Expression Of Env Protein Research Articles

Abstract 4899: Inhibition of murine tumor growth by decitabine is correlated with enhanced expression of the mouse mammary tumor virus

Abstract The purpose of this study is to clarify the mechanisms of the antineoplastic effect of decitabine (DAC), a DNA methyltransferase inhibitor. We previously reported that DAC inhibited the proliferation of the 4T1 mammary cancer cell line in vitro while inhibiting MMTV env and pol expression in the same cells. Here we present findings in vivo using two murine tumor models. We inoculated 4T1 cells into BALB/c mice from which the cell line was derived. In addition, we inoculated the murine colon carcinoma cell line, MC38, into C57BL/6 mice, the mouse strain of its origin. We treated the mice with DAC when tumors became palpable. DAC inhibited tumor growth in both tumor models and inhibited metastasis of 4T1 cells into the lungs. Quantitative PCR detected elevated expression of MMTV genes in the tumor tissues of DAC-treated mice in both models. The levels of MMTV env RNA in tumor tissues were negatively correlated with tumor mass in both models, so were the levels of the MMTV Env protein in 4T1 tumors. DAC also significantly mitigated tumor-induced splenomegaly. Hepatotoxicity of DAC was observed in C57BL/6 mice. Our data suggest the possibility that decitabine inhibits tumor growth in vivo and in vitro via different mechanisms. The findings also support the current understanding of decitabine upregulating endogenous retroviruses (ERVs) which subsequently activate an interferon response. The negative correlation between tumor mass and MMTV Env protein expression suggests a possible role of ERV proteins serving as tumor-associated antigens and decitabine enhancing adaptive immunity against cancer by stimulating ERV expression on tumor cells. Further studies of the interaction between decitabine and MMTV using immunodeficient mice may help us understand the possible involvement of adaptive immunity in the pharmacodynamics of decitabine as well as the role of ERVs in tumor development. Citation Format: Jieyu Zhang, Savannah Higgins, Riley Smith, Tania Reginald, Andrew Qi, Jiayi Li, Anna King, Yingguang Liu. Inhibition of murine tumor growth by decitabine is correlated with enhanced expression of the mouse mammary tumor virus. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4899.

Abstract P4-08-25: Unravelling the role of human endogenous retrovirus K (HERV-K) in inducible nitric oxide synthase (iNOS) mediated triple negative breast cancer progression

Abstract Human endogenous retrovirus K (HERV-K) belongs to a family of endogenous retroviruses that are present in our genome with similarities to present day exogenous retroviruses. This virus can express several proteins but our knowledge of HERV-K expression in human cancers is mainly limited to the envelope (Env) protein. Elevated HERV-K env protein expression has been shown in breast cancer both in in vitro and in vivo studies. This project aimed to decipher mechanisms of HERV-K induction in triple negative breast cancer (TNBC), and the impact of HERV-K targeting shRNA mediated knockdown on TNBC characteristics including cell migration, invasion and proliferative capacity in 2D and 3D cell culture models. Our results show that a role for inducible nitric oxide synthase (iNOS) in the induction of HERV-K in TNBC cell lines. RNA seq and bioinformatics analysis was performed to identify key molecular processes regulated by HERV-K and nitric oxide (NO) in MDA-MB-231 TNBC cells. Reduced rates of migration, invasion and proliferation in HERV-K knockdowns points towards the essential role of HERV-K in tumorigenesis and metastasis. HERV-K knockdown also modulated key gene expression signatures traditionally associated with the basal and mesenchymal phenotypes in breast cancer, cellular senescence and MHC class gene regulation. The co-expression of iNOS and HERV-K was assessed in over 150 TNBC cases and the association with patient outcomes assessed. Taken together, our findings indicate that NO and HERV-K may be a useful molecular target for the treatment of TNBC. Citation Format: Dibyangana Bhattacharyya, Eoin Dervan, Sharon Glynn, Grace Callagy. Unravelling the role of human endogenous retrovirus K (HERV-K) in inducible nitric oxide synthase (iNOS) mediated triple negative breast cancer progression [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-25.

