Final Report Summary - LASSA VIRUS RECEPTOR (The interaction of Lassa virus with its cellular receptor alpha-dystroglycan)
Background and project objectives:
The Old World arenavirus Lassa virus (LASV) causes a severe hemorrhagic fever with high mortality in man and is one of the most devastating human pathogenic viruses. Every year, LASV causes several hundred thousand infections in Western Africa with thousands of deaths. Considering the number of people affected, LASV belongs arguably to the most neglected tropical pathogens. There is currently no licensed vaccine against LASV and therapeutic options are limited resulting in high mortality among hospitalised Lassa fever patients.
The LASV life cycle starts with the attachment of the virion to its cellular receptor and subsequent internalisation. The main receptor for LASV is dystroglycan (DG), a versatile cellular receptor for proteins of the extracellular matrix that is essential for normal cell-matrix interaction. Our studies on the binding of LASV to cellular DG revealed that the virus closely mimics the interactions of ECM proteins with the receptor. As a consequence, virus binding may alter the function of the cellular receptor affecting hoes cell function and contributing to disease. In a first part of our study, we investigated the consequences of the interaction of LASV with cellular DG on DG's function in the host cell.
Upon receptor binding, LASV is internalised by endocytosis and delivered to acidified endosomes where the virus undergoes fusion. Our initial characterization of LASV cell entry revealed that LASV and the related prototypic Old World arenavirus lymphocytic choriomeningitis virus (LCMV) use a novel and unusual pathway of endocytosis that is independent of classical regulatory factors such as clathrin, caveolin, and dynamin. Moreover, the virus seems to use a pathway of trafficking that is distinct from incoming vesicular traffic to early endosomal compartments. Intrigued by its unusual nature, we sought to further characterize this pathway and identify cellular factors involved.
Results
Several lines of evidence indicated that in the host cell, DG can associate with signaling molecules, including components of the MEK/ERK signaling pathway, Grb2, MEK, and ERK. Our studies revealed that engagement of cellular DG by LASV resulted in significant recruitment of Grb2 and MEK1 by the DG complex. Interestingly, we were unable to detect activation of the MEK/ERK signaling upon virus binding at any point, suggesting recruitment of Grb2 and MEK1 into an inactive signaling complex. An important role for such an inactive signaling complex for viral entry seemed rather unlikely. We therefore investigated the effect of these virus-induced changes on DG's association with Grb2 and MEK1 on the known crosstalk between DG and beta 1-integrins, another important class of ECM receptors. Indeed, binding of LASV to cells markedly reduced the phosphorylation of MEK and ERK in response to the ECM protein laminin. Together, our data provide evidence that high affinity LASV binding to cellular DG perturbs the signaling cross-talk between DG and integrins, shifting the normal signaling equilibrium towards inhibition of the MEK/ERK pathway. Intrigued by the observation that binding of LASV to cells can modulate cellular MEK/ERK signaling, we investigated the role of this pathway for LASV cell entry. Inhibition of MEK, which is the only kinase that can phosphorylate ERK, did not affect infection of cells with LASV, making a role of this pathway in LASV cell entry rather unlikely.
A second major goal of our studies was the identification of cellular factors and signaling pathways involved in LASV cell entry. In the second funding period of this grant, we investigated the largely unknown molecular mechanisms of cell entry of LASV and the related LCMV with the goal to identify host cell factors involved. We found that cell entry of LASV and LCMV required functional cytoskeletal structures, in particular microtubules that are involved in long-distance traffic of cargo in mammalian cells. Productive infection with LASV and LCMV infection depended on an intact multivesicular endosome (MVB), which is involved in sorting of cargo within the endosomal trafficking system. We found that efficient cell entry of LASV and LCMV also depends on endosomal sorting complex required for transport (ESCRT), in particular the components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Cell invasion by LASV and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in the membrane dynamics of late endosomes.
Conclusions
Since DG and integrins are co-expressed on a wide variety of human cell types involved in LASV pathogenesis in man like epithelial cells, endothelial cells, and macrophages the impact of LASV binding on DG-mediated signaling may affect normal cell function in LASV infected individuals. In late stages of fatal human Lassa fever, virus loads often exceed 109 infectious particles per ml of blood and similar virus loads are found in many tissues. In this situation, extensive binding of virus particles to cellular DG may result in significant perturbation of DG-mediated signaling that may contribute to cellular dysfunctions that are associated with the Lassa shock syndrome. In fatal Lassa fever there is surprisingly little inflammation and tissue destruction. The absence of classical hallmarks of immunopathology in fatal disease suggests that the direct interaction of the virus with host cells may contribute to some aspects of pathogenesis, such as vascular leakage. Among other mechanisms, the virus-induced perturbation of ECM-induced cell signaling shown in our studies, may contribute to the functional alterations of epithelial and vascular endothelial cells that precede shock and death. This type of LASV-induced receptor signaling reported here is likely due to the extensive mimicry of endogenous ligand binding by the pathogen. The consequent perturbation of cell signaling appears as a 'collateral damage' inflicted on the cell that may contribute to viral pathogenesis.
