Correlation analysis of cancer stem cell marker CD133 and human endogenous retrovirus (HERV)-K env in SKOV3 ovarian cancer cells.

BACKGROUND: Human endogenous retrovirus (HERV)-K is a type of retrovirus that is present in the human genome, and its expression is usually silenced in healthy tissues. The precise mechanism by which HERV-K env influences cancer stemness is not fully understood, but it has been suggested that HERV-K env may activate various signaling pathways that promote stemness traits in cancer cells. OBJECTIVE: To establish the connection between HERV-K env expression and cancer stemness in ovarian cancer cells, we carried out correlation analyses between HERV-K env and the cancer stem cell (CSC) marker known as the cluster of differentiation 133 (CD133) gene in SKOV3 ovarian cancer cells. METHOD: To perform correlation analysis between HERV-K env and CSCs, ovarian cancer cells were cultured in a medium designed for cancer stem cell induction. The expression of HERV-K env and CD133 genes was verified using quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Additionally, the expression of stemness-related markers, such as OCT-4 and Nanog, was also confirmed using RT-qPCR. RESULTS: In the stem cell induction medium, the number of tumorsphere-type SKOV3 cells increased, and the expression of CD133 and HERV-K env genes was up-regulated. Additionally, other stemness-related markers like OCT-4 and Nanog also exhibited increased expression when cultured in the cancer stem cell induction medium. However, when HERV-K env knockout (KO) SKOV3 cells were cultured in the same cancer stem cell induction medium, there was a significant decrease in the number of tumorsphere-type cells compared to mock SKOV3 cells subjected to the same conditions. Furthermore, the expression of CD133, Nanog, and OCT-4 did not show a significant increase in HERV-K env KO SKOV3 cells compared to mock SKOV3 cells cultured in the same cancer stem cell induction medium. CONCLUSION: These findings indicate that the expression of HERV-K env increased in SKOV3 cells when cultured in cancer stem cell induction media, and cancer stem cell induction was inhibited by KO of HERV-K env in SKOV3 cells. These results suggest a strong association between HERV-K env and stemness in SKOV3 ovarian cancer cells.

Molecular analysis of the env, LTR, and pX regions of bovine leukemia virus in dairy cattle of Türkiye.

Bovine leukemia virus is a retrovirus that causes enzootic bovine leukosis and is associated with global economic losses in the livestock industry. The aim of this study was to investigate the genotype determination of BLVs from cattle housed in 6 different farms in Türkiye and the characterization of their LTR and pX (tax, rex, R3, and G4 gene) regions. For this purpose, blood samples from 48 cattle infected with BLV were used. The phylogenetic analysis based on the env gene sequences revealed that all BLVs were clustered in genotype 1 (G1), and the sequences of the LTR (n = 48) and the pX region (n = 33) of BLVs were obtained. Also, analysis of these nucleic acid and amino acid sequences allowed assessments similar to those reported in earlier studies to be relevant to transactivation and pathogenesis. This study reports the molecular analysis of the LTR and pX region of BLVs in Türkiye for the first time.

IFITM1 and IFITM3 Proteins Inhibit the Infectivity of Progeny HIV-1 without Disrupting Envelope Glycoprotein Clusters.

Human interferon-induced transmembrane (IFITM) proteins inhibit the fusion of a broad spectrum of enveloped viruses, both when expressed in target cells and when present in infected cells. Upon expression in infected cells, IFITMs incorporate into progeny virions and reduce their infectivity by a poorly understood mechanism. Since only a few envelope glycoproteins (Envs) are present on HIV-1 particles, and Env clustering has been proposed to be essential for optimal infectivity, we asked if IFITM protein incorporation modulates HIV-1 Env clustering. The incorporation of two members of the IFITM family, IFITM1 and IFITM3, into HIV-1 pseudoviruses correlated with a marked reduction of infectivity. Super-resolution imaging of Env distribution on single HIV-1 pseudoviruses did not reveal significant effects of IFITMs on Env clustering. However, IFITM3 reduced the Env processing and incorporation into virions relative to the control and IFITM1-containing viruses. These results show that, in addition to interfering with the Env function, IFITM3 restricts HIV-1 Env cleavage and incorporation into virions. The lack of notable effect of IFITMs on Env clustering supports alternative restriction mechanisms, such as modification of the properties of the viral membrane.