Abstract P3-06-05: Human endogenous retrovirus-K (HERV-K) is aberrantly expressed in triple negative breast cancer (TNBC) and associated with increased distant metastasis: Impact of HERV-K knockdown on gene expression patterns and invasive potential of mesenchymal TNBC

Abstract Background: Human endogenous retrovirus K (HERV-K) belongs to a family of endogenous retroviruses that are present in our genome with similarities to present day exogenous retroviruses. HERV-K, like other endogenous retroviruses, is transmitted vertically in a Mendelian fashion through the human genome. This virus can express several proteins but our knowledge of HERV-K expression in human cancers is mainly limited to the envelope (Env) protein. Elevated HERV-K env protein expression has been shown in breast cancer both in in vitro and in vivo studies. Previous work from this laboratory has shown increased HERV-K expression in blood is predictive of a prostate cancer diagnosis. Aims & Experimental Approach: This project aimed to decipher whether individual HERV-K proteins (Env and Gag) display differential association with histological type, molecular subtype and patient outcomes in an Irish cohort of triple negative breast cancer patients (n=177). Additionally we aimed to assess the impact of HERV-K targeting shRNA mediated knockdown on TNBC characteristics including cell migration, invasion and proliferative capacity in 2D and 3D cell culture models. RNA seq and bioinformatics analysis was performed to identify key molecular processes regulated by HERV-K in TNBC. Results: HERV-K Env expression showed a trend towards an association with poor survival in TNBC (Log rank test, p=0.105), while HERV-K Gag was not found to be associated with survival. HERV-K Env positive metastatic patients had an increased involvement of the bone and lungs compared to HERV-K Env negative patients, while HERV-K gag positive patients showed reduced liver metastasis involvement. Knockdown of HERV-K in TNBC cell lines MDA-MB-231 and MDA-MB-468 reduced their proliferative, migratory and invasive potential. Preliminary screens using a cancer drug target array shows significantly reduced expression of HDAC4, a regulator of cell growth and the anti-apoptotic protein. GSEA analysis of RNAseq data from MDA-MB-231 HERV-K shRNA knockdown demonstrated enrichment for genes associated with both the Charafe downregulated in Luminal A versus Mesenchymal gene signature (p= 5.14x10-133), and the Charafe downregulated in Luminal A versus Basal gene signature (p= 7.03x10-110), indicating that HERV-K may play an important role in mesenchymal and basal phenotype TNBC. Additionally pathway analysis points toward a role in the regulation of many different pathways and cellular process, including focal adhesion (p= 9.77x10-7), cellular senescence (p= 7.63x10-7) and viral signalling pathways (p= 6.86x10-14). Discussion: Reduced rates of migration, invasion and proliferation in HERV-K knockdowns points towards the essential role of HERV-K in tumorigenesis and metastasis. HERV-K knockdown also modulated key gene expression signatures traditionally associated with the basal and mesenchymal phenotypes in breast cancer. Taken together, our findings indicate that HERV-K may be a useful molecular target for the treatment of TNBC. Citation Format: Sharon A Glynn, Dibyangana Bhattacharyya, Shauna Lambe, Emma Kerr, Simon McDade, Faizan H Khan, Eoin Dervan, Feng Wang-Johanning, Sean Hynes, Grace Callagy. Human endogenous retrovirus-K (HERV-K) is aberrantly expressed in triple negative breast cancer (TNBC) and associated with increased distant metastasis: Impact of HERV-K knockdown on gene expression patterns and invasive potential of mesenchymal TNBC [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-06-05.

Expression of Human Endogenous Retroviruses in the Human Thymus Along T Cell Development

Human Endogenous Retroviruses (HERVs) constitute up to 8% of the human genome and have been emerging as important modulators of the immune system, being associated with cancer, autoimmunity and infectious diseases. Here, we investigated the expression of three HERV families in the human thymus. HERV-K, -W, and -R envelope (env) and HERV-Kgagtranscriptional levels were quantified in the main thymocyte subsets, thymic epithelial cells (TECs), B cells and myeloid populations, and Env protein expression was studied in thymic tissue. We found that HERV mRNA decreased with T cell development, which was in agreement with the identification of HERV-K Env protein in CD3 negative cortical cells. These results suggest a distinct regulation of HERV expression along T cell development, prompting us to evaluate the interplay with host restriction factors and potential underlying pathways. The transcriptional levels of some HERVs were found to positively correlate with the expression of the host restriction factors APOBEC3G and SLFN11, and, conversely, a negative correlation was found with SAMHD1. Moreover, IFN-α and IFN-γ induced the upregulation of HERV-Kenvandgagin purified CD4 single-positive thymocytes. Additionally, we found high levels of HERV mRNAs in TECs. Overall, our data support a tight regulation of HERV expression during human T cell development, with possible implications for the process of T cell selection.

