Ελληνικό Ινστιτούτο Παστέρ

1. Viral and Host Determinants Shape the Outcome of Viral Infection and Persistence

Viruses are highly diverse microorganisms, whose heterogeneous genetic and structural features together with a battery of diverse replication strategies and infection mechanisms, ensure evolvability and survival. The biological diversity exhibited by RNA viruses is a combination of their short generation times, fast evolutionary rates and rapid adaptation against selective pressures imposed by evolving antiviral therapies, the environment, hosts and their immune system. On the other hand, DNA viruses are believed to have more stable genomes, however, this notion can be widely challenged by single-stranded or smaller DNA viral species. Overall, this wondrous genetic polyphony, is often matched by equivalent host genetic determinants, resulting in a well-defined symbiosis between virus and the host immune system, or, if left unchallenged, leads to viral persistence and disease manifestation.

1.1.  Defective Viral Genomes, Exosomes and Liver Disease Progression

HCV intrinsic genetic variability is portrayed by the existence of 8 genotypes, 84 subtypes and the recently discovered defective genomes (DGs) of unknown function. Our studies explore the impact of genetic variability on HCV persistence, pathogenesis, disease progression and treatment effectiveness.

HCV DGs lack the envelope region, replicate independently and can be trans-packaged into infectious virions in the presence of full length viral genome. We have isolated and clone DGs from sera samples and liver tissues of patients infected with HCV genotypes 1b, 2a and 3a and demonstrated by in vitro studies that co-expression of DGs with full length HCV genomes results in enhanced viral replication. We reported for the first time that exosomal RNA isolated from HCV sera samples contains HCV DGs. We also provided evidence that the presence of HCV DGs affects both viral replication and release. It is possible that HCV DGs exploit exosomes as means of transport, a way to evade the immune system, to spread more efficiently and possibly maintain persistent infection (Karamichali et al., Front. Microbiol., 2018).

Laboratory Members involved: E. Karamichali, P. Foka, A. Kakkanas, U. Georgopoulou (PI)

Part of this project is a collaboration with Dr. S. Benjelloum and Dr.H. Chihab (Institut Pasteur Maroc), Dr.P. Pineau (Institut Pasteur Paris) and Prof. J. Koskinas (Athens School of Medicine, NKUA)

1.2.  Host Genetic Polymorphisms and Persistence of Hepatitis Viral Infections

Hepatitis B virus (HBV)-specific CD8+ T cells play an important role in the clearance of HBV infection.

PD-1 mRNA expression analysis has revealed that chronic HBV carriers with GG and GA displayed higher levels of PD-1 mRNA compared with corresponding genotypes in resolved subjects suggesting that HBV-infected patients carrying PD-1 GG and GA genotypes at rs10204525 have high PD-1 mRNA expression and may be more prone to the establishment of viral chronicity (Chihab et al., J. Med. Virol., 2018).

Laboratory Members involved: U. Georgopoulou, E. Karamichali, P. Foka (PI)

This project is a collaboration with Dr. S. Benjelloum and Dr. H. Chihab (Institut Pasteur Maroc)

Genetic polymorphisms within the promoter of interferon-α receptor type-1 (IFNAR1) have been associated with the susceptibility to and the outcome of chronic hepatitis B virus (HBV) infection. The majority of modifications of the Interferome coincided with the production of the truncated IFNAR-1 transcript (Karamitros et al., PNAS, 2018).

Laboratory Members involved: E. Karamichali, U. Georgopoulou (PI)

This project is a collaboration with Dr. T. Karamitros and Dr. A. Mentis (HPI Diagnostics Department)

1.3. The effect of HCV core genetic variability in the activation of pro-tumourigenic molecular mechanisms

The study is focused on the deregulation of the Wnt/ β-catenin pathway in the development of HCV-associated HCC.

In this context, we examined the role of HCV core nucleocapsid protein genetic variability in the activation of the Wnt/ β-catenin pathway, using HCV core proteins isolated from clinical samples of patients chronically infected with HCV genotypes 1a, 4a and 4f. Transcriptional profiling of several genes belonging to this pro-tumourigenic pathway revealed varied levels of expression and activation depending on HCV genotype (Aicher et al., Sci. Rep., 2018).

