Upregulation of Human Endogenous Retroviruses in Bronchoalveolar Lavage Fluid of COVID-19 Patients.

Severe COVID-19 pneumonia has been associated with the development of intense inflammatory responses during the course of infections with SARS-CoV-2. Given that human endogenous retroviruses (HERVs) are known to be activated during and participate in inflammatory processes, we examined whether HERV dysregulation signatures are present in COVID-19 patients. By comparing transcriptomes of bronchoalveolar lavage fluid (BALF) of COVID-19 patients and healthy controls, and peripheral blood monocytes (PBMCs) from patients and controls, we have shown that HERVs are intensely dysregulated in BALF of COVID-19 patients compared to those in BALF of healthy control patients but not in PBMCs. In particular, upregulation in the expression of specific HERV families was detected in BALF samples of COVID-19 patients, with HERV-FRD being the most highly upregulated family among the families analyzed. In addition, we compared the expression of HERVs in human bronchial epithelial cells (HBECs) without and after senescence induction in an oncogene-induced senescence model in order to quantitatively measure changes in the expression of HERVs in bronchial cells during the process of cellular senescence. This apparent difference of HERV dysregulation between PBMCs and BALF warrants further studies in the involvement of HERVs in inflammatory pathogenetic mechanisms as well as exploration of HERVs as potential biomarkers for disease progression. Furthermore, the increase in the expression of HERVs in senescent HBECs in comparison to that in noninduced HBECs provides a potential link for increased COVID-19 severity and mortality in aged populations. IMPORTANCE SARS-CoV-2 emerged in late 2019 in China, causing a global pandemic. Severe COVID-19 is characterized by intensive inflammatory responses, and older age is an important risk factor for unfavorable outcomes. HERVs are remnants of ancient infections whose expression is upregulated in multiple conditions, including cancer and inflammation, and their expression is increased with increasing age. The significance of this work is that we were able to recognize dysregulated expression of endogenous retroviral elements in BALF samples but not in PBMCs of COVID-19 patients. At the same time, we were able to identify upregulated expression of multiple HERV families in senescence-induced HBECs in comparison to that in noninduced HBECs, a fact that could possibly explain the differences in disease severity among age groups. These results indicate that HERV expression might play a pathophysiological role in local inflammatory pathways in lungs afflicted by SARS-CoV-2 and their expression could be a potential therapeutic target.

Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV-W Family Using PNA Strand Invasion into Double-Stranded DNA.

In the presented assay, we elaborated a method for distinguishing sequences that are genetically closely related to each other. This is particularly important in a situation where a fine balance of the allele abundance is a point of research interest. We developed a peptide nucleic acid (PNA) strand invasion technique for the differentiation between multiple sclerosis-associated retrovirus (MSRV) and ERVWE1 sequences, both molecularly similar, belonging to the human endogenous retrovirus HERV-W family. We have found that this method may support the PCR technique in screening for minor alleles which, in certain conditions, may be undetected by the standard PCR technique. We performed the analysis of different ERVWE1 and MSRV template mixtures ranging from 0 to 100% of ERVWE1 in the studied samples, finding the linear correlation between template composition and signal intensity of final reaction products. Using the PNA strand invasion assay, we were able to estimate the relative ERVWE1 expression level in human specimens such as U-87 MG, normal human astrocytes cell lines and placental tissue. The results remained in concordance with those obtained by semi-quantitative or quantitative PCR.

Analysis of Protein Expression in Human Cells Cocultured with Porcine Peripheral Blood Mononuclear Cells.

