scholarly journals Endoplasmic Reticulum-Shaping Atlastin Proteins Facilitate KSHV Replication

2022 ◽  
Vol 11 ◽  
Author(s):  
Wen-ying Long ◽  
Guo-hua Zhao ◽  
Yao Wu
Keyword(s):  
Endoplasmic Reticulum ◽  
Life Cycle ◽  
Er Stress ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Regulatory Role ◽  
Biological Processes ◽  
Kshv Infection ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Er Membrane

Kaposi’s sarcoma-associated herpesvirus (KSHV) has two life cycle modes: the latent and lytic phases. The endoplasmic reticulum (ER) is the site for KSHV production. Furthermore, ER stress can trigger reactivation of KSHV. Little is known about the nature of the ER factors that regulate KSHV replication. Atlastin proteins (ATLs which include ATL1, ATL2, and ATL3) are large dynamin-related GTPases that control the structure and the dynamics of the ER membrane. Here, we show that ATLs can regulate KSHV lytic activation and infection. Overexpression of ATLs enhances KSHV lytic activation, whereas ATLs silence inhibits it. Intriguingly, we find that silencing of ATLs impairs the response of cells to ER stress, and ER stress can promote the lytic activation of KSHV. Our study establishes that ATLs plays a critically regulatory role in KSHV infection, thus expanding the known scope of biological processes controlled by ATLs to include KSHV infection.

scholarly journals Activation of the Unfolded Protein Response by 2-Deoxy-d-Glucose Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication and Gene Expression

2012 ◽  
Vol 56 (11) ◽  
pp. 5794-5803 ◽  
Author(s):  
Howard J. Leung ◽  
Elda M. Duran ◽  
Metin Kurtoglu ◽  
Samita Andreansky ◽  
Theodore J. Lampidis ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Unfolded Protein ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Protein Response ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTLytic replication of the Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for the maintenance of both the infected state and characteristic angiogenic phenotype of Kaposi's sarcoma and thus represents a desirable therapeutic target. During the peak of herpesvirus lytic replication, viral glycoproteins are mass produced in the endoplasmic reticulum (ER). Normally, this leads to ER stress which, through an unfolded protein response (UPR), triggers phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α), resulting in inhibition of protein synthesis to maintain ER and cellular homeostasis. However, in order to replicate, herpesviruses have acquired the ability to prevent eIF2α phosphorylation. Here we show that clinically achievable nontoxic doses of the glucose analog 2-deoxy-d-glucose (2-DG) stimulate ER stress, thereby shutting down eIF2α and inhibiting KSHV and murine herpesvirus 68 replication and KSHV reactivation from latency. Viral cascade genes that are involved in reactivation, including the master transactivator (RTA) gene, glycoprotein B, K8.1, and angiogenesis-regulating genes are markedly decreased with 2-DG treatment. Overall, our data suggest that activation of UPR by 2-DG elicits an early antiviral response via eIF2α inactivation, which impairs protein synthesis required to drive viral replication and oncogenesis. Thus, induction of ER stress by 2-DG provides a new antiherpesviral strategy that may be applicable to other viruses.

scholarly journals The Kaposi’s Sarcoma-Associated Herpesvirus ORF34 Protein Interacts and Stabilizes HIF-2α via Binding to the HIF-2α bHLH and PAS Domains

