Overexpression of centromeric proteins has been identified in a number of human malignancies, but the functional and mechanistic contributions of these proteins to disease progression have not been characterized. The centromeric histone H3 variant centromere protein A (CENPA) is an epigenetic mark that determines centromere identity. Here, using an array of approaches, including RNA-sequencing and ChIP-sequencing analyses, immunohistochemistry-based tissue microarrays, and various cell biology assays, we demonstrate that CENPA is highly overexpressed in prostate cancer in both tissue and cell lines and that the level of CENPA expression correlates with the disease stage in a large cohort of patients. Gain-of-function and loss-of-function experiments confirmed that CENPA promotes prostate cancer cell line growth. The results from the integrated sequencing experiments suggested a previously unidentified function of CENPA as a transcriptional regulator that modulates expression of critical proliferation, cell-cycle, and centromere/kinetochore genes. Taken together, our findings show that CENPA overexpression is crucial to prostate cancer growth. Overexpression of centromeric proteins has been identified in a number of human malignancies, but the functional and mechanistic contributions of these proteins to disease progression have not been characterized. The centromeric histone H3 variant centromere protein A (CENPA) is an epigenetic mark that determines centromere identity. Here, using an array of approaches, including RNA-sequencing and ChIP-sequencing analyses, immunohistochemistry-based tissue microarrays, and various cell biology assays, we demonstrate that CENPA is highly overexpressed in prostate cancer in both tissue and cell lines and that the level of CENPA expression correlates with the disease stage in a large cohort of patients. Gain-of-function and loss-of-function experiments confirmed that CENPA promotes prostate cancer cell line growth. The results from the integrated sequencing experiments suggested a previously unidentified function of CENPA as a transcriptional regulator that modulates expression of critical proliferation, cell-cycle, and centromere/kinetochore genes. Taken together, our findings show that CENPA overexpression is crucial to prostate cancer growth. Centromeres are cellular structures that are necessary for the propagation of hereditary information (1Cleveland D.W. Mao Y. Sullivan K.F. 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The centromere–kinetochore–microtubule interaction facilitates separation of the sister chromatids as mitosis proceeds from metaphase to anaphase. Centromeres are thus essential to ensuring faithful segregation of chromosomes in actively dividing cells. Efforts to study human centromeres have focused on the epigenetics that drive centromere assembly (3Hayashi T. Fujita Y. Iwasaki O. Adachi Y. Takahashi K. Yanagida M. Mis16 and Mis18 are required for CENP-A loading and histone deacetylation at centromeres.Cell. 2004; 118 (15369671): 715-72910.1016/j.cell.2004.09.002Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 4Kim I.S. Lee M. Park K.C. Jeon Y. Park J.H. Hwang E.J. Jeon T.I. Ko S. Lee H. Baek S.H. Kim K.I. Roles of Mis18α in epigenetic regulation of centromeric chromatin and CENP-A loading.Mol. 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Black B.E. The octamer is the major form of CENP-A nucleosomes at human centromeres.Nat. Struct. Mol. Biol. 2013; 20 (23644596): 687-69510.1038/nsmb.2562Crossref PubMed Scopus (141) Google Scholar). Proper CENPA localization is an ubiquitin E3 ligase–dependent process requiring ubiquitination of lysine 124 for engagement with the CENPA-specific chaperone HJURP (7Niikura Y. Kitagawa R. Ogi H. Abdulle R. Pagala V. Kitagawa K. CENP-A K124 ubiquitylation is required for CENP-A deposition at the centromere.Dev. Cell. 2015; 32 (25727006): 589-60310.1016/j.devcel.2015.01.024Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). HJURP subsequently facilitates the incorporation of newly synthesized CENPA into nucleosomes occupying replicated α-satellite DNA (8Barnhart M.C. Kuich P.H. Stellfox M.E. Ward J.A. Bassett E.A. Black B.E. Foltz D.R. HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore.J. 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Diseases of uncontrolled cell division, particularly cancer, are thus compelling to examine from the epigenetic perspective of centromere biology, primarily as it pertains to the key epigenetic mark CENPA. A number of studies have identified aberrant expression of centromeric/kinetochore proteins in cancers, where overexpression is predictive of survival and response to therapy, although their mechanistic contribution to cancer pathogenesis remains elusive (13Thiru P. Kern D.M. McKinley K.L. Monda J.K. Rago F. Su K.