The Role of Human Endogenous Retrovirus (HERV)-K119 env in THP-1 Monocytic Cell Differentiation.

Human endogenous retrovirus (HERV)-K was reportedly inserted into the human genome millions of years ago and is closely related to various diseases, including cancer and immune regulation. In our previous studies, CRISPR-Cas9-enabled knockout (KO) of the HERV-K env gene was found to potentially reduce cell proliferation, cell migration, and invasion in colorectal and ovarian cancer cell lines. The immune response involves the migration and invasion of cells and is similar to cancer; however, in certain ways, it is completely unlike cancer. Therefore, we induced HERV-K119 env gene KO in THP-1, a monocytic cell that can be differentiated into a macrophage, to investigate the role of HERV-K119 env in immune regulation. Cell migration and invasion were noted to be significantly increased in HERV-K119 env KO THP-1 cells than in MOCK, and these results were contrary to those of cancer cells. To identify the underlying mechanism of HERV-K119 env KO in THP-1 cells, transcriptome analysis and cytokine array analysis were conducted. Semaphorin7A (SEMA7A), which induces the production of cytokines in macrophages and monocytic cells and plays an important role in immune effector cell activation during an inflammatory immune response, was significantly increased in HERV-K119 env KO THP-1 cells. We also found that HERV-K119 env KO THP-1 cells expressed various macrophage-specific surface markers, suggesting that KO of HERV-K119 env triggers the differentiation of THP-1 cells from monocytic cells into macrophages. In addition, analysis of the expression of M1 and M2 macrophage markers showed that M1 macrophage marker cluster of differentiation 32 (CD32) was significantly increased in HERV-K119 env KO cells. These results suggest that HERV-K119 env is implicated in the differentiation of monocytic cells into M1 macrophages and plays important roles in the immune response.

Analysis of PERV-C superinfection resistance using HA-tagged viruses.

BACKGROUND: Using pigs as organ donors has advanced xenotransplantation to the point that it is almost ready for clinical use. However, there is still a zoonotic risk associated with xenotransplantation, and the potential transmission of porcine endogenous retroviruses needs to be surveyed. Despite significant attempts to eliminate this risk, by the selection of PERV-C free pigs with low expression of PERV-A, -B, and by the genome-wide inactivation of PERV using CRISPR/Cas9, the impact of superinfection resistance (SIR) was not investigated. SIR is a viral trait that prevents reinfection (superinfection). For PERV, the underlying mechanism is unclear, whether and how cells, that harbor functional PERV, are protected. Using PERV-C(5683) as a reference virus, we investigated SIR in a newly developed in vitro model to pursue the mechanism and confirm its protective effect. RESULTS: We developed three PERV-C constructs on the basis of PERV-C(5683), each of which carries a hemagglutinin tag (HA-tag) at a different position of the envelope gene (SP-HA, HA-VRA, and RPep-HA), to distinguish between primary infection and superinfection. The newly generated PERV-C(5683)-HA viruses were characterized while quantifying the viral RNA, reverse transcriptase activity, protein expression analysis, and infection studies. It was demonstrated that SP-HA and RPep-HA were comparable to PERV-C(5683), whereas HA-VRA was not replication competent. SP-HA and RPep-HA were chosen to challenge PERV-C(5683)-positive ST-IOWA cells demonstrating that PERV-C-HA viruses are not able to superinfect those cells. They do not integrate into the genome and are not expressed. CONCLUSIONS: The mechanism of SIR applies to PERV-C. The production of PERV-C particles serves as a defense mechanism from superinfection with exogenous PERV-C. It was demonstrated by newly generated PERV-C(5683)-HA clones that might be used as a cutting-edge tool. The HA-tagging of PERV-C is novel, providing a blueprint for the tagging of other human tropic PERV viruses. The tagged viruses are suitable for additional in vitro and in vivo infection studies and will contribute, to basic research on viral invasion and pathogenesis. It will maintain the virus safety of XTx.