2019 ◽  
Vol 93 (17) ◽  
Author(s):  
Muzammel Haque ◽  
K. G. Kousoulas
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Late Gene Expression ◽  
Lytic Gene ◽  
Lytic Gene Expression ◽  
Proteasome Pathway ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTHypoxia and hypoxia inducible factors (HIFs) play important roles in the Kaposi’s sarcoma-associated herpesvirus (KSHV) life cycle. KSHV is the causative agent of Kaposi’s sarcoma (KS) and other AIDS-related malignancies. Kaposi’s sarcoma is a highly vascular tumor, which preferentially develops in the lower extremities of the body where blood vessels are often poorly oxygenated. The main cellular responses to hypoxia are mediated mainly by two isoforms of HIF, HIF-1α and HIF-2α. HIF-1α and HIF-2α have common as well as distinct functions, although they are similar in structure and function. Previously, we showed that the KSHV ORF34 protein binds HIF-1α and facilitates its degradation through the ubiquitin-proteasome pathway causing negative regulation of HIF-1α-dependent genes (Haque and Kousoulas, J Virol 87:2164-2173, 2013, https://www.doi.org/10.1128/JVI.02460-12). Herein, we show that theORF34gene is involved in the regulation of KSHV lytic gene expression, since deletion ofORF34resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. Coimmunoprecipitation experiments revealed that the ORF34 protein physically interacted with HIF-2α in transfected as well as in KSHV-infected cells. Utilization of ORF34 truncations revealed that three distinct domains bind HIF-2α and that both bHLH and PAS domains of HIF-2α interacted with ORF34. Unlike HIF-1α, dose-dependent coexpression of ORF34 stabilized the HIF-2α protein, ensuring HIF-2α-dependent transcriptional activity. The ORF34 protein enhanced HIF-2α ubiquitination at the bHLH and PAS domains. The results show that the KSHV ORF34 protein is involved in the KSHV life cycle by regulating the expression of HIF-1α and HIF-2α proteins.IMPORTANCEHypoxia inducible factor 1α (HIF-1α) and HIF-2α are transcription factors which play important roles in the Kaposi’s sarcoma-associated herpesvirus (KSHV) latent and lytic gene replication. Herein, we show that theORF34gene is involved in the regulation of KSHV lytic gene expression, since deletion ofORF34resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. In addition, we demonstrate that the KSHV ORF34 protein binds and stabilizes HIF-2α, in contrast to its role in binding HIF-1α and causing its degradation via the proteasome pathway. Thus, the KSHV ORF34 protein plays a regulatory role in the KSHV life cycle by regulating HIF-1α and HIF-2α expression.

scholarly journals Structure of the Open Reading Frame 49 Protein Encoded by Kaposi's Sarcoma-Associated Herpesvirus

2016 ◽  
Vol 91 (2) ◽  
Author(s):  
Kelly Hew ◽  
Saranya Veerappan ◽  
Daniel Sim ◽  
Tobias Cornvik ◽  
Pär Nordlund ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Life Cycle ◽  
Dna Binding ◽  
Protein Interaction ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Herpesviruses alternate between the latent and the lytic life cycle. Switching into the lytic life cycle is important for herpesviral replication and disease pathogenesis. Activation of a transcription factor replication and transcription activator (RTA) has been demonstrated to govern this switch in Kaposi's sarcoma-associated herpesvirus (KSHV). The protein encoded by open reading frame 49 from KSHV (ORF49KSHV) has been shown to upregulate lytic replication in KSHV by enhancing the activities of the RTA. We have solved the crystal structure of the ORF49KSHV protein to a resolution of 2.4 Å. The ORF49KSHV protein has a novel fold consisting of 12 alpha-helices bundled into two pseudodomains. Most notably are distinct charged patches on the protein surface, which are possible protein-protein interaction sites. Homologs of the ORF49KSHV protein in the gammaherpesvirus subfamily have low sequence similarities. Conserved residues are mainly located in the hydrophobic regions, suggesting that they are more likely to play important structural roles than functional ones. Based on the identification and position of three sulfates binding to the positive areas, we performed some initial protein-DNA binding studies by analyzing the thermal stabilization of the protein in the presence of DNA. The ORF49KSHV protein is stabilized in a dose-responsive manner by double-stranded oligonucleotides, suggesting actual DNA interaction and binding. Biolayer interferometry studies also demonstrated that the ORF49KSHV protein binds these oligonucleotides. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a tumorigenic gammaherpesvirus that causes multiple cancers and lymphoproliferative diseases. The virus exists mainly in the quiescent latent life cycle, but when it is reactivated into the lytic life cycle, new viruses are produced and disease symptoms usually manifest. Several KSHV proteins play important roles in this reactivation, but their exact roles are still largely unknown. In this study, we report the crystal structure of the open reading frame 49 protein encoded by KSHV (ORF49KSHV). Possible regions for protein interaction that could harbor functional importance were found on the surface of the ORF49KSHV protein. This led to the discovery of novel DNA binding properties of the ORF49KSHV protein. Evolutionary conserved structural elements with the functional homologs of ORF49KSHV were also established with the structure.

scholarly journals Disintegrin-like domain of glycoprotein B regulates Kaposi’s sarcoma-associated herpesvirus infection of cells