-C. Tsinman T. Yarar D. Bell G.W. Cheeseman I.M. Kinetochore genes are coordinately up-regulated in human tumors as part of a FoxM1-related cell division program.Mol. Biol. Cell. 2014; 25 (24829384): 1983-199410.1091/mbc.e14-03-0837Crossref PubMed Google Scholar, 14Tomonaga T. Matsushita K. Ishibashi M. Nezu M. Shimada H. Ochiai T. Yoda K. Nomura F. 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Cell. 2014; 53 (24530302): 631-64410.1016/j.molcel.2014.01.018Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Ectopic localization of endogenously overexpressed CENPA has also been shown in colon cancer cell lines (26Athwal R.K. Walkiewicz M.P. Baek S. Fu S. Bui M. Camps J. Ried T. Sung M.-H. Dalal Y. CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human cancer cells.Epigenetics Chromatin. 2015; 8 (25788983): 210.1186/1756-8935-8-2Crossref PubMed Scopus (86) Google Scholar). The phenotypic consequences of such mislocalization in malignancy have yet to be elucidated, although ectopic binding to sites marked by DNase hypersensitivity and CCCTC-binding factor (CTCF) transcription factor affinity has hinted at a potential role in regulating gene transcription (25Lacoste N. Woolfe A. Tachiwana H. Garea A.V. Barth T. Cantaloube S. Kurumizaka H. Imhof A. Almouzni G. Mislocalization of the centromeric histone variant CenH3/CENP-A in human cells depends on the chaperone DAXX.Mol. Cell. 2014; 53 (24530302): 631-64410.1016/j.molcel.2014.01.018Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 26Athwal R.K. Walkiewicz M.P. Baek S. Fu S. Bui M. Camps J. Ried T. Sung M.-H. Dalal Y. CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human cancer cells.Epigenetics Chromatin. 2015; 8 (25788983): 210.1186/1756-8935-8-2Crossref PubMed Scopus (86) Google Scholar). Here we report that CENPA is highly overexpressed in prostate cancer (PCA) and that disease progression correlates with CENPA expression within a large patient cohort. CENPA knockdown markedly decreases proliferation of prostate cancer cells but not that of benign prostate cells and increased expression of CENPA causes benign prostate epithelial cells to proliferate more rapidly. Interestingly, CENPA appears to affect proliferation of prostate cancer cells by acting as a transcriptional regulator that modulates expression of genes critical to proliferation, cell cycle progression, and centromere/kinetochore integrity in addition to its role in the centromere. The significance of centromeres to cell division suggests that centromeric components may play important roles in development and in diseases involving cell division gone awry, particularly in cancer. Previous work identified a centromere-kinetochore (CEN/KT) signature that was associated with aggressive, treatment-refractory malignancy (16Zhang W. Mao J.-H. Zhu W. Jain A.K. Liu K. Brown J.B. Karpen G.H. Centromere and kinetochore gene misexpression predicts cancer patient survival and response to radiotherapy and chemotherapy.Nat. Commun. 2016; 7 (27577169): 1261910.1038/ncomms12619Crossref PubMed Scopus (122) Google Scholar). We therefore profiled the transcriptomes of different types of malignancies across a compiled catalogue of publicly available RNA-sequencing (RNA-seq) databases (n = 10,848) (27Iyer M.K. Niknafs Y.S. Malik R. Singhal U. Sahu A. Hosono Y. Barrette T.R. Prensner J.R. Evans J.R. Zhao S. Poliakov A. Cao X. Dhanasekaran S.M. Wu Y.-M. Robinson D.R. et al.The landscape of long noncoding RNAs in the human transcriptome.Nat. Genet. 2015; 47 (25599403): 199-20810.1038/ng.3192Crossref PubMed Scopus (1875) Google Scholar). We found that CENPA is ubiquitously overexpressed in malignant tissue relative to respective normal counterparts (Fig. S1A and Table S1). These observations, combined with the well-characterized contributions of centromeric components like CENPA to cell division, suggested conducting a more focused interrogation of these components in cancers that display poor prognosis in the context of high proliferation indices. Prostate cancer is one such disease, where a high proliferation index is predictive of poor outcomes (28Fisher G. Yang Z.H. Kudahetti S. Møller H. Scardino P. Cuzick J. Berney D.M. Transatlantic Prostate Group Prognostic value of Ki-67 for prostate cancer death in a conservatively managed cohort.Br. J. Cancer. 