YTHDC1 regulates distinct post-integration steps of HIV-1 replication and is important for viral infectivity

BackgroundThe recent discovery of the role of m6A methylation in the regulation of HIV-1 replication unveiled a novel layer of regulation for HIV gene expression. This epitranscriptomic modification of HIV-1 RNAs is under the dynamic control of specific writers and erasers. In addition, cytoplasmic readers of the m6A mark are recruited to the modified viral RNAs and regulate HIV-1 replication. Yet, little is known about the effects of m6A writers and readers on the biogenesis of HIV-1 RNAs.ResultsWe showed that the METTL3/14 m6A methyltransferase complex and the m6A YTHDF2 cytoplasmic reader down regulates the abundance of HIV-1 RNAs in infected cells. We also identified the m6A nuclear reader YTHDC1 as a novel regulator of HIV-1 transcripts. In HIV-1 producer cells, we showed that knocking down YTHDC1 increases the levels of unspliced and incompletely spliced HIV-1 RNAs, while levels of multiply spliced transcripts remained unaffected. In addition, we observed that depletion of YTHDC1 has no effect on the nuclear cytoplasmic distribution of viral transcripts. YTHDC1 binds specifically to HIV-1 transcripts in a METTL3-dependent manner. Knocking down YTHDC1 reduces the expression of Env and Vpu viral proteins in producer cells and leads to the incorporation of unprocessed Env gp160 in virus particles, resulting in the decrease of their infectivity.ConclusionsOur findings indicate that, by controlling HIV-1 RNA biogenesis and protein expression, the m6A nuclear reader YTHDC1 is required for efficient production of infectious viral particles.Graphical

Endogenous Retroviral-K Envelope Is a Novel Tumor Antigen and Prognostic Indicator of Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is one of the ten most common cancers for men and women with an approximate 75% overall 5-year survival. Sixteen histological tumor subtypes exist and the most common are papillary, chromophobe and clear cell renal cell carcinoma (ccRCC) representing 85% of all RCC. Although epigenetically silenced, endogenous retroviral (ERV) genes become activated in tumors and function to ignite immune responses. Research has intensified to understand ERV protein function and their role as tumor antigens and targets for cancer (immune) therapy. ERV-K env is overexpressed and implicated as a therapeutic target for breast cancer, however studies in RCC are limited. In this investigation a human RCC tissue microarray (TMA) (n=374) predominantly consisting of the most common histological tumor subtypes was hybridized with an ERV-K env antibody and correlated with patient clinical data. TMA results showed the highest amount of ERV-K env protein expression and the strongest significant membrane expression in ccRCC versus other RCC subtypes. High ERV-K env total protein expression of all tumor subtypes significantly correlated with low tumor grading and a longer disease specific survival using multivariable analyses. Cell proliferation and invasion were assayed using the kidney cell lines HEK293 with wild-type p53 and a ccRCC cell line MZ1257RC mutated for p53. Transfecting these cell lines with a codon optimized ERV-K113 env overexpressing CMV vector was performed with or without 5’-Aza-2’-deoxycytidine (Aza) treatment to sustain promoter de-methylation. MZ1257RC showed induction of ERV-K113 expression and significantly increased both proliferation and invasion in the presence or absence of Aza. HEK293 cells demonstrated a restriction of ERV-K113 env expression and invasion with no changes in proliferation in the absence of Aza. However, in the presence of Aza despite increased ERV-K113 env expression, an inhibition of HEK293 proliferation and a further restriction of invasion was found. This study supports ERV-K env as a single prognostic indicator for better survival of RCC, which we propose represents a new tumor antigen. In addition, ERV-K env significantly regulates proliferation and invasion depending on p53 status and Aza treatment.

HIV-1 Lethality and Loss of Env Protein Expression Induced by Single Synonymous Substitutions in the Virus Genome Intronic-Splicing Silencer.