OBJECTIVE: Porcine endogenous retroviruses (PERV) involved in pig to human xenotransplantation have raised great concerns because of their ubiquitous nature in pigs and their ability of infecting human cells in vitro. Although no significant cytopathic effect attributed to PERV was evident on PERV-infected human embryonic kidney 293 (HEK293) cells, we did proteomic analysis to investigate the differences of protein profile in order to further characterize the effect of PERV infection. METHODS: HEK293 cells were cocultured with porcine peripheral blood mononuclear cells (PBMCs). Protein profiles of PERV-infected and -noninfected HEK293 cells were analyzed by two-dimensional gel electrophoresis (2-DE). Protein spots with at least 1.5-fold alteration were identified by high-definition mass spectrometry (HDMS) analysis. Then real-time RT-PCR and Western blotting were performed to validate the proteomic results. RESULTS: Differential analysis of PERV-infected and -noninfected HEK293 cells by 2-DE revealed ten differentially regulated proteins. The proteins identified by HDMS were involved in various cellular pathways including signal transduction, cell apoptosis, and protein synthesis. CONCLUSION: The results of this study revealed differentially expressed proteins in HEK293 cells cocultured with porcine PBMCs and implied that these changes were probably induced by PERV infection. These results provide clues and potential links to understanding the molecular effect of the infection by human-tropic PERV.

The EGF epidermal growth factor counteracts Tat modulation of human endogenous retroviruses of the W family in astrocytes.

Human astrocyte cells were exposed to HIV-Tat and/or epidermal growth factor (EGF), to monitor the expression of the neuropathogenic MSRV and Syncytin-1 elements of the HERV-W family of endogenous retroviruses and of TNFα. The results indicate that EGF counteracts Tat regulation of HERV-W/MSRVenv/Syncytin-1, throughout EGFR activation; this effect occurs by interfering with the induction of TNFα production by Tat. The novel effect of EGF counteraction of Tat-mediated regulation of the neuropathogenic HERV-Ws could be neuro-protective, but its actual role in the brain remains to be elucidated.

Pushing the envelope.

Primates have co-opted a viral gene to produce an envelope protein that prevents infection by the HERV-T virus and likely contributed to the extinction of this virus.

Expression patterns of endogenous avian retrovirus ALVE1 and its response to infection with exogenous avian tumour viruses.

Endogenous retroviruses (ERVs) are genomic elements that are present in a wide range of vertebrates and have been implicated in a variety of human diseases, including cancer. However, the characteristic expression patterns of ERVs, particularly in virus-induced tumours, is not fully clear. DNA methylation was analysed by bisulfite pyrosequencing, and gene expression was analysed by RT-qPCR. In this study, we first found that the endogenous avian retrovirus ALVE1 was highly expressed in some chicken tissues (including the heart, bursa, thymus, and spleen) at 2 days of age, but its expression was markedly decreased at 35 days of age. In contrast, the CpG methylation level of ALVE1 was significantly lower in heart and bursa at 2 days than at 35 days of age. Moreover, we found that the expression of ALVE1 was significantly inhibited in chicken embryo fibroblast cells (CEFs) and MSB1 cells infected with avian leukosis virus subgroup J (ALVJ) and reticuloendotheliosis virus (REV) at the early stages of infection. In contrast, the expression of the ALVE1 env gene was significantly induced in CEFs and MSB1 cells infected with Marek's disease virus (MDV). However, the methylation and expression levels of the ALVE1 long terminal repeat (LTR) did not show obvious alterations in response to viral infection. The present study revealed the expression patterns of ALVE1 in a variety of chicken organs and tissues and in chicken cells in response to avian tumour virus infection. These findings may be of significance for understanding the role and function of ERVs that are present in the host genome.

Cellular Prion Protein Combined with Galectin-3 and -6 Affects the Infectivity Titer of an Endogenous Retrovirus Assayed in Hippocampal Neuronal Cells.

Prion diseases are infectious and fatal neurodegenerative diseases which require the cellular prion protein, PrPC, for development of diseases. The current study shows that the PrPC augments infectivity and plaque formation of a mouse endogenous retrovirus, MuLV. We have established four neuronal cell lines expressing mouse PrPC, PrP+/+; two express wild type PrPC (MoPrPwild) and the other two express mutant PrPC (MoPrPmut). Infection of neuronal cells from various PrP+/+ and PrP-/- (MoPrPKO) lines with MuLV yielded at least three times as many plaques in PrP+/+ than in PrP-/-. Furthermore, among the four PrP+/+ lines, one mutant line, P101L, had at least 2.5 times as many plaques as the other three PrP+/+ lines. Plaques in P101L were four times larger than those in other PrP+/+ lines. Colocalization of PrP and CAgag was seen in MuLV-infected PrP+/+ cells. In the PrP-MuLV interaction, the involvement of galectin-3 and -6 was observed by immunoprecipitation with antibody to PrPC. These results suggest that PrPC combined with galectin-3 and -6 can act as a receptor for MuLV. P101L, the disease form of mutant PrPC results suggest the genetic mutant form of PrPC may be more susceptible to viral infection.

