Human Endogenous Retroviruses (HERV) are derived from exogenous retroviruses that infected inheritable germline tissues millions of years ago and account for 8% of the human genome. Like other retroviruses HERVs encode Gag, Pol and sometimes Env proteins. During a retroviral infection, retroviral Gag recruits the hosts Endosomal Sorting Complex Required for Transport (ESCRT) and associated proteins (ALIX and TSG101) to produce precisely sized viruses from endosomes or the plasma membrane. The ESCRT machinery is also involved in cytokinesis and control growth factor receptor signalling. HERV-K is the most recent HERV family to insert into the genome and is still able to produce mostly intact transcripts, including Gag. When expressed, Gag causes cells to release Virus-Like Particles (VLP) that lack HERV genomes. These retroviral VLP are remarkably similar to a sub-category of extracellular vesicles (EVs) called exosomes. Exosomes require ALIX, TSG101 and the ESCRT machinery for their production. It is possible that HERV-K Gag is required for exosome production or that HERV VLPs are a major contaminant of exosome preparations that account for many of the functions attributed to exosomes. Our data shows that HERV-K Gag over-expression or knockdown did not change the number of EVs released per cell in two cell lines. As well there was no difference in the amount of ALIX and TSG101 in the EVs in these conditions. The most intriguing observation made was the increase of cell number with expression of HERV-K Gag and decrease when HERV-K Gag was knocked down in HEK293T. We are currently unable to conclude the role of HERV-K Gag on EV production and content. We speculate that HERV-K Gag might affect cells through controlling cell proliferation or death, for example by competing with ESCRT machinery to impact signalling through growth factor receptors. This study begins to outline the potential effects HERV-K Gag might have on EV release and cell proliferation.

Environ 8% du génome humain est constitué de rétrovirus endogènes (HERV). La famille de bétarétrovirus HERV-K(HML2), l'une des plus actives chez l'homme, est entrée il y a 45 millions d'années dans le génome des primates et s'est amplifiée efficacement depuis, et ce malgré l'existence de nombreuses protéines cellulaires, appelées facteurs de restriction, qui s'opposent à la réplication du virus dans la cellule hôte. La Tetherin/BST2, l'un d'entre eux, est une protéine membranaire capable de bloquer le relargage des virions dans le milieu extracellulaire et est active sur la plupart des virus enveloppés testés jusqu'à présent, en particulier HERV-K(HML2). Nous avons tout d'abord mis en évidence que l'enveloppe (Env) de la famille HML2 est un antagoniste de la Tetherin, propriété qui a pu contribuer au succès de l'amplification de la famille HERV¬K(HML2) dans les génomes. Plusieurs domaines de l'enveloppe coopèrent pour s'opposer à l'action du facteur de restriction : la SU (domaine d'interaction), ainsi que la partie transmembranaire, alors que la queue cytoplasmique n'est pas indispensable. Le mécanisme de cette inhibition n'a pas été encore complètement élucidé, mais l'on sait, comme pour la glycoprotéine d'Ebola, que l'Env HERV-K(HML2) n'induit ni relocalisation, ni dégradation de la Tetherin. Etant donné le grand polymorphisme insertionnel de la famille HERV-K(1-IML2), il est très probable que cette activité anti-Tetherin endogène soit variable entre les individus, ce qui pourrait avoir des conséquences dans les pathologies où les éléments HERV-K(HML2) sont spécifiquement induits. Parmi ces pathologies, les cancers de la peau, du sein et de la lignée germinale présentent une association particulièrement forte avec l'expression de l'Env HERV-K(HML2), que nous avons voulu mieux comprendre dans la suite de ces travaux de thèse. Nous avons dans un premier temps montré que l'expression de l'Env dans des cellules humaines non transformées de l'épithélium de sein (MCF10A), induit la transition vers un phénotype mésenchymateux (EMT, transition épithélio-mésenchymateuse), caractéristique de l'apparition de métastases dans les cancers. Cette transition est associée à une augmentation de la mobilité des cellules (mise en évidence dans des tests Transwell), à un changement de morphologie des cellules et à une modification du profil d'expression de quelques marqueurs moléculaires caractéristiques (E-cadherin, N-cadherin, vimentin, fibronectin). Grâce à une étude transcriptomique en cellules 293T, nous avons mis en évidence que l'expression de l'Env HERV-K induit fortement plusieurs facteurs de transcription : ETV4, ETVS, ainsi que EGR1, qui ont été identifiés comme des marqueurs du processus de tumorigénèse dans différents modèles. Nous avons également montré que l'Env HERV-K active la voie des MAP kinases via ERK 1/2 —dérégulée dans un grand nombre de cancers- en amont de la kinase Raf. Ces phénomènes d'induction de la transduction de signal requièrent la présence de la queue cytoplasmique de l'enveloppe. De façon remarquable, seule l'enveloppe du bétarétrovirus de mouton JSRV, oncogénique in vivo, est capable d'activer les mêmes voies de signalisation, ce qui renforce l'hypothèse d'une implication de l'Env HERV-K(HML2) dans la tumorigenèse. / Human endogenous retroviruses (HERV) represent about 8% of our genomic content. HERV-K(HML2) betaretroviral family is one of the most active in humans. Although it entered 45 million years ago in the primate genomes, its members have amplified quite recently despite the existence of restriction factors, which are host proteins blocking viral replication in cells. Tetherin/BST2 is one of them and acts by keeping the viral particles attached to the cell surface. It targets most enveloped viruses tested so far including HERV-K(HML2). We show that the envelope protein (Env) of HML2 family is an antagonist of Tetherin retriction, property that probably helped the endogenous retrovirus to efficiently amplify in the genomes. We mapped several domains required for antagonism : the surface subunit of Env (SU), which interacts with Tetherin, and the transmembrane. We also show that the cytoplasmic tail is dispensable for counteraction. Similar to Ebola glycoprotein, HERV-K(HML2) Env does not mediate Tetherin degradation or cell surface removal; therefore, it uses a yet-undescribed mechanism to inactivate the restriction factor. Due to their recent amplification, HERV-K(HML2) elements are extremely polymorphic in the human population, and it is likely that individuals will not all possess the same anti-Tetherin potential. This could have functional consequences in pathologies where HERV-K(HML2) is specifically induced. Among them, melanomas, breast cancers and germ line tumors display a strong association with HML2 Env expression, that we wanted to better analyse. We first show that Env expression in a model of epithelial human breast cancer cells induces the so-called EMT (epithelial mesenchymal transition), critical for cancer progression and the process of metastasis. This includes enhanced migratory capacities (shown by transwell assays), changes in cell morphology and characteristic modifications in a set of molecular markers (e.g. E-cadherin, N-cadherin, vimentin, fibronectin). Microarray experiments performed in 293T cells revealed that HERV-K(HML2) Env is a strong inducer of several transcription factors, namely ETV4, ETVS and EGRI, which have been associated with cellular transformation. Importantly, we also show that HERV-K(HML2) Env activates the MAP kinase pathway via ERK 1/2, key player in numerous cancers. This induction occurs upstream of the kinase Raf and involves the cytoplasmic tail of HERV-K(HML2) Env. In addition, this phenomenon is very specific, being absent with every other Env tested, except for JSRV Env which is already known to have transforming properties in vivo.

Human Endogenous Retroviruses (HERV) are segments of the human genome that are viral in origin and occupy approximately 8% of the human genome, which is nearly 3 times as much as functional protein coding genes (3%). Although most are defective due to accumulation of post insertional mutations, Human Endogenous Retrovirus Type K (HERV-K) retains the ability to produce functional particles and is activated during progression of malignant disease. The resulting proviral products have been associated tumorigenesis through their presumed role in malignant cell production. While therapeutics that focus on HERV-K inhibition have not been manufactured, current Federal Drug Administration (FDA) approved antiretroviral therapies are capable of decreasing expression of HERV-K in cancer cells. In summary, antiretroviral drugs may serve as a promising new anticancer drug by targeting and decreasing expression of HERV-K proteins.