Synonymous genome recoding has been widely used to study different aspects of virus biology. Codon usage affects the temporal regulation of viral gene expression. In this study, we performed synonymous codon mutagenesis to investigate whether codon usage affected HIV-1 Env protein expression and virus viability. We replaced the codons AGG, GAG, CCU, ACU, CUC, and GGG of the HIV-1 env gene with the synonymous codons CGU, GAA, CCG, ACG, UUA, and GGA, respectively. We found that recoding the Env protein gp120 coding region (excluding the Rev response element [RRE]) did not significantly affect virus replication capacity, even though we introduced 15 new CpG dinucleotides. In contrast, changing a single codon (AGG to CGU) located in the gp41 coding region (HXB2 env position 2125 to 2127), which was included in the intronic splicing silencer (ISS), completely abolished virus replication and Env expression. Computational analyses of this mutant revealed a severe disruption in the ISS RNA secondary structure. A variant that restored ISS secondary RNA structure also reestablished Env production and virus viability. Interestingly, this codon variant prevented both virus replication and Env translation in a eukaryotic expression system. These findings suggested that disrupting mRNA splicing was not the only means of inhibiting translation. Our findings indicated that synonymous gp120 recoding was not always deleterious to HIV-1 replication. Importantly¸ we found that disrupting an external ISS loop strongly affected HIV-1 replication and Env translation.IMPORTANCE Synonymous substitutions can influence virus phenotype, replication capacity, and virulence. In this study, we explored how synonymous codon mutations impacted HIV-1 Env protein expression and virus replication capacity. We changed a single codon, AGG to CGU, which was located in the gp41 coding region (env nucleotide residues 2125 to 2127) and was included in the HIV-1 intronic splicing silencer. This change completely abolished virus replication and Env expression. We also found that changing codon usage in the gp120 region by including an increased number of CpG dinucleotides did not significantly affect Env expression or virus viability. Our findings showed that synonymous recoding was useful for altering viral phenotype and exploring virus biology.

Analysis of KAP1 expression patterns and human endogenous retrovirus Env proteins in ovarian cancer.

Human endogenous retroviruses (HERVs) constitute around 8% of the human genome and have important roles in human health and disease, including cancers. Previous studies showed that HERV envelope (Env) proteins are highly expressed in cancer tissues and co-related with cancer progression. KAP1 has been reported to play a key role in regulating retrotransposons, including HERV-K, through epigenetic silencing. The relationship between KAP-1 and HERV Envs expressions was analyzed only in tumor cell lines and has not yet been studied in cancer tissues. In this study, we analyzed the expression patterns and relationship between KAP1 and HERV Env proteins in ovarian cancer tissues. The expression patterns of KAP-1 and HERV Env proteins, including HERV-K and HERV-R, were analyzed in ovarian cancer tissue microarrays that contained 80 surgical specimens, including normal ovary and malignant ovarian cancers. The expression of HERV-R Env and KAP1 proteins is significantly higher in ovarian cancer compared with normal ovary tissues. However, the expression of HERV-K Env did not change significantly in cancer tissues. The expression patterns of HERV-K Env and HERV-R Env significantly increased in early stages of cancer and KAP1 expression was higher in certain stage and types of cancers. However, the expression of HERV-K Env, HERV-R Env, and KAP1 did not change in different age groups. The correlation between the expression of KAP1 and HERV-Env, including HERV-K and HERV-R, was not significantly correlated. The results of this study showed that there was no significant correlation between the expression of KAP1 and HERV Env proteins in ovarian cancer tissues, unlike studies with cell lines in vitro. These results suggest that the actual expression of HERV Env proteins in ovarian cancer tissues may be regulated through various complex factors as well as KAP1.

A cell line resistant to avian leukosis virus subgroup B infection

ABSTRACTThe expression of env proteins that bind to viral cell receptors on avian leukosis virus (ALV)-susceptible cells can block ALV infection. In this study, we constructed a cell line (DF-1/B) by expressing the ALV-B env protein in DF-1 cells. PCR, immune fluorescence assay, Western blot, and immune electron microscopy results showed that the env gene can be stably expressed in DF-1cells and the env protein could be detected on the DF-1 cell membrane. An antiviral experiment concluded that the DF-1/B cell line could be resistant to 1 × 104 TCID50 ALV-B virus infection, but had no inhibitory effect on other subgroup ALV. This means that the DF-1/B cell line is specifically resistant to ALV-B and can be used as a tool for ALV-B diagnosis.

Astrocyte Infection Is Required for Retrovirus-Induced Spongiform Neurodegeneration Despite Suppressed Viral Protein Expression.