Long Terminal Repeats: From Parasitic Elements to Building Blocks of the Transcriptional Regulatory Repertoire.

The life cycle of endogenous retroviruses (ERVs), also called long terminal repeat (LTR) retrotransposons, begins with transcription by RNA polymerase II followed by reverse transcription and re-integration into the host genome. While most ERVs are relics of ancient integration events, "young" proviruses competent for retrotransposition-found in many mammals, but not humans-represent an ongoing threat to host fitness. As a consequence, several restriction pathways have evolved to suppress their activity at both transcriptional and post-transcriptional stages of the viral life cycle. Nevertheless, accumulating evidence has revealed that LTR sequences derived from distantly related ERVs have been exapted as regulatory sequences for many host genes in a wide range of cell types throughout mammalian evolution. Here, we focus on emerging themes from recent studies cataloging the diversity of ERV LTRs acting as important transcriptional regulatory elements in mammals and explore the molecular features that likely account for LTR exaptation in developmental and tissue-specific gene regulation.

"Reverse genomics" and human endogenous retroviruses.

Over millions of years, actively replicating retroviruses entered the human genome and through time became a stable and substantial part of the inherited genetic material. A remarkable 8% of the human genome is accounted for by endogenous retroviruses, whose biological importance has not yet been elucidated. In studying the RNA of these endogenous retroviruses in the blood of living human subjects with HIV infection, we have discovered a whole new family of these viruses that had been hidden in the centromeres of specific human chromosomes. These retroviruses have specific sequences that can elucidate their chromosome of origin. As centromeres represent the most substantial remaining frontier of human genomics, these viral sequences can provide a "bar-code" that can be used to study the role of centromeres in biology and in disease. This work also highlights the efficacy of using "reverse genomics" to understand and annotate the human genome.

A new approach to establish a cell line with reduced risk of endogenous retroviruses.

Endogenous retroviruses (ERVs) are integrated as DNA proviruses in the genomes of all mammalian species. Several ERVs are replication-competent and produced as fully infectious viruses from host cell. Thus, live-attenuated vaccines and biological substances have been prepared using the cell lines which may produce ERV. Indeed, we recently reported that several commercial live-attenuated vaccines for pets were contaminated with the infectious feline endogenous retrovirus, RD-114. In this study, to establish a cell line for vaccine manufacture with reduced risk of ERVs, we generated a cell line stably expressing human tetherin (Teth-CRFK cells). The release of infectious ERV from Teth-CRFK cells was suppressed to undetectable levels, while the production of parvovirus in Teth-CRFK cells was similar to that in parental CRFK cells. These observations suggest that Teth-CRFK cells will be useful as a cell line for the manufacture of live-attenuated vaccines or biological substances with reduced risk of ERV.

Resurrection of endogenous retroviruses in antibody-deficient mice.

The mammalian host has developed a long-standing symbiotic relationship with a considerable number of microbial species. These include the microbiota on environmental surfaces, such as the respiratory and gastrointestinal tracts, and also endogenous retroviruses (ERVs), comprising a substantial fraction of the mammalian genome. The long-term consequences for the host of interactions with these microbial species can range from mutualism to parasitism and are not always completely understood. The potential effect of one microbial symbiont on another is even less clear. Here we study the control of ERVs in the commonly used C57BL/6 (B6) mouse strain, which lacks endogenous murine leukaemia viruses (MLVs) able to replicate in murine cells. We demonstrate the spontaneous emergence of fully infectious ecotropic MLV in B6 mice with a range of distinct immune deficiencies affecting antibody production. These recombinant retroviruses establish infection of immunodeficient mouse colonies, and ultimately result in retrovirus-induced lymphomas. Notably, ERV activation in immunodeficient mice is prevented in husbandry conditions associated with reduced or absent intestinal microbiota. Our results shed light onto a previously unappreciated role for immunity in the control of ERVs and provide a potential mechanistic link between immune activation by microbial triggers and a range of pathologies associated with ERVs, including cancer.