Structures and immune recognition of Env trimers from two Asia prevalent HIV-1 CRFs.

Structure-guided immunofocusing HIV-1 vaccine design entails a comprehensive understanding of Envs from diverse HIV-1 subtypes, including circulating recombinant forms (CRFs). Here, we present the cryo-EM structures of Envs from two Asia prevalent CRFs (CRF01_AE and CRF07_BC) at 3.0 and 3.5 Å. We compare the structures and glycosylation patterns of Envs from different subtypes and perform cross-clade statistical analyses to reveal the unique features of CRF01_AE V1 region, which are associated with the resistance to certain bNAbs. We also solve a 4.1 Å cryo-EM structure of CRF01_AE Env in complex with F6, the first bNAb from CRF01_AE-infected individuals. F6 recognizes a gp120-gp41 spanning epitope to allosterically destabilize the Env trimer apex and weaken inter-protomer packing, which in turn hinders the receptor binding and induces Env trimer disassembly, demonstrating a dual mechanism of neutralization. These findings broaden our understanding of CRF Envs and shed lights on immunofocusing HIV-1 vaccine design.

Decoding the mystery of MMTV-like virus and its relationship with breast cancer metastasis.

BACKGROUND: MMTV causes mammary tumors in mice, and it is associated with invasive and aggressive forms of breast cancer in humans. However, the underlying mechanisms are yet unknown. We aimed to determine the MMTV-like virus (MMTV-LV) association with histological types of breast cancer, nodal involvement, and metastasis. METHODS: First, 105 breast cancer biopsies and 15 disease-free biopsies were collected. Details of clinicopathological characteristics were retrieved from patients' records. The status of MMTV-LV was already known for these biopsy samples. Associations of MMTV-LV prevalence with LNM status and metastatic history were determined. Next, quantitative PCR (qPCR) was used to quantify env gene mRNA in biopsies positive for MMTV-LV. Expression of the env gene was compared against different histopathological types of mammary tumor, LNM status, and metastasis by performing Ordinary One Way ANOVA followed by Tukey's multiple comparisons test. RESULTS: MMTV-LV prevalence was found to have no significant association with LNM or metastatic history. As compared to normal control, expression of the env gene was significantly higher (>2.8 folds) in invasive samples (P-value: < 0.01). Expression was also higher (3.28 and 2.89 folds) in patient samples with LNM (P-value: 0.0006) or metastatic history (P-value: < 0.0001), respectively. CONCLUSION: We conclude that MMTV-LV prevalence is not associated with LNM status or breast cancer metastasis; samples with invasive phenotypes, nodal involvement, and metastasis exhibit significantly higher expression of the MMTV-like env gene.

Interferon-gamma plasma levels and presence of mouse mammary tumor virus-like env gene: Implications on the pathogenesis of breast cancer.