2013; 108 (23329234): 271-27710.1038/bjc.2012.598Crossref PubMed Scopus (84) Google Scholar, 29Li R. Heydon K. Hammond M.E. Grignon D.J. Roach 3rd, M. Wolkov H.B. Sandler H.M. Shipley W.U. Pollack A. Ki-67 staining index predicts distant metastasis and survival in locally advanced prostate cancer treated with radiotherapy: an analysis of patients in Radiation Therapy Oncology Group protocol 86-10.Clin Cancer Res. 2004; 10 (15217948): 4118-412410.1158/1078-0432.CCR-1052-03Crossref PubMed Scopus (92) Google Scholar). New treatment strategies are much needed for prostate cancer, which remains the most diagnosed malignancy in men and the second leading cause of cancer-related death in men (30American Cancer Society Cancer Facts & Figures (2018) https://www.cancer.org/research/cancer-facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2018.html (accessed June 4, 2018).Google Scholar). Although hormonal therapy and chemotherapeutic options are available, resistant metastatic disease and life-altering side effects, such as urinary incontinence and erectile dysfunction, are everlasting concerns (31Cooperberg M.R. Prostate cancer: a new look at prostate cancer treatment complications.Nat. Rev. Clin. Oncol. 2014; 11 (24687033): 304-30510.1038/nrclinonc.2014.58Crossref PubMed Scopus (8) Google Scholar). In view of the above considerations, we performed sample set enrichment analysis (SSEA) in the prostate tissue type cohort containing RNA-seq data from 685 tissue samples (27Iyer M.K. Niknafs Y.S. Malik R. Singhal U. Sahu A. Hosono Y. Barrette T.R. Prensner J.R. Evans J.R. Zhao S. Poliakov A. Cao X. Dhanasekaran S.M. Wu Y.-M. Robinson D.R. et al.The landscape of long noncoding RNAs in the human transcriptome.Nat. Genet. 2015; 47 (25599403): 199-20810.1038/ng.3192Crossref PubMed Scopus (1875) Google Scholar). Gene expression of numerous centromeric components exhibited strong enrichments in prostate cancer tissue relative to their normal counterparts (Fig. 1A and Table S2). Our analysis corroborates on a significantly greater scale previous reports that characterize some of these components as part of the centromere-kinetochore (CEN/KT) signature that is strongly associated with poor disease outcomes (16Zhang W. Mao J.-H. Zhu W. Jain A.K. Liu K. Brown J.B. Karpen G.H. Centromere and kinetochore gene misexpression predicts cancer patient survival and response to radiotherapy and chemotherapy.Nat. Commun. 2016; 7 (27577169): 1261910.1038/ncomms12619Crossref PubMed Scopus (122) Google Scholar). We selected CENPA from this panel of genes for further assessment, given its central role in centromere biology, importance for development, and highly conserved function, and found a significant increase in expression with disease progression (Fig. 1B) (32Smith C.L. Blake J.A. Kadin J.A. Richardson J.E. Bult C.J. Mouse Genome Database Group Mouse Genome Database (MGD)-2018: knowledgebase for the laboratory mouse.Nucleic Acids Res. 2018; 46 (29092072): D836-D84210.1093/nar/gkx1006Crossref PubMed Scopus (145) Google Scholar). This in silico finding was validated at the protein level through prostate tissue microarrays stained for CENPA, notably demonstrating marked overexpression of CENPA that increased with disease severity (n = 58 total tissues, n = 174 cores) (Fig. 1C). Importantly, receiver operator characteristic analysis of the CENPA-stained prostate tissue microarray produced an area under the curve of 0.89, orthogonally demonstrating a strong association between elevated CENPA expression and metastatic prostate cancer (Fig. S1B). Assessment of CENPA expression was also examined in cancer cell line models to determine feasibility for more focused molecular inquiry. We verified robust overexpression of CENPA in prostate cancer cell lines, as compared with benign prostatic epithelial lines (Fig. 1D). The PNT2 benign cell line was a notable exception, likely because of its rapid proliferation rate relative to other cell lines we tested (Fig. S1C). Taken together, CENPA is a functionally conserved, developmentally important factor abundant in prostate cancer tissue, as seen in a large number of patients and in prostate cancer cell lines, and an increase in its expression at the RNA and protein levels is highly correlated with more aggressive disease. The abundance of CENPA in prostate cancer raised the question of whether overexpression plays a functional role in disease pathogenesis and progression. We thus first conducted a comparative analysis of CENPA expression relative to the remaining transcriptome in prostate cancer to identify associations with biological concepts that could computationally guide functional assessments. Our efforts to profile transcriptomes in human cancer and normal tissue facilitates performing transcriptome-wide correlations against nominated genes of interest in a tissue-specific manner within a large catalogue of samples (n = 685). We thus correlated CENPA mRNA levels to the expression levels of all other protein coding elements (Data Set S1) to deconvolute its relative contribution to prostate cancer progression. CENPA expression tracks tightly with a number of previously identified prostate cancer pathogenesis factors including CENPF, UBE2C, and EZH2 (Fig. 2A; Fig. S2, C and D; and Data Set S1). MKI67 (gene encoding proliferation marker Ki67) also performed well