The ability of retroviruses (RVs) to cause neurodegeneration is critically dependent upon two activities of the envelope protein (Env). First, Env facilitates viral genome delivery to CNS target cells through receptor binding and membrane fusion. Second, Env expression within one or more targets indirectly alters the physiology of certain neurons. Although the major Env expressing CNS cell types have been identified for many neurovirulent RVs, it remains unresolved, which targets play a causal role in neuropathogenesis. Moreover, this issue is complicated by the potential for post-infection virus suppression. To address these questions we explored herein, whether and how cryptic neurotropism differences between ecotropic and amphotropic murine leukemia viruses (MLVs) impacted neurovirulence. Neurotropism was first explored ex vivo using (1) acute primary glial cell cultures and (2) neural progenitor cell (NPC)- neural stem cell (NSC) neural sphere (NPH) chimeras. These experiments indicated that primary astrocytes and NPCs acutely restrict amphotropic but not ecotropic virus entry. CNS tropism was investigated using NSC transplant-based Cre-vector pseudotyping wherein mTmG transgenic fluorescent protein reporter mice revealed both productive and suppressed infection. Cre-pseudotyping with FrCasE, a prototypic neurovirulent ecotropic virus, identified glia and endothelia, but not neurons, as targets. Almost two-thirds (62%) of mGFP+ cells failed to show Env expression, suggesting widespread virus suppression. To circumvent RV superinfection interference confounds, targets were also identified using ecotropic packaging NSCs. These experiments identified known ecotropic targets: microglia, oligodendrocyte progenitor cells (OPCs) and endothelia. Additionally, one third of mGFP+ cells were identified as protoplasmic astrocytes, cells that rarely express virus in vivo. A CNS targeting comparison between isogenic ecotropic (FrCasE) and amphotropic (FrAmE) viruses showed a fourfold higher astrocyte targeting by FrCasE. Since ecotropic Env pseudotyping of amphotropic virus in the CNS dramatically exacerbates neurodegeneration, these results strongly suggest that astrocyte infection is a major disease requirement. Moreover, since viral Env protein expression is largely subdetectable in astrocytes, minimal viral protein expression appears sufficient for affecting neuronal physiology. More broadly, these findings raise the specter that subdetectable astrocyte expression of exogenous or endogenous RVs could play a major role in human and animal neurodegenerative diseases.

Genetically Intact but Functionally Impaired HIV-1 Env Glycoproteins in the T-Cell Reservoir.

HIV-infected subjects under antiretroviral treatment (ART) harbor a persistent viral reservoir in resting CD4+ T cells, which accounts for the resurgence of HIV replication after ART interruption. A large majority of HIV reservoir genomes are genetically defective, but even among intact proviruses few seem able to generate infectious virus. To understand this phenomenon, we examined the function and expression of HIV envelope glycoproteins reactivated from the reservoir of four HIV-infected subjects under suppressive ART. We studied full-length genetically intact env sequences from both replicative viruses and cell-associated mRNAs. We found that these Env proteins varied extensively in fusogenicity and infectivity, with strongest functional defects found in Envs from cell-associated mRNAs. Env functional impairments were essentially explained by defects in Env protein expression. Our results support the idea that defects in HIV Env expression, preventing cytopathic or immune HIV clearance, contribute to the persistence of the HIV T-cell reservoir in vivoIMPORTANCE In most individuals, evolution of HIV infection is efficiently controlled on the long-term by combination antiviral therapies. These treatments, however, fail to eradicate HIV from the infected subjects, a failure that results both in resurgence of virus replication and in resumption of HIV pathogenicity when the treatment is stopped. HIV resurgence, in these instances, is widely assumed to emerge from a reservoir of silent virus integrated in the genomes of a small number of T lymphocytes. The silent HIV reservoir is mostly composed of heavily deleted or mutated HIV DNA. Moreover, among the seemingly intact remaining HIV, only very few are actually able to efficiently propagate in tissue culture. In this study, we find that intact HIV in the reservoir often carry strong defects in their capacity to promote fusion to neighboring cells and infection of target cells, a defect related to the function and expression of the HIV envelope glycoprotein. Impaired envelope glycoprotein expression and function could explain why cells harboring these viruses tend to remain undetected and unharmed in the reservoir.

Downregulation of Human Endogenous Retrovirus Type K (HERV-K) Viral env RNA in Pancreatic Cancer Cells Decreases Cell Proliferation and Tumor Growth.