Mouse mammary tumor virus (MMTV) is a retrovirus that has been associated with the development of breast cancer (BC) in mice. The identification of a 95% homologous gene sequence to MMTV in human BC samples has increased interest in this hypothesis. This virus in humans received the name of mouse mammary tumor virus-like (MMTV-like). Several cytokines may be involved in the interactions between MMTV and the immune system, such as interferon-gamma (IFN-γ), which can enhance Th1-mediated antitumor immune response but it can also play a protumorigenic role by transmitting antiapoptotic and proliferative signals. Little is known about the antiviral immune response in a microenvironment with the presence of MMTV-like in BC patients. Therefore, the purpose of the present study was to quantify the plasma levels of IFN-γ in the peripheral blood of 123 neoplasia-free donors and 98 BC patients of different molecular subtypes, by enzyme-linked immunosorbent assay (ELISA), and evaluate the association of these plasma levels with the detection of the MMTV-like env gene in tumor tissue. Correlation analyzes involving IFN-γ plasma levels and clinical-pathological parameters were performed by Kendall Tau-c test. In our study, a decrease in IFN-γ levels was observed in the group of BC patients (30.85 ± 57.49 pg/ml) compared to the control group (115.00 ± 176.80 pg/ml) (p < 0.0001). In the analysis by stratified BC molecular subtypes, Luminal-A (30.79 ± 61.04 pg/ml; p < 0.0001), Luminal-B (24.74 ± 25.78 pg/ml; p = 0.0188) and triple-negative (23.95 ± 40.45 pg/ml; p = 0.0005) had a lower plasma level compared to control group. There was no significant difference between IFN-γ plasma levels of MMTV-like DNA positive samples compared to MMTV-negative samples (p = 0.2056). In general BC, patients with larger tumor size had higher IFN-γ plasma levels (Tau-c = 0.202; p = 0.019). By analyzing the MMTV-like env negative samples, we could identify that IFN-γ plasma levels were higher in larger tumor size (Tau-c = 0.222; p = 0.020) and with greater lymph node involvement (Tau-c = 0.258; p = 0.042). Also, higher IFN-γ plasma levels were observed in patients with higher histopathological grades (Tau-c = 0.384; p = 0.019) in MMTV-like env positive samples. For the first time, we assessed the association between plasma levels of IFN-γ and the presence of the MMTV-like env gene in BC samples. However, more studies are needed to clarify whether the high levels of IFN-γ in MMTV-like env positive samples are reflecting a possible antiviral immune response or whether this cytokine is promoting tumor growth.

Dynamic Evolution of Retroviral Envelope Genes in Egg-Laying Mammalian Genomes.

Independently acquired envelope (env) genes from endogenous retroviruses have contributed to the placental trophoblast cell-cell fusion in therian mammals. Egg-laying mammals (monotremes) are an important sister clade for understanding mammalian placental evolution, but the env genes in their genomes have yet to be investigated. Here, env-derived open reading frames (env-ORFs) encoding more than 400 amino acid lengths were searched in the genomes of two monotremes: platypus and echidna. Only two env-ORFs were present in the platypus genome, whereas 121 env-ORFs were found in the echidna genome. The echidna env-ORFs were phylogenetically classified into seven groups named env-Tac1 to -Tac7. Among them, the env-Tac1 group contained only a single gene, and its amino acid sequence showed high similarity to those of the RD114/simian type D retroviruses. Using the pseudotyped virus assay, we demonstrated that the Env-Tac1 protein utilizes echidna sodium-dependent neutral amino acid transporter type 1 and 2 (ASCT1 and ASCT2) as entry receptors. Moreover, the Env-Tac1 protein caused cell-cell fusion in human 293T cells depending on the expression of ASCT1 and ASCT2. These results illustrate that fusogenic env genes are not restricted to placental mammals, providing insights into the evolution of retroviral genes and the placenta.

Pregnancy Is Associated with Impaired Transcription of Human Endogenous Retroviruses and of TRIM28 and SETDB1, Particularly in Mothers Affected by Multiple Sclerosis.

Accumulating evidence highlights the pathogenetic role of human endogenous retroviruses (HERVs) in eliciting and maintaining multiple sclerosis (MS). Epigenetic mechanisms, such as those regulated by TRIM 28 and SETDB1, are implicated in HERV activation and in neuroinflammatory disorders, including MS. Pregnancy markedly improves the course of MS, but no study explored the expressions of HERVs and of TRIM28 and SETDB1 during gestation. Using a polymerase chain reaction real-time Taqman amplification assay, we assessed and compared the transcriptional levels of pol genes of HERV-H, HERV-K, HERV-W; of env genes of Syncytin (SYN)1, SYN2, and multiple sclerosis associated retrovirus (MSRV); and of TRIM28 and SETDB1 in peripheral blood and placenta from 20 mothers affected by MS; from 27 healthy mothers, in cord blood from their neonates; and in blood from healthy women of child-bearing age. The HERV mRNA levels were significantly lower in pregnant than in nonpregnant women. Expressions of all HERVs were downregulated in the chorion and in the decidua basalis of MS mothers compared to healthy mothers. The former also showed lower mRNA levels of HERV-K-pol and of SYN1, SYN2, and MSRV in peripheral blood. Significantly lower expressions of TRIM28 and SETDB1 also emerged in pregnant vs. nonpregnant women and in blood, chorion, and decidua of mothers with MS vs. healthy mothers. In contrast, HERV and TRIM28/SETDB1 expressions were comparable between their neonates. These results show that gestation is characterized by impaired expressions of HERVs and TRIM28/SETDB1, particularly in mothers with MS. Given the beneficial effects of pregnancy on MS and the wealth of data suggesting the putative contribution of HERVs and epigenetic processes in the pathogenesis of the disease, our findings may further support innovative therapeutic interventions to block HERV activation and to control aberrant epigenetic pathways in MS-affected patients.