in our analysis, further suggesting a role for CENPA in cellular proliferation (Fig. 2B). Of note, CENPA does not tightly correlate with ACTB (housekeeping gene), AMACR (prostate cancer biomarker), or AR (Fig. S2, A and B, and Data Set S1), suggesting a pathogenic process that is independent of androgen signaling, a pharmacologically relevant molecular pathway that is frequently targeted in prostate cancer treatment. Strong associations with cellular proliferation genes and select pathogenesis factors independent of AR implicate CENPA as a contributor to a biological process that is involved in androgen refractory prostate cancer progression. In fact, we found that AR signaling actually represses CENPA expression in cell culture (Fig. S3A). We additionally used the Database for Annotation, Visualization, and Integrated Discovery (DAVID) to conduct ontology assessments on the highest-performing genes from our transcriptome-wide correlation against CENPA expression in prostate cancer (r > 0.8) (33Huang D.W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat. Protoc. 2009; 4 (19131956): 44-5710.1038/nprot.2008.211Crossref PubMed Scopus (25346) Google Scholar). Our analysis revealed a correlation between CENPA gene expression and biological concept clusters that highlight centromeres, kinetochores, mitosis, and cell division (Fig. 2C and Fig. S3B). Concepts that include genes encoding components of the CCAN were additionally captured by our analysis. Of note, CDC25C, CDCA5, TOP2A, and CENPU, genes known to play roles in cellular proliferation, cell cycle progression, and centromere/kinetochore integrity, were included in these biological concepts. Preranked gene set enrichment analysis (GSEA) independently confirmed enrichments in gene signatures important for cell cycle, cell division, and mitosis (Fig. 2D and Fig. S3C). Taken together, CENPA expression is strongly linked to gene signatures that underlie processes that govern proliferation, cell cycle progression, and centromere/kinetochore integrity in prostate cancer. Significant association between CENPA and proliferation signatures is expected given the role CENPA plays in the structural integrity of the centromere. There is limited evidence, however, concerning CENPA function in human malignancy. We therefore performed loss-of- and gain-of-function experiments in cell lines stably expressing either doxycycline-inducible short hairpin RNAs against CENPA or EF1A promoter–driven full-length CENPA. Doxycycline administration at 2 μg/ml was sufficient to produce robust knockdown of CENPA after 72 h (Fig. 3A and Fig. S4, A and B). CENPA depletion led to a profound growth-inhibitory effect on 22Rv1, LnCaP, and DU145 prostate cancer cells (Fig. 3, B–D, and Fig. S4, C and D). CENPA depletion in prostate cancer cells results in an accumulation of cells in G1 that seem to be unable to progress through the cell cycle (Fig. 3E and Fig. S4, E and F). Conversely, overexpression of CENPA in the 957E-hTERT benign prostate epithelial cell line leads to a profound growth-promoting effect (Fig. 3, F and G). Interestingly, benign 957E-hTERT cells depleted of CENPA do not demonstrate significant proliferative changes, consistent with previous reports that nonmalignant cells can proliferate with low levels of CENPA (Fig. 3G) (34Black B.E. Jansen L.E. Maddox P.S. Foltz D.R. Desai A.B. Shah J.V. Cleveland D.W. Centromere identity maintained by nucleosomes assembled with histone H3 containing the CENP-A targeting domain.Mol. Cell. 2007; 25 (17244537): 309-32210.1016/j.molcel.2006.12.018Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Taken together, our data show that CENPA is an essential factor for progression through the cell cycle and that overexpression drives proliferation of prostate cancer cells. Given prior reports of ectopic deposition in the setting of CENPA overexpression and the marked overexpression of CENPA in prostate cancer, we performed native ChIP followed by sequencing to identify noncentromeric and potentially regulatory binding sites for CENPA in prostate cancer. As expected, four α-satellites were enriched relative to the IgG control antibodies using a PCR assay we previously devised that can distinguish chromosome-specific α-satellite DNA from any given centromere, verifying the validity of the CENPA ChIP (Fig. S5A) (35Contreras-Galindo R. Fischer S. Saha A.K. Lundy J.D. Cervantes P.W. Mourad M. Wang C. Qian B. Dai M. Meng F. Chinnaiyan A. Omenn G.S. Kaplan M.H. Markovitz D.M. Rapid molecular assays to study human centromere genomics.Genome Res. 2017; 27 (29141960): 2040-204910.1101/gr.219709.116Crossref PubMed Scopus (13) Google Scholar). CENPA-directed ChIP-seq identified 569 noncentromeric binding sites in the VCaP prostate cancer cell line within three experimental replicates (Fig. 4A; Fig. S5, C and D; and Data Set S2). One example of such a CENPA-binding site is present in the promoter region of CDC25C (Fig. 4B), a cell cycle phosphatase that is critical for progression through anaphase that was also identified in our comparative gene expression analysis described above (Fig. 2A). Intriguingly, the promoter of CENPA itself was also bound by CENPA, consistent with previously reported results, hinting that CENPA might regulate its own transcription (26Athwal R.K. Walkiewicz M.P. Baek S. Fu S. Bui M. Camps J. Ried T. Sung M.