Laboratory Members involved: A. Kakkanas (co-PI), P. Mavromara (PI, past member / collaborating faculty)

1.4. YXXΦ motifs dictate endocytosis of the viral capsid in HCV infection

The nucleocapsid HCV core protein possesses highly conserved ΥΧΧΦ and distal di-leucine motifs that confer primary endocytosis signals. We have showed that the YXXΦ motif is crucial for maintaining the architectural integrity of HCV non-enveloped capsids (ne), as it helps them retain their particulate form.

HCVne clathrin-mediated endocytosis redirects HCVne to the endosomal-lysosomal pathway through induction of AP-2 clathrin adaptors. Their ensuing co-localization with lipid droplets and the replication complex eventually enhances viral replication (Karamichali et al., Infect. Genet. Evol., 2017).

Laboratory Members involved: E. Karamichali, P. Foka, U. Georgopoulou (PI)

1.5. Biochemical Analysis of structural and regulatory features of SARS-CoV and SARS-CoV-2 proteins

We have been carrying out comparative biochemical and structural prediction analyses for the identification of putative post-translational modifications and other structural features on specific viral proteins of the SARS-CoV and SARS-CoV-2 emerging viruses.

Prediction of putative alterations in regulatory sequences and antigenicity could reveal gain- and loss-of-function properties, significant for enhanced propagation, survival fitness and virulence of the new virus.

Laboratory Members involved: A. Kakkanas (co-PI), E. Karamichali, U. Georgopoulou, P. Foka (PI)

2. Dissecting the Metabolic, Epigenetic and Signalling Mechanisms implicated in Virus-Host Interactions and the Progression to Virus-related Cancer

Hepatitis C (HCV) and B virus (HBV) infections often lead to severe liver disease, including cirrhosis and hepatocellular carcinoma (HCC). HCC accounts for approximately 90% of the incidence of all primary liver cancers, and it is the 5^th most prevalent cancer worldwide and the 4^th leading cause of death globally. Recently, direct-acting anti-viral (DAAs) therapies have achieved to eliminate HCV in the majority of cases. However, low barriers to resistance-associated mutations, reduced efficacy in the treatment of difficult patient groups, inability to prevent HCV-induced HCC after viral eradication, recurrence of HCV or latent HBV post-treatment and the lack of an effective vaccine, all underscore the need for better understanding of (a) the viral biology and persistence, (b) host-virus interactions, (c) monitoring disease progression and HCC onset through the identification of molecular signatures/biomarkers  so that (d) novel therapies are discovered.

2.1. The Role of Host Iron Homeostasis in HCV infection and Persistence

Chronic HCV infection is characterised by hepatic iron overload, leading to cirrhosis and HCC.

The hepatic hormone hepcidin regulates host iron homeostasis through degradation of the only known Fe exporter, ferroportin. We have shown that HCV core protein through a complex signaling network involving CK2 kinase (Foka et al., CMLS, 2014). Later, we provided evidence that HCV control over host iron homeostasis occurs both by intracellular iron sequestration, through hepcidin, and intercellular iron mobilisation via ferritin, as means towards enhanced viral replication. We used an HCV-infected hepatoma/macrophage co-culture system to show that viral persistence could be achieved through HCV-induced changes in macrophagic iron that ultimately support low replication levels (Foka et al., Virulence, 2016).

During HCV infection, the multifunctional core and NS5A viral proteins regulate hepcidin expression antagonistically. While hepcidin is enhanced by core‐mediated activation of BMP/SMAD and STAT3 pathways, it is down‐regulated by NS5A independently of these factors. We showed that NS5A disturbs the MTF‐1/Zn2+/hepcidin positive regulation axis, by mopping up Zn2+, thereby preserving the structural integrity of NS5A dimmers (Dimitriadis et al., FEBS Open Bio, 2021).

Laboratory Members involved: P. Foka, E. Karamichali, U. Georgopoulou (PI)

This project is a collaboration with Dr A. Mamalaki (Laboratory of Molecular Biology and Immunobiotechnology, HPI).