Viral and cellular requirements for the budding of feline endogenous retrovirus RD-114.

BACKGROUND: RD-114 virus is a feline endogenous retrovirus and produced as infectious viruses in some feline cell lines. Recently, we reported the contamination of an infectious RD-114 virus in a proportion of live attenuated vaccines for dogs and cats. It is very difficult to completely knock out the RD-114 proviruses from cells, as endogenous retroviruses are usually integrated multiply into the host genome. However, it may be possible to reduce the risk of contamination of RD-114 virus by regulating the viral release from cells. RESULTS: In this study, to understand the molecular mechanism of RD-114 virus budding, we attempted to identify the viral and cellular requirements for RD-114 virus budding. Analyses of RD-114 L-domain mutants showed that the PPPY sequence in the pp15 region of Gag plays a critical role in RD-114 virus release as viral L-domain. Furthermore, we investigated the cellular factors required for RD-114 virus budding. We demonstrated that RD-114 virus release was inhibited by overexpression of dominant negative mutants of Vps4A, Vps4B, and WWP2. CONCLUSIONS: These results strongly suggest that RD-114 budding utilizes the cellular multivesicular body sorting pathway similar to many other retroviruses.

Susceptibility and production of a feline endogenous retrovirus (RD-114 virus) in various feline cell lines.

RD-114 virus is a replication-competent feline endogenous retrovirus that has been classified as a xenotropic virus. In this study, we examined the expression of the receptors for RD-114 virus in feline cell lines by conducting a pseudotype virus infection assay. Six out of eight feline cell lines were susceptible to the RD-114 pseudotype virus and two cell lines (MCC and FER cells) were resistant. The two resistant cell lines and one cell line (CRFK cells) weakly sensitive to the RD-114 pseudotype virus were found to produce replication-competent RD114-like viruses by the LacZ marker rescue assay and the interference assay. These data strongly suggest that RD-114 virus is polytropic and resistance to RD-114 virus in certain cell lines is due to receptor interference but not polymorphism of the RD-114 receptors. In addition, we determined the amino acid sequences of the envelope region of RD-114-like viruses produced from MCC, FER and CRFK cells. The sequences were identical with the authentic RD-114 virus. Because many feline cell lines are used to manufacture live attenuated vaccines for companion animals, attention should be paid to contamination of the RD-114 virus in vaccines.

Acutely transforming retrovirus expressing Nras generated from HT-1080 fibrosarcoma cells infected with the human retrovirus XMRV.

Virus from HT-1080 fibrosarcoma cells infected with the human retrovirus XMRV (xenotropic murine leukemia virus-related virus) can induce rare foci of transformation in rat 208F fibroblasts. Characterization of three such foci revealed that one produced an acutely transforming virus at a high titer. The virus consists of a mutant Nras cDNA from the HT-1080 cells inserted into a retroviral vector (added to the HT-1080 cells as a marker for infection) in place of internal vector sequences. These results show that XMRV can generate acutely transforming viruses at a low rate, as is typical of other replication-competent retroviruses, and reveal the potential for transforming virus contamination of retroviral vectors made from transformed cell lines.

Interplay between ovine bone marrow stromal cell antigen 2/tetherin and endogenous retroviruses.