Our studies on LASV cell entry identified cellular factors that are essential for the invasion of the host cell by LASV and shed light on the intracellular traffic route hijacked by the pathogen. Based on our data, we propose a new model for cell entry of LASV and related Old World arenaviruses. The requirement for the MVB/late endosome for cell entry of LASV and LCMV found here is further reminiscent to earlier reports on the cellular entry of the Anthrax toxin, the major virulence factor of Bacillus anthracis, suggesting that very different pathogens may use similar strategies to invade the host cell. A hallmark of fatal LASV infection in humans is the inability of the host cell's innate immune system to detect and contain the virus, resulting in uncontrolled infection. Instead of being recognised as a foreign antigen, LASV escapes innate pathogen detection and establish a productive infection without inducing an interferon response. An important class of cellular pathogen-recognition receptors that allow early detection of incoming viruses are transmembrane receptors of the Toll-like receptor family localised in the early endosome. The ability of LASV to use a pathway of endocytosis bypassing classical routes of incoming endosomal trafficking followed by direct delivery to late endosomes may contribute to LASV's ability to escape detection by endosomal receptors of innate anti-viral defense, a hypothesis we are currently testing.
Socio-economic impact of the project:
LASV is one of the most devastating human pathogens known and is still rampant in large parts of the African continent. The virus is currently endemic from Senegal to Cameroon and effects circa 180 million people. Considering the socio-economic impact of emerging arenaviruses on several of the poorest countries on the African continent and the magnitude of the humanitarian problem caused by LASV, novel anti-viral therapeutics are urgently needed. Considering the limited information available on the structure of the virus, therapeutic interventions that target cellular factors essential for viral infection appear promising. In our project, we uncovered important new aspects of LASV-receptor binding and the consequent effects on the host cell. We demonstrated that virus-receptor binding affects normal function of the host cell, contributing to pathogenesis. In our second aim, we were able to identify cellular factors that are essential for the invasion of the host cell by LASV and shed light on the intracellular traffic route hijacked by the pathogen. This work provides us with candidate cell factors that will be evaluated in novel strategies of anti-viral therapeutic intervention. The development of novel drugs against emerging human pathogenic arenaviruses, in particular LASV represents an important line of our research. Novel therapeutics against LASV will significantly improve the current situation and ameliorate the quality of life in wide parts of Western Africa. To improve dissemination of the result of our studies, in particular in the second part of the project, we published the results of our studies in a high-ranking open access journal of the Public Library of Science (PLOS): Pasqual, G., Rojek, J.M. Masin, M., Chatton, J.-Y. and Kunz, S. (2011) Old World arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport. PLOS Pathogens Sep;7(9):e1002232.
The Old World arenavirus Lassa virus (LASV) causes a severe hemorrhagic fever with high mortality in man and is one of the most devastating human pathogenic viruses. Every year, LASV causes several hundred thousand infections in Western Africa with thousands of deaths. Considering the number of people affected, LASV belongs arguably to the most neglected tropical pathogens. There is currently no licensed vaccine against LASV and therapeutic options are limited resulting in high mortality among hospitalised Lassa fever patients.
The LASV life cycle starts with the attachment of the virion to its cellular receptor and subsequent internalisation. The main receptor for LASV is dystroglycan (DG), a versatile cellular receptor for proteins of the extracellular matrix that is essential for normal cell-matrix interaction. Our studies on the binding of LASV to cellular DG revealed that the virus closely mimics the interactions of ECM proteins with the receptor. As a consequence, virus binding may alter the function of the cellular receptor affecting hoes cell function and contributing to disease. In a first part of our study, we investigated the consequences of the interaction of LASV with cellular DG on DG's function in the host cell.
Upon receptor binding, LASV is internalised by endocytosis and delivered to acidified endosomes where the virus undergoes fusion. Our initial characterization of LASV cell entry revealed that LASV and the related prototypic Old World arenavirus lymphocytic choriomeningitis virus (LCMV) use a novel and unusual pathway of endocytosis that is independent of classical regulatory factors such as clathrin, caveolin, and dynamin. Moreover, the virus seems to use a pathway of trafficking that is distinct from incoming vesicular traffic to early endosomal compartments. Intrigued by its unusual nature, we sought to further characterize this pathway and identify cellular factors involved.
Results
Several lines of evidence indicated that in the host cell, DG can associate with signaling molecules, including components of the MEK/ERK signaling pathway, Grb2, MEK, and ERK. Our studies revealed that engagement of cellular DG by LASV resulted in significant recruitment of Grb2 and MEK1 by the DG complex. Interestingly, we were unable to detect activation of the MEK/ERK signaling upon virus binding at any point, suggesting recruitment of Grb2 and MEK1 into an inactive signaling complex. An important role for such an inactive signaling complex for viral entry seemed rather unlikely. We therefore investigated the effect of these virus-induced changes on DG's association with Grb2 and MEK1 on the known crosstalk between DG and beta 1-integrins, another important class of ECM receptors. Indeed, binding of LASV to cells markedly reduced the phosphorylation of MEK and ERK in response to the ECM protein laminin. Together, our data provide evidence that high affinity LASV binding to cellular DG perturbs the signaling cross-talk between DG and integrins, shifting the normal signaling equilibrium towards inhibition of the MEK/ERK pathway. Intrigued by the observation that binding of LASV to cells can modulate cellular MEK/ERK signaling, we investigated the role of this pathway for LASV cell entry. Inhibition of MEK, which is the only kinase that can phosphorylate ERK, did not affect infection of cells with LASV, making a role of this pathway in LASV cell entry rather unlikely.
