Type I interferons (IFN-I) are pleiotropic cytokines that were originally identified for their antiviral properties and are now recognized for playing key roles in the defense against a range of... Show moreType I interferons (IFN-I) are pleiotropic cytokines that were originally identified for their antiviral properties and are now recognized for playing key roles in the defense against a range of other microorganisms as well as cancer. Their production should be well-controlled to be of benefit to the host, as excessive or chronic IFN-I expression leads to adverse effects such as immunosuppression or the induction of severe immunopathology.The studies presented in this thesis are aimed at uncovering mechanisms that regulate the production of IFN-I. The obtained knowledge on the involved molecular processes, may aid the development of targeted therapies that enhance or intercept IFN-I responses for maximum host protection while minimizing damage. Show less
T cell exhaustion is a state of T cell hypofunction arising during persistent viral infections and cancer. Recent advances in the field of immunology uncover the roles of cytokines in regulating T... Show moreT cell exhaustion is a state of T cell hypofunction arising during persistent viral infections and cancer. Recent advances in the field of immunology uncover the roles of cytokines in regulating T cell responses. Using LCMV Clone-13 as a model of persistent viral infection, this thesis investigates the roles of IL-27 and IFN-I in regulating T cells during infection. In addition, the thesis explores the potential of JAK inhibitor in rescuing T cell exhaustion during persistent viral infection and cancer. Show less
Replication of positive-stranded RNA viruses requires the activity of proteases that cleave the viral replicase polyproteins. For Middle East respiratory coronavirus (MERS-CoV), the virus-encoded... Show moreReplication of positive-stranded RNA viruses requires the activity of proteases that cleave the viral replicase polyproteins. For Middle East respiratory coronavirus (MERS-CoV), the virus-encoded papain-like protease (PLpro) is one of such proteases. This protease also functions as a deubiquitinating enzyme (DUB) that removes ubiquitin from substrates, most likely to suppress the ubiquitin-dependent activation of the innate immune response. The work described in this thesis provides novel insights in the interaction between PLpro and ubiquitin. The crystal structure of the PLpro-ubiquitin complex facilitated the design of substitutions in PLpro that selectively disrupted its DUB activity. DUB-negative MERS-CoV induced enhanced immune responses compared to wild-type virus, while showing similar replication in infected cells. Relative to wild-type virus, the virulence of DUB-negative MERS-CoV was reduced in mice and earlier, better-regulated immune responses were measured in their lungs. In the search for novel antivirals, ubiquitin sequence variants were selected that bound with very high affinity to MERS-CoV PLpro. Expression of those ubiquitin variants affected the activity of PLpro and concomitantly inhibited virus replication resulting in severely less virus progeny. Collectively, the gained knowledge can be used to design novel coronavirus vaccines or further develop ubiquitin variants as antiviral agents against viruses that encode DUBs. Show less
The impact of respiratory virus infections on the health of children and adults can be very significant. Yet, in contrast to most other childhood infections as well as other viral and bacterial... Show moreThe impact of respiratory virus infections on the health of children and adults can be very significant. Yet, in contrast to most other childhood infections as well as other viral and bacterial diseases, prophylactic vaccines or effective antiviral treatments against viral respiratory infections are either still not available, or provide only limited protection. Given the widespread prevalence, a general lack of natural sterilizing immunity, and/or high morbidity and lethality rates of diseases caused by influenza, respiratory syncytial virus, coronaviruses, and rhinoviruses, this difficult situation is a genuine societal challenge. A thorough understanding of the virus-host interactions during these respiratory infections will most probably be pivotal to ultimately meet these challenges. This review attempts to provide a comparative overview of the knowledge about an important part of the interaction between respiratory viruses and their host: the arms race between host innate immunity and viral innate immune evasion. Many, if not all, viruses, including the respiratory viruses listed above, suppress innate immune responses to gain a window of opportunity for efficient virus replication and setting-up of the infection. The consequences for the host's immune response are that it is often incomplete, delayed or diminished, or displays overly strong induction (after the delay) that may cause tissue damage. The affected innate immune response also impacts subsequent adaptive responses, and therefore viral innate immune evasion often undermines fully protective immunity. In this review, innate immune responses relevant for respiratory viruses with an RNA genome will briefly be summarized, and viral innate immune evasion based on shielding viral RNA species away from cellular innate immune sensors will be discussed from different angles. Subsequently, viral enzymatic activities that suppress innate immune responses will be discussed, including activities causing host shut-off and manipulation of stress granule formation. Furthermore, viral protease-mediated immune evasion and viral manipulation of the ubiquitin system will be addressed. Finally, perspectives for use of the reviewed knowledge for the development of novel antiviral strategies will be sketched. Show less
The work described in this thesis provides novel insights into the structural and (multi)functional characteristics of arterivirus PLP2. This enzyme plays an essential role in the viral replication... Show moreThe work described in this thesis provides novel insights into the structural and (multi)functional characteristics of arterivirus PLP2. This enzyme plays an essential role in the viral replication cycle by cleaving the viral replicase polyproteins. In addition, there were indications that PLP2 is able to influence certain cellular processes by cleaving ubiquitin. We have now shown that PLP2 indeed functions as a deubiquitinating enzyme (DUB) and that this activity is important for the suppression of the innate immune response in the cell. To be able to separate both functions of PLP2 we have solved the crystal structure of this enzyme in complex with ubiquitin. Based on this structure, we were able to design mutations in PLP2 that selectively disrupt the interaction with ubiquitin, without interfering with cleavage of the viral polyproteins. Using these mutants, we have demonstrated for the first time the importance of a viral DUB in the evasion of innate immunity in the context of an infection. The acquired knowledge can now be applied to the design of improved arterivirus vaccines and studies of other viral DUBs, including those encoded by the zoonotic coronaviruses that cause SARS and MERS. Show less