Endogenous betaretroviruses (enJSRVs) of sheep are expressed abundantly in the female reproductive tract and play a crucial role in conceptus development and placental morphogenesis. Interestingly, the colonization of the sheep genome by enJSRVs is likely still ongoing. During early pregnancy, enJSRV expression correlates with the production of tau interferon (IFNT), a type I IFN, by the developing conceptus. IFNT is the pregnancy recognition signal in ruminants and possesses potent antiviral activity. In this study, we show that IFNT induces the expression of bone marrow stromal cell antigen 2 (BST2) (also termed CD317/tetherin) both in vitro and in vivo. The BST2 gene is duplicated in ruminants. Transfection assays found that ovine BST2 proteins (oBST2A and oBST2B) block release of viral particles produced by intact enJSRV loci and of related exogenous and pathogenic jaagsiekte sheep retrovirus (JSRV). Ovine BST2A appears to restrict enJSRVs more efficiently than oBST2B. In vivo, the expression of BST2A/B and enJSRVs in the endometrium increases after day 12 and remains high between days 14 and 20 of pregnancy. In situ hybridization analyses found that oBST2A is expressed mainly in the endometrial stromal cells but not in the luminal and glandular epithelial cells, in which enJSRVs are highly expressed. In conclusion, enJSRVs may have coevolved in the presence of oBST2A/B by being expressed in different cellular compartments of the same organ. Viral expression in cells unable to express BST2 may be one of the mechanisms used by retroviruses to escape restriction.

Studies on long-term infection of human cells with Porcine endogenous retrovirus.

A major concern in pig-to-human xenotransplantations is the potential risk of transmission of Porcine endogenous retroviruses (PERVs) integrated in the pig genome. Our previous work has shown that PERV provirus genes and gag protein can be detected in human embryonic kidney HEK-293 cells during a long-term infection with PERV (Yu et al., Transplant. Proc. 37, 496-499, 2005). In this study, we continued studying the long-term (>6 months) PERV infection of HEK-293 cells. The results showed no significant differences in morphology, growth, apoptosis, and [(3)H]-thymidine incorporation between PERV-infected and uninfected cells. The PERV LTR sequence showed only an insignifcant mutation after the long-term infection. PERV infection had no effect on the transcription of genes of Human endogenous retrovirus (HERV) naturally occurring in HEK-293 cells. Summing up, this study indicated that a long-term PERV infection of HEK-293 cells in vitro does not result in any significant changes in host cells as well as in PERV LTR sequence.

Mobilization of endogenous retroviruses in mice after infection with an exogenous retrovirus.

Mammalian genomes harbor a large number of retroviral elements acquired as germ line insertions during evolution. Although many of the endogenous retroviruses are defective, several contain one or more intact viral genes that are expressed under certain physiological or pathological conditions. This is true of the endogenous polytropic retroviruses that generate recombinant polytropic murine leukemia viruses (MuLVs). In these recombinants the env gene sequences of exogenous ecotropic MuLVs are replaced with env gene sequences from an endogenous polytropic retrovirus. Although replication-competent endogenous polytropic retroviruses have not been observed, the recombinant polytropic viruses are capable of replicating in numerous species. Recombination occurs during reverse transcription of a virion RNA heterodimer comprised of an RNA transcript from an endogenous polytropic virus and an RNA transcript from an exogenous ecotropic MuLV RNA. It is possible that homodimers corresponding to two full-length endogenous RNA genomes are also packaged. Thus, infection by an exogenous virus may result not only in recombination with endogenous sequences, but also in the mobilization of complete endogenous retrovirus genomes via pseudotyping within exogenous retroviral virions. We report that the infection of mice with an ecotropic virus results in pseudotyping of intact endogenous viruses that have not undergone recombination. The endogenous retroviruses infect and are integrated into target cell genomes and subsequently replicate and spread as pseudotyped viruses. The mobilization of endogenous retroviruses upon infection with an exogenous retrovirus may represent a major interaction of exogenous retroviruses with endogenous retroviruses and may have profound effects on the pathogenicity of retroviral infections.

Expression of the human endogenous retrovirus-K transmembrane envelope, Rec and Np9 proteins in melanomas and melanoma cell lines.

