < 0.01). Examining HERV-K gag expression in peripheral blood mononuclear cells (PBMC) from 294 cases and 135 healthy men, we found that the abundance of HERV-K gag message was significantly higher in cases than controls and was associated with increased plasma interferon-γ. Men with gag expression in the highest quartile had >12-fold increased odds {odds ratio = 12.87 [95% confidence interval 6.3-26.25]} of being diagnosed with prostate cancer than those in the lowest quartile. Moreover, our results showed that HERV-K expression may perform better as a disease biomarker in older than younger men (whereas the sensitivity of prostate-specific antigen (PSA) testing decreases with age) and in men with a smoking history compared with never smokers. Combining non-invasive HERV-K testing with PSA testing may improve the efficacy of prostate cancer detection specifically among older men and smokers who tend to develop a more aggressive disease.

De novo DNA methylation of endogenous retroviruses is shaped by KRAB-ZFPs/KAP1 and ESET.

Endogenous retroviruses (ERVs) undergo de novo DNA methylation during the first few days of mammalian embryogenesis, although the factors that control the targeting of this process are largely unknown. We asked whether KAP1 (KRAB-associated protein 1) is involved in this mechanism because of its previously defined role in maintaining the silencing of ERVs through the histone methyltransferase ESET and histone H3 lysine 9 trimethylation. Here, we demonstrate that introduced ERV sequences are sufficient to direct rapid de novo methylation of a flanked promoter in embryonic stem (ES) cells. This mechanism requires the presence of an ERV sequence-recognizing KRAB zinc-finger protein (ZFP) and both KAP1 and ESET. Furthermore, this process can also take place on a strong cellular promoter and leads to methylation signatures that are subsequently maintained in vivo throughout embryogenesis. Finally, we show that methylation of ERVs residing in the genome is affected by knockout of KAP1 in early embryos. KRAB-ZFPs, KAP1 and ESET are thus likely to be responsible for the early embryonic instatement of stable epigenetic marks at ERV-containing loci.

Detection of RD-114 virus by a reverse transcriptase assay based on product enhancement.

RD-114 virus is a feline endogenous retrovirus that exists in the genome of all cats. It can be assumed that feline and canine live vaccines manufactured by culturing cells of feline origin are contaminated with the virus. The current study is attempted to develop a product enhanced reverse transcriptase (PERT) assay to detect replication-competent RD-114 virus. Since culture supernatants of Crandell-Rees feline kidney (CRFK) cells do not have detectable reverse transcriptase activity in case of do not passage on other cell lines, these results raise the possibility that RD-114 virus grow efficiently in other retrovirus producing cell lines. For the PERT assay, RD-114 virus isolated from CRFK cells may need to be passaged more than four times on 293T cells to be expressed at appreciable levels. The PERT assay described here provides an accurate method for determining the presence of RD-114 in culture supernatants and will help monitor live vaccines produced in endogenous virus producing cell lines.

Protective effect of human endogenous retrovirus K dUTPase variants on psoriasis susceptibility.

Previous genetic and functional studies have implicated the human endogenous retrovirus K (HERV-K) dUTPase located within the PSORS1 locus in the major histocompatibility complex region as a candidate psoriasis gene. Here, we describe a variant discovery and case-control association study of HERV-K dUTPase variants in 708 psoriasis cases and 349 healthy controls. Five common HERV-K dUTPase variants were found to be highly associated with psoriasis, with the strongest association occurring at the missense single-nucleotide polymorphism (SNP) rs3134774 (K158R, P=3.28 × 10(-15), odds ratio =2.36 (95% confidence interval: 1.91-2.92)). After adjusting the association of the HERV-K dUTPase variants for the potential confounding effects of HLA alleles associated with psoriasis, the HERV-K SNPs rs9264082 and rs3134774 remained significantly associated. Haplotype analysis revealed that HERV-K haplotypes containing the non-risk alleles for rs3134774 and rs9264082 significantly reduced the risk of psoriasis. Functional testing showed higher antibody responses against recombinant HERV-K dUTPase in psoriasis patients compared with controls (P<0.05), as well as higher T-cell responses against a single HERV-K dUTPase peptide (P<0.05). Our data support an independent role for the HERV-K dUTPase on psoriasis susceptibility, and suggest the need for additional studies to clarify the role of this dUTPase in the pathogenesis of psoriasis.

A human endogenous retrovirus K dUTPase triggers a TH1, TH17 cytokine response: does it have a role in psoriasis?