Purpose: We investigated the role of the human endogenous retrovirus type K (HERV-K) envelope (env) gene in pancreatic cancer.Experimental Design: shRNA was employed to knockdown (KD) the expression of HERV-K in pancreatic cancer cells.Results: HERV-K env expression was detected in seven pancreatic cancer cell lines and in 80% of pancreatic cancer patient biopsies, but not in two normal pancreatic cell lines or uninvolved normal tissues. A new HERV-K splice variant was discovered in several pancreatic cancer cell lines. Reverse transcriptase activity and virus-like particles were observed in culture media supernatant obtained from Panc-1 and Panc-2 cells. HERV-K viral RNA levels and anti-HERV-K antibody titers were significantly higher in pancreatic cancer patient sera (N = 106) than in normal donor sera (N = 40). Importantly, the in vitro and in vivo growth rates of three pancreatic cancer cell lines were significantly reduced after HERV-K KD by shRNA targeting HERV-K env, and there was reduced metastasis to lung after treatment. RNA-Seq results revealed changes in gene expression after HERV-K env KD, including RAS and TP53. Furthermore, downregulation of HERV-K Env protein expression by shRNA also resulted in decreased expression of RAS, p-ERK, p-RSK, and p-AKT in several pancreatic cancer cells or tumors.Conclusions: These results demonstrate that HERV-K influences signal transduction via the RAS-ERK-RSK pathway in pancreatic cancer. Our data highlight the potentially important role of HERV-K in tumorigenesis and progression of pancreatic cancer, and indicate that HERV-K viral proteins may be attractive biomarkers and/or tumor-associated antigens, as well as potentially useful targets for detection, diagnosis, and immunotherapy of pancreatic cancer. Clin Cancer Res; 23(19); 5892-911. ©2017 AACR.

Differences in Env and Gag protein expression patterns and epitope availability in feline immunodeficiency virus infected PBMC compared to infected and transfected feline model cell lines

Env and Gag are key components of the FIV virion that are targeted to the plasma membrane for virion assembly. They are both important stimulators and targets of anti-FIV immunity. To investigate and compare the expression pattern and antigenic changes of Gag and Env in various research models, infected PBMC (the natural FIV host cells) and GFox, and transfected CrFK were stained over time with various Env and Gag specific MAbs. In FIV infected GFox and PBMC, Env showed changes in epitope availability for antibody binding during processing and trafficking, which was not seen in transfected CrFK. Interestingly, epitopes exposed on intracellular Env and Env present on the plasma membrane of CrFK and GFox seem to be hidden on plasma membrane expressed Env of FIV infected PBMC. A kinetic follow up of Gag and Env expression showed a polarization of both Gag and Env expression to specific sites at the plasma membrane of PBMC, but not in other cell lines. In conclusion, mature trimeric cell surface expressed Env might be antigenically distinct from intracellular monomeric Env in PBMC and might possibly be unrecognizable by feline humoral immunity. In addition, Env expression is restricted to a small area on the plasma membrane and co-localizes with a large moiety of Gag, which may represent a preferred FIV budding site, or initiation of virological synapses with direct cell-to-cell virus transmission.

Expression profiles of HERV-K Env protein in normal and cancerous tissues

Human endogenous retroviruses (HERVs) have been proposed as etiological cofactors in chronic diseases such as cancer, autoimmunity and neurological disease. HERV RNA expression is also increased in several autoimmune diseases and cancers. However, only a few studies have analyzed the expression levels of HERV-derived proteins in normal and diseased tissues. Here, we investigated the expression profiles of HERV-K Env protein, which is the most well-known pathogen, from normal and cancer tissue microarray. Normal and cancer tissue microarray slides containing 59 normal or 60 tumor surgical specimens, respectively were immunostained with mouse monoclonal antibody to HERV-K and staining intensity was scored. The expression of HERV-K Env protein is generally low in normal tissues but high in specific tissues including bronchus submucosal gland, salivary gland acini, pancreas acini, testis seminiferous tubule, uterine cervix epithelium, ovary stroma, skin epidermis, and heart. The expression of HERV-K Env protein in tumors was usually higher than normal tissues and specifically high in breast, liver, stomach, prostate, and ovarian cancer. This study provides protein expression profiles of HERV-K Env protein in human normal and cancer tissues and provides a good reference for further studies on the expression patterns and roles of HERV-K Env protein in normal organs and various cancers. Further studies with specific organs or cancers and a statistically significant numbers of samples are necessary.