The interplay between nidoviruses and the infected host cell was investigated. Arterivirus RNA-synthesising activity was shown to depend on intact membranes and on a cytosolic host protein which... Show moreThe interplay between nidoviruses and the infected host cell was investigated. Arterivirus RNA-synthesising activity was shown to depend on intact membranes and on a cytosolic host protein which does not cosediment with the RTC. Furthermore, the immunosuppressant drug cyclosporin A (CsA) blocks replication of EAV and the swine arterivirus PRRSV in cell culture. Cyclophilin A appears to be an important host factor for EAV replication. CsA may be a nidovirus-wide inhibitor of replication since this compound also blocked replication of the coronaviruses SARS-CoV, HCoV-229E, and MHV. We further described a kinase siRNA library screen that identified ninety antiviral and forty proviral hits and signalling pathways involved in the SARS-CoV replicative cycle. PKR (antiviral) and COPB2 (proviral) were validated in follow-up experiments. We also investigated MERS-CoV replication characteristics and we described an assay t o screen for compounds that block MERS-CoV infection. CsA and pegylated IFN-_ (PEG-IFN) significantly inhibited infection, and MERS-CoV was shown to be much more sensitive to PEG-IFN treatment than SARS-CoV, an observation that may have implications for the treatment of MERS-CoV infection. The data presented in this thesis might contribute to better understand virus replication and hopefully provide additional starting points for the development of antiviral strategies. Show less
Paus, R.A. de; Crevel, R. van; Beek, R. van; Sahiratmadja, E.; Alisjahbana, B.; Marzuki, S.; ... ; Vosse, E. van de 2013
Type I immune responses play an essential role in the control of mycobacterial infections. Mutations in the genes involved in the type I cytokine pathway were found in patients with Mendelian... Show moreType I immune responses play an essential role in the control of mycobacterial infections. Mutations in the genes involved in the type I cytokine pathway were found in patients with Mendelian susceptibility to mycobacterial diseases. These patients are highly susceptible to infections with non-tuberculous mycobacteria (NTM), which are usually poorly pathogenic. The first part of this thesis focuses on the relation between the genotype and phenotype in the cause of an impaired immunity leading to the susceptibility to NTM infections. The role of genetic factors in the control of infections with more virulent tuberculous mycobacteria is less evident. The second part of this thesis focuses on putative non-genetic causes of an impaired immunity in the control of tuberculous mycobacterial diseases. In tuberculosis patients type I immune responses regulated by interferon-_ are also repressed. Virally induced interferons, other than interferon-_, may be involved in this repression, thereby influencing the immunopathogenesis of tuberculosis. Show less
Coronaviruses (CoV) are positive-stranded RNA viruses with a genome of approximately 30 kb. Most human coronaviruses cause upper respiratory tract infections. Here we applied microarray analysis to... Show moreCoronaviruses (CoV) are positive-stranded RNA viruses with a genome of approximately 30 kb. Most human coronaviruses cause upper respiratory tract infections. Here we applied microarray analysis to assess global changes in cellular expression profiles during acute cytopathic coronavirus infection. We demonstrated that MHV infection in fibroblast-like cells triggers very few changes in cellular mRNA levels in the first hours of infection and that infection of fibroblasts with MHV and SARS-CoV does not result in a significant induction of IFN production through intracellular detection by RNA helicases. Data from additional experiments suggest that the absence of IRF-3 activation is a general characteristic of group 2 coronaviruses. Furthermore we show that MHV and SARS-CoV do not actively block IRF-3 activation via either RIG-I or MDA-5 as they are unable to inhibit IRF-3 translocation upon Sendai virus infection or poly (I:C) transfection, respectively. Finally, we show that SARS-CoV and MHV induce ER stress and chemokine mRNA transcription during infection in vitro. Expression of the viral spike protein significantly induced ER stress and Cxcl2 mRNA upregulation. However, translation of these cellular mRNAs is severely reduced, resulting in no significant CXCL2 and HERPUD1 protein synthesis in infected cells despite their increased mRNA concentrations. Show less
Monoclonal proteinaemia (M-proteinemia) is associated with multiple myeloma (MM) or other hematological malignancies. In the absence of these diseases the term MGUS (Monoclonal Gammopathy of... Show moreMonoclonal proteinaemia (M-proteinemia) is associated with multiple myeloma (MM) or other hematological malignancies. In the absence of these diseases the term MGUS (Monoclonal Gammopathy of Undetermined Significance) is used. During 1991-1993 1464 patients with newly diagnosed M-proteinemia in the mid-western of The Netherlands (1.6 million inhabitants) were registered in a database and followed annually. Information on characte_ristics, laboratory and bone marrow test results, skeletal-X-rays as well as M-protein related diagnosis were obtained. At diagnosis potential discriminatory serum markers like interleukin-6 and syndecan-1 proved to be of no informative value. The reported correlation between solid tumors and M-proteinemia was rather based on a diagnostic selection bias than on a causal role. Long-term follow-up (median 9.0 years for those alive) showed that survival of MGUS patients is reduced compared to an age-matched cohort of the Dutch population. Age, gender and serum albumen were important factors for survival; the annual 0.4% risk for malignant transformation was influenced by M-protein level and IgA-isotype. In MM early response to melphalan-prednisone chemotherapy as measured by the rate of serum M-protein decrement was of prognostic value. Interferon maintenance therapy in MM-patients prolonged progression free survival, but not overall survival without affecting quality of life. Show less