2014 ◽  
Vol 95 (8) ◽  
pp. 1770-1782 ◽  
Author(s):  
Lia R. Walker ◽  
Hosni A. M. Hussein ◽  
Shaw M. Akula
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Glycoprotein B ◽  
Binding Motif ◽  
Wild Type ◽  
Infection Rates ◽  
Herpesvirus Infection ◽  
Kshv Infection ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) glycoprotein B (gB) is a lytic structural protein expressed on the envelope of mature virions and on the membrane of cells supporting lytic infection. In addition to this viral glycoprotein’s interaction with integrins via its RGD (Arg-Gly-Asp) motif, KSHV gB possesses a disintegrin-like domain (DLD), which binds integrins as well. Prior to this study, there has been minimal research involving the less common integrin-binding motif, DLD, of gB as it pertains to herpesvirus infection. By using phage display peptide library screening and molecular biology techniques, the DLD of KSHV gB was shown to interact specifically with non-RGD binding α9β1 integrins. Similarly, monitoring wild-type infection confirmed α9β1:DLD interactions to be critical to successful KSHV infection of human foreskin fibroblast (HFF) cells and human dermal microvascular endothelial cells (HMVEC-d) compared with 293 cells. To further demonstrate the importance of the DLD of gB in KSHV infection, two recombinant virus constructs were generated using a bacterial artificial chromosome (BAC) system harbouring the KSHV genome (BAC36): BAC36ΔD-KSHV (lacking a functionally intact DLD of gB and containing an introduced tetracycline cassette) and BAC36.T-KSHV (containing an intact DLD sequence and an introduced tetracycline cassette). Accordingly, BAC36ΔD-KSHV presented significantly lower infection rates in HFF and HMVEC-d cells compared with the comparable infection rates achieved by wild-type BAC36-KSHV and BAC36.T-KSHV. Thus, the present report has delineated a critical role for the DLD of gB in KSHV infection, which may lead to a broader knowledge regarding the sophisticated mechanisms utilized by virus-encoded structural proteins in KSHV entry and infection.

scholarly journals Focal Adhesion Kinase Is Critical for Entry of Kaposi's Sarcoma-Associated Herpesvirus into Target Cells

2006 ◽  
Vol 80 (3) ◽  
pp. 1167-1180 ◽  
Author(s):  
Harinivas H. Krishnan ◽  
Neelam Sharma-Walia ◽  
Daniel N. Streblow ◽  
Pramod P. Naranatt ◽  
Bala Chandran
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Viral Entry ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Target Cells ◽  
Viral Dna ◽  
Kshv Infection ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) interacts with cell surface α3β1 integrin early during in vitro infection of human endothelial cells and fibroblasts and activates the focal adhesion kinase (FAK) that is immediately downstream in the outside-in signaling pathway by integrins, leading to the activation of several downstream signaling molecules. In this study, using real-time DNA and reverse transcription-PCR assays to measure total internalized viral DNA, viral DNA associated with infected nuclei, and viral gene expression, we examined the stage of infection at which FAK plays the most significant role. Early during KSHV infection, FAK was phosphorylated in FAK-positive Du17 mouse embryonic fibroblasts. The absence of FAK in Du3 (FAK−/−) cells resulted in about 70% reduction in the internalization of viral DNA, suggesting that FAK plays a role in KSHV entry. Expression of FAK in Du3 (FAK−/−) cells via an adenovirus vector augmented the internalization of viral DNA. Expression of the FAK dominant-negative mutant FAK-related nonkinase (FRNK) in Du17 cells significantly reduced the entry of virus. Virus entry in Du3 cells, albeit in reduced quantity, delivery of viral DNA to the infected cell nuclei, and expression of KSHV genes suggested that in the absence of FAK, another molecule(s) may be partially compensating for FAK function. Infection of Du3 cells induced the phosphorylation of the FAK-related proline-rich tyrosine kinase (Pyk2) molecule, which has been shown to complement some of the functions of FAK. Expression of an autophosphorylation site mutant of Pyk2 in which Y402 is mutated to F (F402 Pyk2) reduced viral entry in Du3 cells, suggesting that Pyk2 facilitates viral entry moderately in the absence of FAK. These results suggest a critical role for KSHV infection-induced FAK in the internalization of viral DNA into target cells.

scholarly journals Modulation of oncogenic signaling networks by Kaposi’s sarcoma-associated herpesvirus