-H. Dalal Y. CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human cancer cells.Epigenetics Chromatin. 2015; 8 (25788983): 210.1186/1756-8935-8-2Crossref PubMed Scopus (86) Google Scholar). CENPA-directed ChIP was additionally conducted in the benign prostatic epithelial cell line 957E-hTERT to determine whether ectopic CENPA binding is a cancer-specific observation (Fig. S5B). CENPA enrichment over the four previously assessed α-satellites was significantly lower than that observed in the VCaP cell line, consistent with each cell line's respective CENPA abundance observed above (Fig. 1D). CENPA-directed ChIP-seq for the 957E-hTERT cell line was thus deferred. We next conducted a global assessment of CENPA-binding sites to obtain a functional taxonomy of CENPA-bound genes. Ontologic assessment of genes whose transcriptional start sites were in close proximity to CENPA-binding sites revealed enrichments in biological concepts that are involved with maintenance of nuclear architecture and organization, such as protein–DNA complex assembly (p = 6.44 × 10−18) and chromosome organization (p = 8.70 × 10−13) (Fig. 4C). Furthermore, binning CENPA-binding sites into categories corresponding to discreet locations within the human genome demonstrates a predilection toward binding regulatory elements such as promoters and CpG islands (Fig. 4D). Comparing the number of peaks present within any two genomic regions reveals significant overlap between loci considered to be regulatory areas (Fig. S5E). Taken together, we show that CENPA localizes to noncanonical genomic loci, with a predilection toward the regulatory elements of genes that control cellular proliferation. Histone variants have been well-characterized as modulators of aberrant gene expression in cancer. H2A.Z.2, macroH2A, and H3.3 are well-documented as key contributors to malignant phenotypes in a number of cancer types (36Sporn J.C. Kustatscher G. Hothorn T. Collado M. Serrano M. Muley T. Schnabel P. Ladurner A.G. Histone macroH2A isoforms predict the risk of lung cancer recurrence.Oncogene. 2009; 28 (19648962): 3423-342810.1038/onc.2009.26Crossref PubMed Scopus (146) Google Scholar, 37Vardabasso C. Hasson D. Ratnakumar K. Chung C.-Y. Duarte L.F. Bernstein E. Histone variants: emerging players in cancer biology.Cell. Mol. Life Sci. 2014; 71 (23652611): 379-40410.1007/s00018-013-1343-zCrossref PubMed Scopus (115) Google Sc
We previously reported finding the RNA of a particle-coding human endogenous retrovirus type K, HERV-K (HML-2), in the plasma of HIV-1 patients. We found that the HERV-K (HML-2) RNA was contained in HERV-K viral particles as observed by immunoelectron microscopy. Surprisingly, a novel HERV-K (HML-2) provirus was discovered, termed K111, which seems to be transcriptionally active exclusively during HIV infection. Using real time RT-PCR specific for the K111 env sequence, we corroborate the detection of K111 RNA in plasma of HIV patients and not in breast cancer or lymphoma patients. K111 RNA was also found in the supernatants of cultured cell lines and PBMCs infected with R5 and X4 HIV strains. A K111 counterpart was found to be present in Chimpanzees. The human K111 provirus was fully amplified and sequenced. K111 proviruses were detected in all human DNA samples tested (140 individuals and 10 cell lines) and may produce only the Np9 oncoprotein. K111 is polymorphic in humans, represented by a minimum of at least 6 copies in each individual. K111 soloLTRs can also be found in the centromeric region. All these variants are integrated into tandemly repetitive D22Z3 sequences, which have been found uniquely in the centromere of the chromosome 22. The existence of more than one centromeric full-length K111 in every single patient and a balanced number of synonymous and non-synonymous mutations in their RNA sequences indicates that K111 has been expanded, possibly by homologous recombination. The degree of variability in the different K111 forms is only appreciated in HIV infected patients, where these proviruses are transcriptionally active. Furthermore, the discovery of the first centromeric HERV-K (HML-2) described so far might have only been possible due to a unique effect of HIV on chromatin remodeling of the centromere of chromosome 22, exposing K111 to active transcription.