Thesis advisor: Welkin Johnson / Endogenous Retroviruses (ERVs) are “fossilized” retroviruses of a once exogenous retrovirus located in the genome of extant vertebrates. Retroviral infection results in a provirus integration into the host genome. An infection of a germline cell could lead to the provirus potentially being inherited by the offspring of the infected individual. Once in the genome, the provirus becomes subject to evolutionary processes and can become either lost or fixed in a population, remaining as “fossils” long after the exogenous retrovirus has gone extinct23. Notably, 8% of the human genome consists of ERVs30. Human Endogenous Retrovirus Type K (HERV-K)(HML-2) family is of particular interest. HERV-K integrations are as old as 30-35 million years, endogenizing before the separation of humans and Old World Monkeys. However, there are human specific insertions, some as young as 150,000 – 250,000 years, making them the youngest insertion in the human genome. There are over 90 insertions in the human genome; the bulk is shared by all humans44,47. Transcripts of HERV-K genes are upregulated in multiple cancer and tumor cell lines 14,39,46, as well as in HIV-1 infected patients 7,11,29. Just as there are human specific insertions of ERV-K, there are also Old World Monkey specific insertions44. I have identified an intact endogenous retroviral envelope open reading frame on chromosome 12 of the rhesus macaque genome. This viral envelope-encoding sequence, which I refer to as rhERV-K env, retains all the canonical features of a retroviral Env protein. An alignment between rhERV-K env and a consensus sequence of HERV-K, HERV-Kcon env, shows a 70% amino acid sequence identity. For experimental purposes, reconstructed HERV-K envelopes have been incorporated into virions of Human Immunodeficiency virus (HIV-1)19,26,49, Murine Leukemia Virus (MLV)12, and Vesicular stomatitis Virus (VSV)26,41,49. While these approaches have illuminated some aspects of HERV-K Env-mediated entry, to date a cell-surface receptor has not been identified for any ERV-K Env. This could be due to its low infectivity levels12,26,49, its seemingly broad cell tropism limiting identification of null cell lines26,49, or possibly the HERV-K consensus reconstructions are not an accurate representation of the progenitor HERV-K virus. I am interested in understanding how the ERV-K retrovirus accessed the human germline (some 150,000 – 250,000 years ago). To do this, I focused specifically on the envelope proteins of HERV-K and rhERV-K, with the goal of analyzing the ERV-K entry process. The identification and inclusion of rhERV-K Env in this study is meant to circumvent the possibility that the previously described consensus reconstructions of human HERV-K Env are not representative, and may also provide a means to compare the endogenization process in the human/ape and old-world monkey lineages. I focused on developing two systems for single-cycle infection, one based on Mason-Pfizer Monkey Virus (MPMV) (which has not been done before), and a second based on MLV, which has previously been reported on. MPMV, like HERV-K, is a betaretrovirus, and I reasoned that possibly using a betaretrovirus would overcome some of the low-infectivity issues associated with prior attempts using HIV and MLV. To develop a system for examining function of the ERV-K Env proteins, I addressed 3 issues: 1. Are the HERV-K Env and rhERV-K Env proteins expressed and properly processed? 2. Can they be incorporated into virions of a heterologous virus? 3. Are ERV-K pseudotyped virions infectious? I have answered these questions in the following thesis. / Thesis (MS) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

No description available.