Psoriasis is a chronic inflammatory immune disease of the skin characterized by a complex interplay between multiple risk genes and their interactions with environmental factors. Recent haplotype analyses have suggested that deoxyuridine triphosphate nucleotidohydrolase (dUTPase) encoded by a human endogenous retrovirus K (HERV-K) may be a candidate gene for the psoriasis susceptibility 1 locus. However, no functional studies have been conducted to determine the role of HERV-K dUTPase in psoriasis. For this purpose, we constructed an HERV-K dUTPase wild-type sequence, as well as specific mutations reflecting the genotype characteristic of high- and low-risk haplotypes, purified the recombinant proteins, and evaluated whether they could modulate innate and/or adaptive immune responses. In this study, we demonstrate that wild-type and mutant HERV-K dUTPase proteins induce the activation of NF-κB through Toll-like receptor 2, independent of enzymatic activity. Proteome array studies revealed that treatment of human primary cells with wild-type and mutant HERV-K dUTPase proteins triggered the secretion of T(H)1 and T(H)17 cytokines involved in the formation of psoriatic plaques, including IL-12p40, IL-23, IL-17, tumor necrosis factor-α, IL-8, and CCL20, in dendritic/Langerhans-like cells and to a lesser extent in keratinocytes. These data support HERV-K dUTPase as a potential contributor to psoriasis pathophysiology.

Genome-wide detection and characterization of endogenous retroviruses in Bos taurus.

Endogenous retroviruses (ERVs) are the proviral phase of exogenous retroviruses that become integrated into a host germ line. They can play an important role in the host genome. Bioinformatic tools have been used to detect ERVs in several vertebrates, primarily primates and rodents. Less information is available regarding ERVs in other mammalian groups, and the source of this information is basically experimental. We analyzed the genome of the cow (Bos taurus) using three different methods. A BLAST-based method detected 928 possible ERVs, LTR_STRUC detected 4,487 elements flanked by long terminal repeats (LTRs), and Retrotector detected 9,698 ERVs. The ERVs were not homogeneously distributed across chromosomes; the number of ERVs was positively correlated with chromosomal size and negatively correlated with chromosomal GC content. The bovine ERVs (BoERVs) were classified into 24 putative families, with 20 of them not previously described. One of these new families, BoERV1, was the most abundant family and appeared to be specific to ruminants. An analysis of representatives of ERV families from rodents, primates, and ruminants showed a phylogenetic relationship following their hosts' relationships. This study demonstrates the importance of using multiple methods when trying to identify new ERVs and shows that the number of bovine ERV families is not as limited as previously thought.

Porcine endogenous retrovirus (PERV) and its transmission characteristics: a study of the New Zealand designated pathogen-free herd.

Previously a strategy for monitoring of pigs intended for cell transplantation was developed and successfully applied to several representative herds in New Zealand. A designated pathogen-free (DPF) herd has been chosen as a good candidate for xenotransplantation. This herd has previously tested free of infectious agents relevant to xenotransplantation and we present here an in depth study of porcine endogenous retrovirus (PERV) transmission. A panel of assays that describes the constraints for the transmission of PERV has been suggested. It includes a) infectivity test in coculture of DPF pig primary cells with both human and pig target cell lines; b) RT activity in supernatant of stimulated primary cells from DPF pigs; c) viral load in donor's blood plasma; d) PERV proviral copy number in DPF pig genome; e) PERV class C prevalence in the herd and its recombination potential. There was no evidence of PERV transmission from DPF pig tissue to either pig or human cells. Additionally, there was no evidence of PERV RNA present in pig blood plasma. PERV copy number differs in individual pigs from as low as 3 copies to 30 copies and the presence of PERV-C varied between animals and breeds. In all DPF pigs tested, a specific locus for PERV-C potentially associated with the recombination of PERV in miniature swine was absent. Presented data on the PERV transmission allows us to classify the DPF potential donors as "null" or noninfectious pigs.

Selective inhibition of porcine endogenous retrovirus replication in human cells by acyclic nucleoside phosphonates.

Several anti-human immunodeficiency virus type 1 reverse transcriptase inhibitors were evaluated for their antiviral activities against porcine endogenous retrovirus in human cells. Among the test compounds, zidovudine was found to be the most active. The order of potency was zidovudine > phosphonylmethoxyethoxydiaminopyrimidine = phosphonylmethoxypropyldiaminopurine > tenofovir > or = adefovir > stavudine.

Tissue specificity of methylation and expression of human genes coding for neuropeptides and their receptors, and of a human endogenous retrovirus K family.

The purpose of the present study was to understand the tissue specificity of DNA methylation and the relationship between methylation and expression of genes with essential roles in neurodevelopment and brain function. We chose dopamine receptor genes (DRD1 and DRD2), NCAM, and COMT as examples of genes with CpG islands around the promoter region, and serotonin receptor genes (HTR2A and HTR3A), HCRT, and DRD3 as genes without CpG islands. Methylation states were investigated in fetal brain, fetal liver, placenta, and in adult peripheral leukocytes from three individuals by Southern blot and bisulfite-modified DNA sequencing. A repetitive sequence, human endogenous retrovirus (HERV)-K was also examined. All genes examined were almost completely unmethylated in brains. The genes with CpG islands were unmethylated regardless of their expression state. In contrast, genes without CpG islands showed various methylation patterns, which did not necessarily reflect the transcriptional activity of the genes. Most HERV-K loci were methylated, but some loci showed relatively low methylation in the placenta and liver. Interestingly, we found inter-individual differences in methylation levels in HTR2A and HCRT in the placenta and in some loci of HERV-K in the placenta and liver. The sample with the lowest methylation levels in the two unique genes showed higher methylation of HERV-K loci than the other samples. These results provide detailed information about the methylation states of the genes analyzed and evidence for inter-individual variations in methylation in both unique and repetitive sequences.