Human endogenous retroviruses (HERV) make up 8% of the human genome. While the youngest of these retroviruses, HERV-K(HML-2), termed HK2, is able to code for all viral proteins and produce virus-like particles, it is not known if these virus particles package and transmit HK2-related sequences. Here, we analyzed the capacity of HK2 for packaging and transmitting HK2 sequences. We created an HK2 probe, termed Bogota, which can be packaged into HK2 viruses, and transfected it into cells that make HK2 particles. Supernatants of the transfected cells, which contained HK2 viral particles, then were added to target cells, and the transmissibility of the HK2 Bogota reporter was tracked by G418 resistance. Our studies revealed that contemporary HK2 virions produced by some teratocarcinoma and breast cancer cell lines, as well as by peripheral blood lymphocytes from lymphoma patients, can package HK2 Bogota probes, and these viruses transmitted these probes to other cells. After transmission, HK2 Bogota transcripts undergo reverse transcription, a step impaired by antiretroviral agents or by introduction of mutations into the probe sequences required for reverse transcription. HK2 viruses were more efficiently transmitted in the presence of HK2 Rec or HIV-1 Tat and Vif. Transmitted Bogota probes formed episomes but did not integrate into the cellular genome. Resistance to integration might explain the relatively low number of HK2 insertions that were acquired during the last 25 million years of evolution. Whether transient transmission of modern HK2 sequences, which encode two putative oncoproteins, can lead to disease remains to be studied.Retroviruses invaded the genome of human ancestors over the course of millions of years, yet these viruses generally have been inactivated during evolution, with only remnants of these infectious sequences remaining in the human genome. One of these viruses, termed HK2, still is capable of producing virus particles, although these particles have been regarded as being noninfectious. Using a genetic probe derived from HK2, we have discovered that HK2 viruses produced in modern humans can package HK2 sequences and transmit them to various other cells. Furthermore, the genetic sequences packaged in HK2 undergo reverse transcription. The transmitted probe circularized in the cell and failed to integrate into the cellular genome. These findings suggest that modern HK2 viruses can package viral RNA and transmit it to other cells. Contrary to previous views, we provide evidence of an extracellular viral phase of modern HK2 viruses. We have no evidence of sustained, spreading infection.
ABSTRACT Human endogenous retroviruses (HERVs) make up 8% of the human genome. The HERV type K (HERV-K) HML-2 (HK2) family contains proviruses that are the most recent entrants into the human germ line and are transcriptionally active. In HIV-1 infection and cancer, HK2 genes produce retroviral particles that appear to be infectious, yet the replication capacity of these viruses and potential pathogenicity has been difficult to ascertain. In this report, we screened the efficacy of commercially available reverse transcriptase inhibitors (RTIs) at inhibiting the enzymatic activity of HK2 RT and HK2 genomic replication. Interestingly, only one provirus, K103, was found to encode a functional RT among those examined. Several nucleoside analogue RTIs (NRTIs) blocked K103 RT activity and consistently inhibited the replication of HK2 genomes. The NRTIs zidovudine (AZT), stavudine (d4T), didanosine (ddI), and lamivudine (3TC), and the nucleotide RTI inhibitor tenofovir (TDF), show efficacy in blocking K103 RT. HIV-1-specific nonnucleoside RTIs (NNRTIs), protease inhibitors (PIs), and integrase inhibitors (IIs) did not affect HK2, except for the NNRTI etravirine (ETV). The inhibition of HK2 infectivity by NRTIs appears to take place at either the reverse transcription step of the viral genome prior to HK2 viral particle formation and/or in the infected cells. Inhibition of HK2 by these drugs will be useful in suppressing HK2 infectivity if these viruses prove to be pathogenic in cancer, neurological disorders, or other diseases associated with HK2. The present studies also elucidate a key aspect of the life cycle of HK2, specifically addressing how they do, and/or did, replicate. IMPORTANCE Endogenous retroviruses are relics of ancestral virus infections in the human genome. The most recent of these infections was caused by HK2. While HK2 often remains silent in the genome, this group of viruses is activated in HIV-1-infected and cancer cells. Recent evidence suggests that these viruses are infectious, and the potential exists for HK2 to contribute to disease. We show that HK2, and specifically the enzyme that mediates virus replication, can be inhibited by a panel of drugs that are commercially available. We show that several drugs block HK2 with different efficacies. The inhibition of HK2 replication by antiretroviral drugs appears to occur in the virus itself as well as after infection of cells. Therefore, these drugs might prove to be an effective treatment by suppressing HK2 infectivity in diseases where these viruses have been implicated, such as cancer and neurological syndromes.