Chimeric antigen receptor T cells targeting HERV-K inhibit breast cancer and its metastasis through downregulation of Ras

We have previously reported that human endogenous retrovirus-K (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) for cancer vaccines, and that its antibodies (mAbs) possess antitumor activity against cancer. In this study, a chimeric antigen receptor (CAR) specific for HERV-K env protein (K-CAR) was generated using anti-HERV-K mAb. K-CAR T cells from peripheral blood mononuclear cells (PBMCs) of 9 breast cancer (BC) patients and 12 normal donors were able to inhibit growth of, and to exhibit significant cytotoxicity toward, BC cells but not MCF-10A normal breast cells. The antitumor effects in cancer cells were significantly reduced when control T cells were used, or the expression of HERV-K was knocked down by an shRNA. Secretion of multiple cytokines, including IFNγ, TNF-α, and IL-2, was significantly enhanced in culture media of BC cells treated with K-CARs. Significantly reduced tumor growth and tumor weight was observed in xenograft models bearing MDA-MB-231 or MDA-MB-435.eB1 BC cells. Importantly, the K-CAR prevented tumor metastasis to other organs. Furthermore, downregulation of HERV-K expression in tumors of mice treated with K-CAR correlated with upregulation of p53 and downregulation of MDM2 and p-ERK. Importantly, the expression of HERV-K env protein in metastatic tumor tissues treated with K-CAR T cells correlated with the expression of Ras. Our results indicate that HERV-K env protein is an oncoprotein and may play an important role in tumorigenesis related to p53 and Ras signaling pathways. Anti-HERV-K treatment, including K-CAR treatment, shows potential for immunotherapy of BC.

Systematic analysis of intracellular trafficking motifs located within the cytoplasmic domain of simian immunodeficiency virus glycoprotein gp41.

Previous studies have shown that truncation of the cytoplasmic-domain sequences of the simian immunodeficiency virus (SIV) envelope glycoprotein (Env) just prior to a potential intracellular-trafficking signal of the sequence YIHF can strongly increase Env protein expression on the cell surface, Env incorporation into virions and, at least in some contexts, virion infectivity. Here, all 12 potential intracellular-trafficking motifs (YXXΦ or LL/LI/IL) in the gp41 cytoplasmic domain (gp41CD) of SIVmac239 were analyzed by systematic mutagenesis. One single and 7 sequential combination mutants in this cytoplasmic domain were characterized. Cell-surface levels of Env were not significantly affected by any of the mutations. Most combination mutations resulted in moderate 3- to 8-fold increases in Env incorporation into virions. However, mutation of all 12 potential sites actually decreased Env incorporation into virions. Variant forms with 11 or 12 mutated sites exhibited 3-fold lower levels of inherent infectivity, while none of the other single or combination mutations that were studied significantly affected the inherent infectivity of SIVmac239. These minor effects of mutations in trafficking motifs form a stark contrast to the strong increases in cell-surface expression and Env incorporation which have previously been reported for large truncations of gp41CD. Surprisingly, mutation of potential trafficking motifs in gp41CD of SIVmac316, which differs by only one residue from gp41CD of SIVmac239, effectively recapitulated the increases in Env incorporation into virions observed with gp41CD truncations. Our results indicate that increases in Env surface expression and virion incorporation associated with truncation of SIVmac239 gp41CD are not fully explained by loss of consensus trafficking motifs.

Infection of monkeys by simian-human immunodeficiency viruses with transmitted/founder clade C HIV-1 envelopes

Simian-human immunodeficiency viruses (SHIVs) that mirror natural transmitted/founder (T/F) viruses in man are needed for evaluation of HIV-1 vaccine candidates in nonhuman primates. Currently available SHIVs contain HIV-1 env genes from chronically-infected individuals and do not reflect the characteristics of biologically relevant HIV-1 strains that mediate human transmission. We chose to develop clade C SHIVs, as clade C is the major infecting subtype of HIV-1 in the world. We constructed 10 clade C SHIVs expressing Env proteins from T/F viruses. Three of these ten clade C SHIVs (SHIV KB9 C3, SHIV KB9 C4 and SHIV KB9 C5) replicated in naïve rhesus monkeys. These three SHIVs are mucosally transmissible and are neutralized by sCD4 and several HIV-1 broadly neutralizing antibodies. However, like natural T/F viruses, they exhibit low Env reactivity and a Tier 2 neutralization sensitivity. Of note, none of the clade C T/F SHIVs elicited detectable autologous neutralizing antibodies in the infected monkeys, even though antibodies that neutralized a heterologous Tier 1 HIV-1 were generated. Challenge with these three new clade C SHIVs will provide biologically relevant tests for vaccine protection in rhesus macaques.