The distribution of pol containing human endogenous retroviruses in non-human primates.

Few human endogenous retroviruses (HERVs) have been extensively studied in non-human primates. Such investigations have demonstrated that several element classes are primate unique, contain members with important biological function, are conserved in specific primate lineages, and have in some cases expanded in copy number. We have examined multiple sub-families of all major groups of HERVs using a DNA microarray based on the reverse transcriptase (RT) domain of the viral polymerase gene (pol). The microarray was used to investigate the distribution of HERVs in non-human primates with particular focus on the differences between New World monkeys (NWMs) and other anthropoids. This is the first study examining most HERV families in multiple non-human primate DNAs using a uniform and sensitive method and suggests that major differences exist between primate groups. The results indicate that a major invasion and expansion of pol containing HERVs occurred after the platyrrhine (NWM) lineage separated from the catarrhines (Old World Monkeys and apes).

Detection of serum reverse transcriptase activity in patients with ALS and unaffected blood relatives.

BACKGROUND: Retroviral involvement in the etiology of sporadic ALS has been suspected for several years since the recognition that both murine and human retroviruses can cause motor neuron disease-like syndromes. In a pilot study, an increased prevalence of a retroviral marker (reverse transcriptase [RT] activity) was demonstrated in the serum of British patients with ALS. The current investigation was designed to confirm and extend these findings in a geographically distinct patient cohort under blinded testing conditions. METHODS: A highly sensitive product-enhanced RT assay was employed to test coded sera obtained from 30 American patients with sporadic ALS and from 14 of their blood relatives, 16 of their spouses, and 28 nonrelated, nonspousal control subjects. RESULTS: Serum RT activity was detected in a higher proportion of ALS patients (47%) than in non-blood-related controls (18%; p = 0.008). The prevalence of RT activity in the serum of spousal controls (13%) was similar to that in other non-blood-related controls. Unexpectedly, the prevalence of serum RT activity in blood relatives of ALS patients (43%) approached that in the ALS patients themselves. CONCLUSIONS: These results confirm that patients with ALS have a significantly higher prevalence of serum reverse transcriptase (RT) activity than that seen in unrelated control subjects. The finding of a similarly increased prevalence in blood relatives of ALS patients raises the possibility that the observed RT activity might be due to an inherited endogenous retrovirus.

Real-time reverse transcriptase PCR for the endogenous koala retrovirus reveals an association between plasma viral load and neoplastic disease in koalas.

Koala retrovirus (KoRV) is a newly described endogenous retrovirus and is unusual in that inserts comprise a full-length replication competent genome. As koalas are known to suffer from an extremely high incidence of leukaemia/lymphoma, the association between this retrovirus and disease in koalas was examined. Using quantitative real-time reverse transcriptase PCR it was demonstrated that KoRV RNA levels in plasma are significantly increased in animals suffering from leukaemia or lymphoma when compared with healthy animals. Increased levels of KoRV were also seen for animals with clinical chlamydiosis. A significant positive association between viral RNA levels and age was also demonstrated. Real-time PCR demonstrated as much as 5 log variation in KoRV proviral DNA levels in genomic DNA extracted from whole blood from different animals. Taken together these data indicate that KoRV is an active endogenous retrovirus and suggests that it may be causally linked to neoplastic disease in koalas.

Spontaneous fusion of cells between species yields transdifferentiation and retroviral transfer in vivo.

Human cells can fuse with damaged or diseased somatic cells in vivo. Whether human cells fuse in vivo in the absence of disease and with cells of disparate species is unknown. Such a question is of current interest because blood exchanges between species through direct physical contact, via insect vectors or parasitism, are thought to underlie the transmission of zoonotic agents. In a model of human-pig chimerism, we show that some human hematopoietic stem cells engrafted in pigs contain both human and porcine chromosomal DNA. These hybrid cells divide, express human and porcine proteins, and contribute to porcine nonhematopoietic tissues. In addition, the hybrid cells contain porcine endogenous retroviral DNA sequences and are able to transmit this virus to uninfected human cells in vitro. Thus, spontaneous fusion can occur in vivo between the cells of disparate species and in the absence of disease. The ability of these cell hybrids to acquire and transmit retroviral elements together with their ability to integrate into tissues could explain genetic recombination and generation of novel pathogens. * differentiation * fusion * retrovirus