ABSTRACT Human endogenous retroviruses (HERVs), which are remnants of ancestral retroviruses integrated into the human genome, are defective in viral replication. Because activation of HERV-K and coexpression of this virus with HIV-1 have been observed during HIV-1 infection, it is conceivable that HERV-K could affect HIV-1 replication, either by competition or by cooperation, in cells expressing both viruses. In this study, we found that the release efficiency of HIV-1 Gag was 3-fold reduced upon overexpression of HERV-K CON Gag. In addition, we observed that in cells expressing Gag proteins of both viruses, HERV-K CON Gag colocalized with HIV-1 Gag at the plasma membrane. Furthermore, HERV-K CON Gag was found to coassemble with HIV-1 Gag, as demonstrated by (i) processing of HERV-K CON Gag by HIV-1 protease in virions, (ii) coimmunoprecipitation of virion-associated HERV-K CON Gag with HIV-1 Gag, and (iii) rescue of a late-domain-defective HERV-K CON Gag by wild-type (WT) HIV-1 Gag. Myristylation-deficient HERV-K CON Gag localized to nuclei, suggesting cryptic nuclear trafficking of HERV-K Gag. Notably, unlike WT HERV-K CON Gag, HIV-1 Gag failed to rescue myristylation-deficient HERV-K CON Gag to the plasma membrane. Efficient colocalization and coassembly of HIV-1 Gag and HERV-K Gag also required nucleocapsid (NC). These results provide evidence that HIV-1 Gag heteromultimerizes with HERV-K Gag at the plasma membrane, presumably through NC-RNA interaction. Intriguingly, HERV-K Gag overexpression reduced not only HIV-1 release efficiency but also HIV-1 infectivity in a myristylation- and NC-dependent manner. Altogether, these results indicate that Gag proteins of endogenous retroviruses can coassemble with HIV-1 Gag and modulate the late phase of HIV-1 replication.
Human Endogenous Retroviruses type K HML-2 (HK2) are integrated into 117 or more areas of human chromosomal arms while two newly discovered HK2 proviruses, K111 and K222, spread extensively in pericentromeric regions, are the first retroviruses discovered in these areas of our genome. We use PCR and sequencing analysis to characterize pericentromeric K111 proviruses in DNA from individuals of diverse ethnicities and patients with different diseases. We found that the 5′ LTR-gag region of K111 proviruses is missing in certain individuals, creating pericentromeric instability. K111 deletion (−/− K111) is seen in about 15% of Caucasian, Asian, and Middle Eastern populations; it is missing in 2.36% of African individuals, suggesting that the −/− K111 genotype originated out of Africa. As we identified the −/−K111 genotype in Cutaneous T-cell lymphoma (CTCL) cell lines, we studied whether the −/−K111 genotype is associated with CTCL. We found a significant increase in the frequency of detection of the −/−K111 genotype in Caucasian patients with severe CTCL and/or Sézary syndrome (n = 35, 37.14%), compared to healthy controls (n = 160, 15.6%) [p = 0.011]. The −/−K111 genotype was also found to vary in HIV-1 infection. Although Caucasian healthy individuals have a similar frequency of detection of the −/− K111 genotype, Caucasian HIV Long-Term Non-Progressors (LTNPs) and/or elite controllers, have significantly higher detection of the −/−K111 genotype (30.55%; n = 36) than patients who rapidly progress to AIDS (8.5%; n = 47) [p = 0.0097]. Our data indicate that pericentromeric instability is associated with more severe CTCL and/or Sézary syndrome in Caucasians, and appears to allow T-cells to survive lysis by HIV infection. These findings also provide new understanding of human evolution, as the −/−K111 genotype appears to have arisen out of Africa and is distributed unevenly throughout the world, possibly affecting the severity of HIV in different geographic areas.