Comparison of a Genotype 1 and a Genotype 2 Macaque Foamy Virus env Gene Indicates Distinct Infectivity and Cell-Cell Fusion but Similar Tropism and Restriction of Cell Entry by Interferon-Induced Transmembrane Proteins.

Foamy viruses (FVs) are naturally found in many different animals and also in primates with the notable exception of humans, but zoonotic infections are common. In several species, two different envelope (env) gene sequence clades or genotypes exist. We constructed a simian FV (SFV) clone containing a reporter gene cassette. In this background, we compared the env genes of the SFVmmu-DPZ9524 (genotype 1) and of the SFVmmu_R289hybAGM (genotype 2) isolates. SFVmmu_R289hybAGM env-driven infection was largely resistant to neutralization by SFVmmu-DPZ9524-neutralizing sera. While SFVmmu_R289hybAGM env consistently effected higher infectivity and cell-cell fusion, we found no differences in the cell tropism conferred by either env across a range of different cells. Infection by both viruses was weakly and non-significantly enhanced by simultaneous knockout of interferon-induced transmembrane proteins (IFITMs) 1, 2, and 3 in A549 cells, irrespective of prior interferon stimulation. Infection was modestly reduced by recombinant overexpression of IFITM3, suggesting that the SFV entry step might be weakly restricted by IFITM3 under some conditions. Overall, our results suggest that the different env gene clades in macaque foamy viruses induce genotype-specific neutralizing antibodies without exhibiting overt differences in cell tropism, but individual env genes may differ significantly with regard to fitness.

What role do endogenous retroviruses play in domestic cats infected with feline leukaemia virus?

Feline leukaemia virus (FeLV) is a retrovirus that infects domestic and wild cats around the world. FeLV infection is associated with the development of neoplasms, bone marrow disorders and immunosuppression. Viral subgroups arise from mutations in the FeLV genome or from recombination of FeLV with ancestral endogenous retroviruses in the cat genome. The retroviral endogenisation process has allowed generation of a diversity of endogenous viruses, both functional and defective. These elements may be part of the normal functioning of the feline genome and may also interact with FeLV to form recombinant FeLV subgroups, enhance pathogenicity of viral subgroups, or inhibit and/or regulate other retroviral infections. Recombination of the env gene occurs most frequently and appears to be the most significant in terms of both the quantity and diversification of pathogenic effects in the viral population, as well as affecting cell tropism and types of disease that occur in infected cats. This review focuses on available information regarding genetic diversity, pathogenesis and diagnosis of FeLV as a result of the interaction between endogenous and exogenous viruses.

Cross-species transmission of an ancient endogenous retrovirus and convergent co-option of its envelope gene in two mammalian orders.