2017 ◽  
Vol 398 (8) ◽  
pp. 911-918 ◽  
Author(s):  
Jason P. Wong ◽  
Blossom Damania
Keyword(s):  
Life Cycle ◽  
Kaposi's Sarcoma ◽  
Primary Effusion Lymphoma ◽  
Kaposi’S Sarcoma ◽  
Host Cells ◽  
Signaling Networks ◽  
Oncogenic Signaling ◽  
Extracellular Signal ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Abstract Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of three human malignancies: Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. To persist and replicate within host cells, KSHV encodes proteins that modulate different signaling pathways. Manipulation of cell survival and proliferative networks by KSHV can promote the development of KSHV-associated malignancies. In this review, we discuss recent updates on KSHV pathogenesis and the viral life cycle. We focus on proteins encoded by KSHV that modulate the phosphatidylinositol-4,5-bisphosphate 3 kinase and extracellular signal-regulated kinases 1/2 pathways to create an environment favorable for viral replication and the development of KSHV malignancies.

scholarly journals NFAT and CREB Regulate Kaposi's Sarcoma-Associated Herpesvirus-Induced Cyclooxygenase 2 (COX-2)

2010 ◽  
Vol 84 (24) ◽  
pp. 12733-12753 ◽  
Author(s):  
Neelam Sharma-Walia ◽  
Arun George Paul ◽  
Kinjan Patel ◽  
Karthic Chandran ◽  
Waseem Ahmad ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Protein Kinase ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Prostaglandin E ◽  
Intracellular Camp ◽  
Kshv Infection ◽  
Cox 2 ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT COX-2 has been implicated in Kaposi's sarcoma-associated herpesvirus (KSHV) latency and pathogenesis (A. George Paul, N. Sharma-Walia, N. Kerur, C. White, and B. Chandran, Cancer Res. 70:3697-3708, 2010; P. P. Naranatt, H. H. Krishnan, S. R. Svojanovsky, C. Bloomer, S. Mathur, and B. Chandran, Cancer Res. 64:72-84, 2004; N. Sharma-Walia, A. G. Paul, V. Bottero, S. Sadagopan, M. V. Veettil, N. Kerur, and B. Chandran, PLoS Pathog. 6:e1000777, 2010; N. Sharma-Walia, H. Raghu, S. Sadagopan, R. Sivakumar, M. V. Veettil, P. P. Naranatt, M. M. Smith, and B. Chandran, J. Virol. 80:6534-6552, 2006). However, the precise regulatory mechanisms involved in COX-2 induction during KSHV infection have never been explored. Here, we identified cis-acting elements involved in the transcriptional regulation of COX-2 upon KSHV de novo infection. Promoter analysis using human COX-2 promoter deletion and mutation reporter constructs revealed that nuclear factor of activated T cells (NFAT) and the cyclic AMP (cAMP) response element (CRE) modulate KSHV-mediated transcriptional regulation of COX-2. Along with multiple KSHV-induced signaling pathways, infection-induced prostaglandin E2 (PGE2) also augmented COX-2 transcription. Infection of endothelial cells markedly induced COX-2 expression via a cyclosporine A-sensitive, calcineurin/NFAT-dependent pathway. KSHV infection increased intracellular cAMP levels and activated protein kinase A (PKA), which phosphorylated the CRE-binding protein (CREB) at serine 133, which probably led to interaction with CRE in the COX-2 promoter, thereby enhancing COX-2 transcription. PKA selective inhibitor H-89 pretreatment strongly inhibited CREB serine 133, indicating the involvement of a cAMP-PKA-CREB-CRE loop in COX-2 transcriptional regulation. In contrast to phosphatidylinositol 3-kinase and protein kinase C, inhibition of FAK and Src effectively reduced KSHV infection-induced COX-2 transcription and protein levels. Collectively, our study indicates that mediation of COX-2 transcription upon KSHV infection is a paradigm of a complex regulatory milieu involving the interplay of multiple signal cascades and transcription factors. Intervention at each step of COX-2/PGE2 induction can be used as a potential therapeutic target to treat KSHV-associated neoplasm and control inflammatory sequels of KSHV infection.

scholarly journals Kaposi’s Sarcoma-Associated Herpesvirus, but Not Epstein-Barr Virus, Co-infection Associates With Coronavirus Disease 2019 Severity and Outcome in South African Patients

2022 ◽  
Vol 12 ◽  
Author(s):  
Melissa J. Blumenthal ◽  
Humaira Lambarey ◽  
Abeen Chetram ◽  
Catherine Riou ◽  
Robert J. Wilkinson ◽  
...  
Keyword(s):  
South Africa ◽  
South African ◽  
Kaposi's Sarcoma ◽  
Epstein Barr Virus ◽  
Kaposi’S Sarcoma ◽  
Kshv Infection ◽  
Barr Virus ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Epstein Barr ◽  
Kaposi’S Sarcoma Associated Herpesvirus