A second major goal of our studies was the identification of cellular factors and signaling pathways involved in LASV cell entry. In the second funding period of this grant, we investigated the largely unknown molecular mechanisms of cell entry of LASV and the related LCMV with the goal to identify host cell factors involved. We found that cell entry of LASV and LCMV required functional cytoskeletal structures, in particular microtubules that are involved in long-distance traffic of cargo in mammalian cells. Productive infection with LASV and LCMV infection depended on an intact multivesicular endosome (MVB), which is involved in sorting of cargo within the endosomal trafficking system. We found that efficient cell entry of LASV and LCMV also depends on endosomal sorting complex required for transport (ESCRT), in particular the components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Cell invasion by LASV and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in the membrane dynamics of late endosomes.
Conclusions
Since DG and integrins are co-expressed on a wide variety of human cell types involved in LASV pathogenesis in man like epithelial cells, endothelial cells, and macrophages the impact of LASV binding on DG-mediated signaling may affect normal cell function in LASV infected individuals. In late stages of fatal human Lassa fever, virus loads often exceed 109 infectious particles per ml of blood and similar virus loads are found in many tissues. In this situation, extensive binding of virus particles to cellular DG may result in significant perturbation of DG-mediated signaling that may contribute to cellular dysfunctions that are associated with the Lassa shock syndrome. In fatal Lassa fever there is surprisingly little inflammation and tissue destruction. The absence of classical hallmarks of immunopathology in fatal disease suggests that the direct interaction of the virus with host cells may contribute to some aspects of pathogenesis, such as vascular leakage. Among other mechanisms, the virus-induced perturbation of ECM-induced cell signaling shown in our studies, may contribute to the functional alterations of epithelial and vascular endothelial cells that precede shock and death. This type of LASV-induced receptor signaling reported here is likely due to the extensive mimicry of endogenous ligand binding by the pathogen. The consequent perturbation of cell signaling appears as a 'collateral damage' inflicted on the cell that may contribute to viral pathogenesis.
Our studies on LASV cell entry identified cellular factors that are essential for the invasion of the host cell by LASV and shed light on the intracellular traffic route hijacked by the pathogen. Based on our data, we propose a new model for cell entry of LASV and related Old World arenaviruses. The requirement for the MVB/late endosome for cell entry of LASV and LCMV found here is further reminiscent to earlier reports on the cellular entry of the Anthrax toxin, the major virulence factor of Bacillus anthracis, suggesting that very different pathogens may use similar strategies to invade the host cell. A hallmark of fatal LASV infection in humans is the inability of the host cell's innate immune system to detect and contain the virus, resulting in uncontrolled infection. Instead of being recognised as a foreign antigen, LASV escapes innate pathogen detection and establish a productive infection without inducing an interferon response. An important class of cellular pathogen-recognition receptors that allow early detection of incoming viruses are transmembrane receptors of the Toll-like receptor family localised in the early endosome. The ability of LASV to use a pathway of endocytosis bypassing classical routes of incoming endosomal trafficking followed by direct delivery to late endosomes may contribute to LASV's ability to escape detection by endosomal receptors of innate anti-viral defense, a hypothesis we are currently testing.
Socio-economic impact of the project:
LASV is one of the most devastating human pathogens known and is still rampant in large parts of the African continent. The virus is currently endemic from Senegal to Cameroon and effects circa 180 million people. Considering the socio-economic impact of emerging arenaviruses on several of the poorest countries on the African continent and the magnitude of the humanitarian problem caused by LASV, novel anti-viral therapeutics are urgently needed. Considering the limited information available on the structure of the virus, therapeutic interventions that target cellular factors essential for viral infection appear promising. In our project, we uncovered important new aspects of LASV-receptor binding and the consequent effects on the host cell. We demonstrated that virus-receptor binding affects normal function of the host cell, contributing to pathogenesis. In our second aim, we were able to identify cellular factors that are essential for the invasion of the host cell by LASV and shed light on the intracellular traffic route hijacked by the pathogen. This work provides us with candidate cell factors that will be evaluated in novel strategies of anti-viral therapeutic intervention. The development of novel drugs against emerging human pathogenic arenaviruses, in particular LASV represents an important line of our research. Novel therapeutics against LASV will significantly improve the current situation and ameliorate the quality of life in wide parts of Western Africa. To improve dissemination of the result of our studies, in particular in the second part of the project, we published the results of our studies in a high-ranking open access journal of the Public Library of Science (PLOS): Pasqual, G., Rojek, J.M. Masin, M., Chatton, J.-Y. and Kunz, S. (2011) Old World arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport. PLOS Pathogens Sep;7(9):e1002232.