The human endogenous retrovirus-K encodes two potential tumor proteins, Rec and Np9. Rec is related to the Rev protein of HIV-1 and has been shown to be associated with tumor development in nude mice. Having shown the expression of human endogenous retrovirus-K in human melanomas and melanoma cell lines, tools were developed to allow the expression of the transmembrane envelope, Rec and Np9 mRNA and proteins to be studied in more detail. The expression of spliced env, rec and np9 was investigated by reverse transcriptase-polymerase chain reaction using a set of primers developed to discriminate between full-length and spliced mRNA. Env-specific, Rec-specific and Np9-specific antisera were produced, characterized and used to study protein expression in melanomas and melanoma cell lines by immunohistochemistry, immunofluorescence and Western blot analyses. Existence of human endogenous retrovirus-K Rec and Np9-specific antibodies in the sera of melanoma patients were analyzed by Western blot of immunofluorescence studies. The expression of both spliced env and rec mRNA was detected in 39% of the melanomas and in 40% of the melanoma cell lines and np9 mRNA was detected in 29 and 21%, respectively. In normal neonatal melanocytes, spliced rec mRNA was detected in the absence of spliced env mRNA. Using antisera specific for Rec and Np9, Rec protein was found in 14% of the melanomas but Np9 in none. In addition, cell surface expression of the putatively immunosuppressive transmembrane envelope protein and release of virus particles were shown. Antibodies specific for neither Rec nor Np9 were detected. The transmembrane envelope protein, Rec and Np9 proteins are expressed in melanoma cells with a pattern similar to that seen in teratocarcinoma cell lines. Additional experiments are needed to determine their involvement, if any, in cell proliferation and tumor progression.

Establishing the reactivity of monoclonal antibodies against porcine endogenous retrovirus envelope protein.

Xenotransplantation of pig organs may be associated with a risk of transmission of microorganisms. Porcine endogenous retroviruses (PERV) are of particular concern since in vitro experiments have demonstrated that human cells are susceptible to such microorganisms. To monitor the transmission of PERV, highly sensitive and specific immunoassays must be developed for clinical surveillance. This report describes the production, preliminary characterization and application of a monoclonal antibody (mAb) against a recombinant PERV envelope (Env) protein. The generated mAb was tested using recombinant PERV Env protein expressed in Escherichia coli, purified PERV virus particles and human 293 cell line infected with PERV. PERV-translated proteins of 15, 70 and 85 kD were recognized specifically using PERV-8E10 mAb and Western blotting. No cross-reactivity was demonstrated with exogenous viral protein (HIV, HTLV and MuLV). Moreover, PERV-8E10 mAb can be applied to localize PERV proteins using an immunoperoxidase assay. This work reveals that recombinant PERV Env protein and mAb may be effective in detecting antibodies against PERV in xenotransplanted patients, or for butchers who have extensive contact with pigs.

Early detection of endogenous retroviruses in chemically induced mouse cells.

Endogenous retroviral sequences are present as an integral part of eukaryotic genomes. Although the majority of these sequences are defective, a few can produce infectious virus, either spontaneously upon long-term culture or by treatment with various chemical or other agents. Early, extensive studies of retrovirus induction were done in mouse cells; however, similar studies have not been done using state-of-the-art virus detection assays and with cells of other mammalian species. To investigate induction and detection of occult retroviruses in cells of different species, especially primate cells that are used in production of biologics, we have initially determined the optimum conditions for retrovirus induction in chemically treated K-BALB mouse cells using highly sensitive product-enhanced reverse transcriptase (PERT) assays as well as transmission electron microscopy (TEM). Retrovirus induction was detected at day 1 post-drug treatment under all test conditions but was optimum using 30 microg ml(-1) of 5-iododeoxyuridine (IdU) for 24 h. Additionally, the combination of IdU and 5-azacytidine specifically enhanced activation of type C particles. RT activity was detected by PERT assays in one microliter equivalent of test sample and retroviral particle production was seen by TEM analysis. The induction of infectious murine leukemia retroviruses was confirmed by infectivity assays and correlated with PERT activity. These results indicate that strategies for detection of occult viral agents should include optimization of induction conditions using multiple viral detection assays to evaluate virus activation.