In South Africa, the Coronavirus Disease 2019 (COVID-19) pandemic is occurring against the backdrop of high Human Immunodeficiency Virus (HIV), tuberculosis and non-communicable disease burdens as well as prevalent herpesviruses infections such as Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV). As part of an observational study of adults admitted to Groote Schuur Hospital, Cape Town, South Africa during the period June–August 2020 and assessed for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, we measured KSHV serology and KSHV and EBV viral load (VL) in peripheral blood in relation to COVID-19 severity and outcome. A total of 104 patients with PCR-confirmed SARS-CoV-2 infection were included in this study. 61% were men and 39% women with a median age of 53 years (range 21–86). 29.8% (95% CI: 21.7–39.1%) of the cohort was HIV positive and 41.1% (95% CI: 31.6–51.1%) were KSHV seropositive. EBV VL was detectable in 84.4% (95% CI: 76.1–84.4%) of the cohort while KSHV DNA was detected in 20.6% (95% CI: 13.6–29.2%), with dual EBV/KSHV infection in 17.7% (95% CI: 11.1–26.2%). On enrollment, 48 [46.2% (95% CI: 36.8–55.7%)] COVID-19 patients were classified as severe on the WHO ordinal scale reflecting oxygen therapy and supportive care requirements and 30 of these patients [28.8% (95% CI: 20.8–38.0%)] later died. In COVID-19 patients, detectable KSHV VL was associated with death after adjusting for age, sex, HIV status and detectable EBV VL [p = 0.036, adjusted OR = 3.17 (95% CI: 1.08–9.32)]. Furthermore, in HIV negative COVID-19 patients, there was a trend indicating that KSHV VL may be related to COVID-19 disease severity [p = 0.054, unstandardized co-efficient 0.86 (95% CI: –0.015–1.74)] in addition to death [p = 0.008, adjusted OR = 7.34 (95% CI: 1.69–31.49)]. While the design of our study cannot distinguish if disease synergy exists between COVID-19 and KSHV nor if either viral infection is indeed fueling the other, these data point to a potential contribution of KSHV infection to COVID-19 outcome, or SARS-CoV-2 infection to KSHV reactivation, particularly in the South African context of high disease burden, that warrants further investigation.

scholarly journals Kaposi’s sarcoma-associated herpesvirus promotes mesenchymal-to-endothelial transition by resolving the bivalent chromatin of PROX1 gene

2021 ◽  
Vol 17 (9) ◽  
pp. e1009847
Author(s):  
Yao Ding ◽  
Weikang Chen ◽  
Zhengzhou Lu ◽  
Yan Wang ◽  
Yan Yuan
Keyword(s):  
Stem Cells ◽  
Kaposi's Sarcoma ◽  
Embryonic Stem ◽  
Kaposi’S Sarcoma ◽  
Kshv Infection ◽  
Invasion And Migration ◽  
Endothelial Lineage ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Msc Differentiation

Increasing evidence suggests that Kaposi’s sarcoma (KS) arises from Kaposi’s sarcoma-associated herpesvirus (KSHV)-infected mesenchymal stem cells (MSCs) through mesenchymal-to-endothelial transition (MEndT). KSHV infection promotes MSC differentiation of endothelial lineage and acquisition of tumorigeneic phenotypes. To understand how KSHV induces MEndT and transforms MSCs to KS cells, we investigated the mechanism underlying KSHV-mediated MSC endothelial lineage differentiation. Like embryonic stem cells, MSC differentiation and fate determination are under epigenetic control. Prospero homeobox 1 (PROX1) is a master regulator that controls lymphatic vessel development and endothelial differentiation. We found that the PROX1 gene in MSCs harbors a distinctive bivalent epigenetic signature consisting of both active marker H3K4me3 and repressive marker H3K27me3, which poises expression of the genes, allowing timely activation upon differentiation signals or environmental stimuli. KSHV infection effectively resolves the bivalent chromatin by decreasing H3K27me3 and increasing H3K4me3 to activate the PROX1 gene. vIL-6 signaling leads to the recruitment of MLL2 and SET1 complexes to the PROX1 promoter to increase H3K4me3, and the vGPCR-VEGF-A axis is responsible for removing PRC2 from the promoter to reduce H3K27me3. Therefore, through a dual signaling process, KSHV activates PROX1 gene expression and initiates MEndT, which renders MSC tumorigenic features including angiogenesis, invasion and migration.

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