Endogenous retroviruses (ERVs) found in vertebrate genomes are remnants of retroviral invasions of their ancestral species. ERVs thus represent molecular fossil records of ancient retroviruses and provide a unique opportunity to study viral-host interactions, including cross-species transmissions, in deep time. While most ERVs contain the mutated remains of the original retrovirus, on rare occasions evolutionary selection pressures lead to the co-option/exaptation of ERV genes for a host function. Here, we report the identification of two ancient related non-orthologous ERV env genes, ARTenvV and CARenvV, that are preserved with large open reading frames (ORFs) in the mammalian orders Artiodactyla and Carnivora, respectively, but are not found in other mammals. These Env proteins lack a transmembrane motif, but phylogenetic analyses show strong sequence preservation and positive selection of the env surface ORF in their respective orders, and transcriptomic analyses show a broad tissue expression pattern for both ARTenvV and CARenvV, suggesting that these genes may be exapted for a host function. Multiple lines of evidence indicate that ARTenvV and CARenvV were derived from an ancient ancestral exogenous gamma-like retrovirus that was independently endogenized in two mammalian orders more than 60 million years ago, which roughly coincides with the K-Pg mass extinction event and subsequent mammalian diversification. Thus, these findings identify the oldest known retroviral cross-ordinal transmission of a gamma-like retrovirus with no known extant infectious counterpart in mammals, and the first discovery of the convergent co-option of an ERV gene derived from the same ancestral retrovirus in two different mammalian orders.

Stealing genes and facing consequences.

The human genome contains a domesticated viral envelope gene with antiviral activity.

SERINC5 Restricts HIV-1 Infectivity by Promoting Conformational Changes and Accelerating Functional Inactivation of Env.

SERINC5 incorporates into HIV-1 particles and inhibits the ability of Env glycoprotein to mediate virus-cell fusion. SERINC5-resistance maps to Env, with primary isolates generally showing greater resistance than laboratory-adapted strains. Here, we examined a relationship between the inhibition of HIV-1 infectivity and the rate of Env inactivation using a panel of SERINC5-resistant and -sensitive HIV-1 Envs. SERINC5 incorporation into pseudoviruses resulted in a faster inactivation of sensitive compared to resistant Env strains. A correlation between fold reduction in infectivity and the rate of inactivation was also observed for multiple Env mutants known to stabilize and destabilize the closed Env structure. Unexpectedly, most mutations disfavoring the closed Env conformation rendered HIV-1 less sensitive to SERINC5. In contrast, functional inactivation of SERINC5-containing viruses was significantly accelerated in the presence of a CD4-mimetic compound, suggesting that CD4 binding sensitizes Env to SERINC5. Using a small molecule inhibitor that selectively targets the closed Env structure, we found that, surprisingly, SERINC5 increases the potency of this compound against a laboratory-adapted Env which prefers a partially open conformation, indicating that SERINC5 may stabilize the closed trimeric Env structure. Our results reveal a complex effect of SERINC5 on Env conformational dynamics that promotes Env inactivation and is likely responsible for the observed restriction phenotype.

Effect of human endogenous retrovirus-K env gene knockout on proliferation of ovarian cancer cells.

BACKGROUND: Among various human endogenous retroviruses (HERVs), the HERV-K (HML-2) group has been reported to be highly related to cancer. In pancreatic cancer cells, shRNA-mediated downregulation of HERV-K env RNA decreases cell proliferation and tumor growth through the RAS-ERK-RSK pathway; in colorectal cancer, CRISPR-Cas9 knockout (KO) of the HERV-K env gene affects tumorigenic characteristics through the nupr-1 gene. OBJECTIVE: The effect of HERV-K env KO has not been studied in ovarian cancer cell lines. In this study, we analyzed the tumorigenic characteristics of ovarian cancer cell lines, including cell proliferation, migration, and invasion, and the expression patterns of related proteins after CRISPR-Cas9 KO of the HERV-K env gene. METHODS: The HERV-K env gene KO was achieved using the CRISPR-Cas9 system in ovarian cancer cell lines SKOV3 and OVCAR3. Tumorigenic characteristics including cell proliferation, migration, and invasion were analyzed, and related protein expression was investigated by western blot analysis. RESULTS: The expression of the HERV-K env gene in KO cells was significantly reduced at RNA and protein levels, and tumorigenic characteristics including cell proliferation, migration, and invasion were significantly reduced. In HERV-K env KO SKOV3 cells, the expression of the RB protein was significantly up-regulated and the cyclin B1 protein level was significantly reduced. In contrast, in HERV-K env KO OVCAR3 cells, the level of phospho-RB protein was significantly reduced, but other protein levels were not changed. CONCLUSION: The results of this study showed that HERV-K env gene KO affects cell proliferation, invasion, and migration of ovarian cells through RB and Cyclin B1 proteins, but the specific regulation pattern can differ by cell line.