Daten des humanen Genomprojektes zeigen, dass ca. 8% des gesamten humanen Genoms aus retroviralen Sequenzen besteht. Der überwiegende Teil dieser Proviren ist aufgrund verschiedener Mutationen defekt. Im Gegensatz zu allen anderen HERV Proviren scheinen einige HERV-K Proviren intakt zu sein und besitzen offene Leserahmen für alle viralen Proteine. Die Familie des humanen endogenen Retrovirus K HML2 umfasst ca. 30 eng verwandte Proviren. Zusätzlich zu den Strukturproteinen Gag und Env und der Reversen Transkriptase, exprimiert HERV-K zwei regulatorische Proteine, Rec und Np9. Beide sind im Nukleus lokalisiert und tumorigene Eigenschaften bzw. eine Expression in Assoziation mit Tumorgeweben wurde nachgewiesen. Neben Zelllinien, wie die Teratokarzinomzelllinie GH und einigen Brustkrebszelllinien, für die die Expression von HERV-K mRNA und die Produktion von Viruspartikeln bekannt ist, konnte die Expression von HERV-K Proteinen und Partikeln für Melanomzellen gezeigt werden. Volllängen mRNA von HERV-K war in allen untersuchten humanen Proben nachweisbar. Gespleißtes env und rec war in 39% der Gewebe und in 38% der Melanomzelllinien exprimiert. Zusätzlich werden HERV-H, -R und -W exprimiert. Von den auf spezifische Antikörper gegen HERV-K Proteine untersuchten Seren der Melanompatienten waren 16% positiv für das transmembrane Hüllprotein, jedoch reagierte kein Serum mit Re oder Np9. Da im Zuge der Entstehung von Tumoren immer auch eine Dedifferenzierung der entarteten Zellen diskutiert wird, wurde die Expression von HERVs in undifferenzierten, embryonalen Stammzellen bestimmt. In den untersuchten embryonalen Stammzellen lässt sich Volllängen mRNA, sowie gespleißte env, rec und np9 mRNA nachweisen. Während der Differenzierung zu neuronalen Vorläuferzellen sinkt die Expression jedoch wieder auf ein mit normalen Zellen vergleichbares Niveau. Obwohl gespleißte RNA und virale Proteine von HERV-K vor allem in Tumoren und Tumorzelllinien exprimiert werden, ist deren Funktion während der Tumorentstehung noch immer ungeklärt. Auch die Bedeutung der HERV-K Expression in humanen Stammzellen ist noch unklar, insbesondere in Hinblick auf eine mögliche Tumorigenität. / In contrast to all other human endogenous retroviruses, proviruses of the human endogenous retrovirus family HERV-K have maintained open reading frames for all viral proteins. Although most proviruses are defective, structural proteins Gag and Env, the reverse transcriptase and two regulatory proteins, Rec and Np9, have been described. Rec resembles the Rev protein of HIV and tumourigenic potential was confirmed. Np9 as well is located in the nucleus and expression in association with tumour tissues was observed. Additionally to cell lines known to produce HERV-K virus particles, such as the teratocarcinoma cell line GH and breast cancer cell lines, recently melanoma cells were described to express HERV-K proteins and particles. In order to study the expression of HERV-K, -H, -R and -W, in melanoma cell lines and biopsies primer sets were used. Antisera specific for HERV-K proteins were used for immunohistochemistry and sera from melanoma patients were investigated for HERV-K specific antibodies. Full length mRNAs of all HERVs were found in all human cells. Spliced env and rec of HERV-K were detected in 39% of the melanoma biopsies and in 38% of the melanoma cell lines. Expression of HERV-K in situ was shown by immunohistochemistry. In addition, 16% of the patients sera tested showed antibodies against the HERV-K transmembrane envelope protein, but no antibodies against Np9 or Rec could be detected. A certain dedifferentiation of cells as a consequence of tumour development is discussed. Therefore the expression of HERV-K in undifferentiated embryonic stem cells was investigated. The investigated stem cells showed expression of HERV-K full length, env, rec and np9 mRNA. Although the expression decreased with differentiation to neuronal precursor cells. Even though HERV-K mRNA and proteins were expressed in a high percentage of melanomas their function in tumour development is still unclear. As well as the meaning of the HERV-K expression in embryonic stem cells, particularly for a tumourigenic potential.