Selection of unadapted, pathogenic SHIVs encoding newly transmitted HIV-1 envelope proteins.

Infection of macaques with chimeric viruses based on SIVMAC but expressing the HIV-1 envelope (Env) glycoproteins (SHIVs) remains the most powerful model for evaluating prevention and therapeutic strategies against AIDS. Unfortunately, only a few SHIVs are currently available. Furthermore, their generation has required extensive adaptation of the HIV-1 Env sequences in macaques so they may not accurately represent HIV-1 Env proteins circulating in humans, potentially limiting their translational utility. We developed a strategy for generating large numbers of SHIV constructs expressing Env proteins from newly transmitted HIV-1 strains. By inoculating macaques with cocktails of multiple SHIV variants, we selected SHIVs that can replicate and cause AIDS-like disease in immunologically intact rhesus macaques without requiring animal-to-animal passage. One of these SHIVs could be transmitted mucosally. We demonstrate the utility of the SHIVs generated by this method for evaluating neutralizing antibody administration as a protection against mucosal SHIV challenge.

Polyadenylation of Friend murine leukemia virus env‐mRNA is affected by its splicing

As splicing was previously found to be important for increasing Friend murine leukemia virus env-mRNA stability and translation, we investigated whether splicing of env-mRNA affected the poly(A) tail length using env expression vectors that yielded unspliced or spliced env-mRNA. Incomplete polyadenylation was detected in a fraction of the unspliced env-mRNA products in an env gene-dependent manner, showing that splicing of Friend murine leukemia virus plays an important role in the efficiency of complete polyadenylation of env-mRNA. These results suggested that the promotion of complete polyadenylation of env-mRNA by splicing might partially explain up-regulation of Env protein expression as a result of splicing.

The potential of peripheral blood HERV-K expression as a biomarker for diagnosis with prostate cancer in older men.

5049 Background: Aberrant expression of human endogenous retrovirus-k (HERV-K) has been observed in prostate cancer (PCa). HERV-K is unique as 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. Methods: We evaluated HERV-K gag mRNA expression in PBMCs of African-American (AA) and European-American (EA) men via qRT-PCR (294 PCa cases and 135 male controls). We examined HERV-K env protein expression in PCa by immunohistochemistry. Fisher's exact test and logistic regression were used to analyze continuous and dichotomized data and calculate odds ratios (ORs). Multivariable models adjusted for age at diagnosis and race/ethnicity. Mann-Whitney test was used to compare HERV-K Ct-based expression values between groups. Results: HERV-K gag expression in PBMCs was significantly higher in PCa cases than controls (Table). Men with gag in the highest quartile had > 12-fold increased odds [OR = 12.87 (95% CI 6.3-26.25)] of PCa than those in the lowest quartile. Additionally, HERV-K may perform better in older than younger men (sensitivity of PSA testing decreases with age). HERV-K envelope protein was up-regulated in PCa, with the most abundant expression observed in AA patients (61% versus 40%, P < 0.01). Conclusions: Combining non-invasive HERV-K testing with PSA testing may improve the efficacy of PCa detection specifically among older men. Association between HERV-K gag mRNA levels in PBMC and a PCa diagnosis. Univariate analysis† Multivariable analysis* OR 95% CI P value n OR 95% CI P value n Logistic regression as a continuous variable (per Ct value) Gag mRNA 1.33 1.24-1.43 <0.0001 429 1.36 1.26-1.47 <0.0001 427 Logistic regression evaluating dose response effect** 1st Quartile 1 48 1 48 2nd Quartile 1.38 0.61-3.11 0.433 57 1.58 0.69-3.62 0.280 57 3rd Quartile 4.14 1.98-8.67 <0.0001 98 5.90 2.69-13.0 <0.0001 96 4th Quartile 12.87 6.30-26.3 <0.0001 226 17.3 8.07-37.0 <0.0001 226 Ptrend <0.0001 Ptrend <0.0001 †294 cases and 135 controls. *Adjusted for age at diagnosis and ethnicity. ***HERV-K gag levels were divided into quartiles based on control population.