Convergent evolution of antiviral machinery derived from endogenous retrovirus truncated envelope genes in multiple species.

Host genetic resistance to viral infection controls the pathogenicity and epidemic dynamics of infectious diseases. Refrex-1 is a restriction factor against feline leukemia virus subgroup D (FeLV-D) and an endogenous retrovirus (ERV) in domestic cats (ERV-DC). Refrex-1 is encoded by a subset of ERV-DC loci with truncated envelope genes and secreted from cells as a soluble protein. Here, we identified the copper transporter CTR1 as the entry receptor for FeLV-D and genotype I ERV-DCs. We also identified CTR1 as a receptor for primate ERVs from crab-eating macaques and rhesus macaques, which were found in a search of intact envelope genes capable of forming infectious viruses. Refrex-1 counteracted infection by FeLV-D and ERV-DCs via competition for the entry receptor CTR1; the antiviral effects extended to primate ERVs with CTR1-dependent entry. Furthermore, truncated ERV envelope genes found in chimpanzee, bonobo, gorilla, crab-eating macaque, and rhesus macaque genomes could also block infection by feline and primate retroviruses. Genetic analyses showed that these ERV envelope genes were acquired in a species- or genus-specific manner during host evolution. These results indicated that soluble envelope proteins could suppress retroviral infection across species boundaries, suggesting that they function to control retroviral spread. Our findings revealed that several mammalian species acquired antiviral machinery from various ancient retroviruses, leading to convergent evolution for host defense.

Identification of Copper Transporter 1 as a Receptor for Feline Endogenous Retrovirus ERV-DC14.

Vertebrates harbor hundreds of endogenous retroviral (ERV) sequences in their genomes, which are considered signs of past infections that occurred during evolution. On rare occasions, ERV genes like env are maintained and coopted by hosts for physiological functions, but they also participate in recombination events with exogenous retroviruses to generate rearranged viruses with novel tropisms. In domestic cats, feline leukemia virus type D (FeLV-D) has been described as a recombinant virus between the infectious FeLV-A and likely the ERV-DC14 env gene that resulted in an extended tropism due to the usage of a new uncharacterized retroviral receptor. Here, we report the identification of SLC31A1 encoding the copper transporter 1 (CTR1) as a susceptibility gene for ERV-DC14 infection. Expression of human CTR1 into nonpermissive cells was sufficient to confer sensitivity to ERV-DC14 pseudotype infection and to increase the binding of an ERV-DC14 Env ligand. Moreover, inactivation of CTR1 by genome editing or cell surface downmodulation of CTR1 by a high dose of copper dramatically decreased ERV-DC14 infection and binding, while magnesium treatment had no effect. We also investigated the role of CTR1 in the nonpermissivity of feline and hamster cells. While feline CTR1 was fully functional for ERV-DC14, we found that binding was strongly reduced upon treatment with conditioned medium of feline cells, suggesting that the observed resistance to infection was a consequence of CTR1 saturation. In contrast, hamster CTR1 was inactive due to the presence of a N-linked glycosylation site at position 27, which is absent in the human ortholog. These results provide evidence that CTR1 is a receptor for ERV-DC14. Along with chimpanzee endogenous retrovirus type 2, ERV-DC14 is the second family of endogenous retrovirus known to have used CTR1 during past infections of vertebrates. IMPORTANCE Receptor usage is an important determinant of diseases induced by pathogenic retroviruses. In the case of feline leukemia viruses, three subgroups (A, B, and C) based on their ability to recognize different cell host receptors, respectively, the thiamine transporter THTR1, the phosphate transporter PiT1, and the heme exporter FLVCR1, are associated with distinct feline diseases. FeLV-A is horizontally transmitted and found in all naturally infected cats, while FeLV-B and FeLV-C have emerged from FeLV-A, respectively, by recombination with endogenous retroviral env sequences or by mutations in the FeLV-A env gene, both leading to a switch in receptor usage and in subsequent in vivo tropism. Here, we set up a genetic screen to identify the retroviral receptor of ERV-DC14, a feline endogenous provirus whose env gene has been captured by infectious FeLV-A to give rise to FeLV-D in a process similar to FeLV-B. Our results reveal that the copper transporter CTR1 was such a receptor and provide new insights into the acquisition of an expanded tropism by FeLV-D.