Human endogenous retroviruses (HERVs) make up 8% of the human genome. The expression of HERV-K (HML-2), the family of HERVs that most recently entered the genome, is tightly regulated but becomes markedly increased after infection with HIV-1. To better understand the mechanisms involved in this activation, we explored the role of the HIV-1 Tat protein in inducing the expression of these endogenous retroviral genes. Administration of recombinant HIV-1 Tat protein caused a 13-fold increase in HERV-K (HML-2) gag RNA transcripts in Jurkat T cells and a 10-fold increase in primary lymphocytes, and the expression of the HERV-K (HML-2) rec and np9 oncogenes was also markedly increased. This activation was seen especially in lymphocytes and monocytic cells, the natural hosts for HIV-1 infection. Luciferase reporter gene assays demonstrated that the effect of Tat on HERV-K (HML-2) expression occurred at the level of the transcriptional promoter. The transcription factors NF-κB and NF-AT contribute to the Tat-induced activation of the promoter, as shown by chromatin immunoprecipitation assays, mutational analysis of the HERV-K (HML-2) long terminal repeat, and treatments with agents that inhibit NF-κB or NF-AT activation. These studies demonstrate that HIV-1 Tat plays an important role in activating expression of HERV-K (HML-2) in the setting of HIV-1 infection.
Human endogenous retroviruses are remnants of ancient germline infections that make up approximately 8% of the modern human genome. The HERV-K (HML-2) family is one of the most recent entrants into the human germline, these viruses appear to be transcriptionally active, and HERV-K viral like particles (VLPs) are found in cell lines from a number of human malignancies. HERV-K VLPs were first found to be produced in teratocarcinoma cell lines, and since then teratocarcinoma has been thought of as the classical model for HERV-Ks, with the NCCIT teratocarcinoma cell line particularly known to produce VLPs. Treatment for teratocarcinoma has progressed since its discovery, with improved prognosis for patients. Since the introduction of platinum based therapy, first year survival has greatly improved even with disseminated disease; however, it is estimated that 20% to 30% of patients present with metastatic germ cell tumor relapse following initial treatments. Also, the toxicity associated with the use of chemotherapeutic agents used to treat germ cell tumors is still a major concern. In this study, we show that the depletion of the HERV-K accessory protein Np9 increases the sensitivity of NCCIT teratocarcinoma cells to bleomycin and cisplatin. While decreasing the expression of Np9 had only a modest effect on the baseline viability of the cells, the reduced expression of Np9 increased the sensitivity of the teratocarcinoma cells to environmental (serum starvation) and chemical (chemotherapeutic) stresses. Np9 is also essential to the migration of NCCIT teratocarcinoma cells: in a wound closure assay, reduced expression of Np9 resulted in cells migrating into the wound at a slower rate, whereas reintroduction of Np9 resulted in NCCIT cells migrating back into the wound in a manner similar to the control. These findings support the implication that the HERV-K accessory protein Np9 has oncogenic potential.
Centromere defects in Systemic Sclerosis (SSc) have remained unexplored despite the fact that many centromere proteins were discovered in patients with SSc. Here we report that lesion skin fibroblasts from SSc patients show marked alterations in centromeric DNA. SSc fibroblasts also show DNA damage, abnormal chromosome segregation, aneuploidy (only in diffuse cutaneous (dcSSc)) and micronuclei (in all types of SSc), some of which lose centromere identity while retaining centromere DNA sequences. Strikingly, we find cytoplasmic "leaking" of centromere proteins in limited cutaneous SSc (lcSSc) fibroblasts. Cytoplasmic centromere proteins co-localize with antigen presenting MHC Class II molecules, which correlate precisely with the presence of anti-centromere antibodies. CENPA expression and micronuclei formation correlate highly with activation of the cGAS-STING/IFN-β pathway as well as markers of reactive oxygen species (ROS) and fibrosis, ultimately suggesting a link between centromere alterations, chromosome instability, SSc autoimmunity, and fibrosis.