Mouse Mammary Tumour Virus (MMTV) in Human Breast Cancer-The Value of Bradford Hill Criteria.

For many decades, the betaretrovirus, mouse mammary tumour virus (MMTV), has been a causal suspect for human breast cancer. In recent years, substantial new evidence has been developed. Based on this evidence, we hypothesise that MMTV has a causal role. We have used an extended version of the classic A. Bradford Hill causal criteria to assess the evidence. 1. Identification of MMTV in human breast cancers: The MMTV 9.9 kb genome in breast cancer cells has been identified. The MMTV genome in human breast cancer is up to 98% identical to MMTV in mice. 2. EPIDEMIOLOGY: The prevalence of MMTV positive human breast cancer is about 35 to 40% of breast cancers in Western countries and 15 to 20% in China and Japan. 3. Strength of the association between MMTV and human breast cancer: Consistency-MMTV env gene sequences are consistently five-fold higher in human breast cancer as compared to benign and normal breast controls. 4. Temporality (timing) of the association: MMTV has been identified in benign and normal breast tissues up to 10 years before the development of MMTV positive breast cancer in the same patient. 5. EXPOSURE: Exposure of humans to MMTV leads to development of MMTV positive human breast cancer. 6. Experimental evidence: MMTVs can infect human breast cells in culture; MMTV proteins are capable of malignantly transforming normal human breast epithelial cells; MMTV is a likely cause of biliary cirrhosis, which suggests a link between MMTV and the disease in humans. 7. Coherence-analogy: The life cycle and biology of MMTV in humans is almost the same as in experimental and feral mice. 8. MMTV Transmission: MMTV has been identified in human sputum and human milk. Cereals contaminated with mouse fecal material may transmit MMTV. These are potential means of transmission. 9. Biological plausibility: Retroviruses are the established cause of human cancers. Human T cell leukaemia virus type I (HTLV-1) causes adult T cell leukaemia, and human immunodeficiency virus infection (HIV) is associated with lymphoma and Kaposi sarcoma. 10. Oncogenic mechanisms: MMTV oncogenesis in humans probably differs from mice and may involve the enzyme APOBEC3B. CONCLUSION: In our view, the evidence is compelling that MMTV has a probable causal role in a subset of approximately 40% of human breast cancers.

Dual Role of HIV-1 Envelope Signal Peptide in Immune Evasion.

HIV-1 Env signal peptide (SP) is an important contributor to Env functions. Env is generated from Vpu/Env encoded bicistronic mRNA such that the 5' end of Env-N-terminus, that encodes for Env-SP overlaps with 3' end of Vpu. Env SP displays high sequence diversity, which translates into high variability in Vpu sequence. This study aimed to understand the effect of sequence polymorphism in the Vpu-Env overlapping region (VEOR) on the functions of two vital viral proteins: Vpu and Env. We used infectious molecular clone pNL4.3-CMU06 and swapped its SP (or VEOR) with that from other HIV-1 isolates. Swapping VEOR did not affect virus production in the absence of tetherin however, presence of tetherin significantly altered the release of virus progeny. VEOR also altered Vpu's ability to downregulate CD4 and tetherin. We next tested the effect of these swaps on Env functions. Analyzing the binding of monoclonal antibodies to membrane embedded Env revealed changes in the antigenic landscape of swapped Envs. These swaps affected the oligosaccharide composition of Env-N-glycans as shown by changes in DC-SIGN-mediated virus transmission. Our study suggests that genetic diversity in VEOR plays an important role in the differential pathogenesis and also assist in immune evasion by altering Env epitope exposure.