2.2 HCV Infection and Host Epigenetic and Pro-metastatic Factors in HCC Development and Progression

Epigenetic changes may enhance the selective advantage of cancer cells and alterations of the epigenome, such as DNA methylation and chromatin modifications, boost uncontrolled cell growth, invasion and metastasis.

The epigenetic factor Lysine-Specific Demethylase 1 (LSD1) is involved in lipid homeostasis and is overexpressed in HCC. We have been investigating the impact of LSD1 (Papadopoulou et al., Invited oral presentation, Hellenic Association for the Study of the Liver,17th Hellenic Liver Congress, 9-11 May 2019) and sister molecule LSD2 in the HCV-mediated regulation of host gene transcription pertinent to lipid metabolism, innate immune response and the modulation of the pro-metastatic factor Emmprin (CD147), which is a matrix metalloproteinase enhancer and regulator of ECM. Ongoing studies indicate an epigenetic element in the manipulation of Emmprin by HCV, thereby providing evidence for the epigenetic control of HCV-directed metastasis.

Laboratory Members involved: E. Karamichali, P. Foka (co-PI), U. Georgopoulou (PI)

Part of this project is a collaboration with the Laboratory of Molecular Biology and Immunobiotechnology (Dr. A.Mamalaki).

2.3 The Role of Host Lipid Metabolism in HCV-associated HCC

HCV uses the host lipid metabolism in various stages of its life cycle. Lipid accumulation causes inflammation and hepatic steatosis, a hallmark of HCV infection observed in the chronically infected patients. ANGPTL-3 and ANGPTL-4 are major lipid metabolism regulators that fine-tune serum lipids via inhibition of tissue lipases.

Recently, both proteins have been implicated in carcinogenesis. We had previously shown that ANGPTL-3 is negatively regulated by the HCV nucleocapsid protein core (Foka et al., J. Hepatol., 2014), while there is no information on the action of HCV on ANGPTL-4. We have lately been working towards the delineation of their role in HCV infection and HCV-induced HCC using sera samples from acute/chronic HCV and HCV-HCC patients. In addition, we have investigated their input in liver disease progression and HCV-induced hepatocarcinogenesis, by generating ANGPTL-3 and -4 overexpression cell lines and using them to identify lipid-related gene alterations with microarray analysis. We have also investigated the role of ANGPTLs in macrophage-regulated lipid clearance and liver steatosis and established an HCV in vitro clearance model to assess their involvement in the emerging “post-cure viral fingerprint” that could predispose susceptible individuals to develop HCC (Valiakou V et al., Int. J. Mol. Sci. 2021).

Laboratory Members involved: E. Karamichali, U. Georgopoulou (co-PI), P. Foka (PI)

Part of this project is in collaboration with Prof. J. Koskinas (Athens School of Medicine, NKUA), and Dr. A. Chroni (Demokritos NCSR)

2.4.  Analysis of Protein Interaction Networks for the Discovery of Hepatitis B and C Biomarkers

Protein interaction networks (PINs) may be used to identify novel mechanisms implicated in host immune responses, liver fibrosis and HCC, in viral hepatitis. 48 overlapping nodes were found with three of them being of higher importance after statistical ranking (Simos et al., IEEE J Biomed Health Inform., 2015). Variations in TNFRSF1A and TMSB4X serum levels were associated with disease stage and warrant further research. (Tsiomita et al., Infect. Genet. Evol., 2018).

Laboratory Members involved: E. Karamichali, U. Georgopoulou (PI)

This project is a collaboration with the Prof. K. Papaloukas and Prof. G.Thyphronitis (University of Ioannina) and Prof. J. Koskinas (Athens School of Medicine, NKUA)

3. Viral Persistence and the Host Immune Response

Depending on whether the virus is eliminated or not, viral infections are either acute or chronic. Chronic infections are further classified into latent infections, where the virus is dormant but occasional viral replication may occur during periodic episodes of reactivation (e.g. HSV-1), or persistent, where viral replication continues to maintain persistent viraemia (e.g. HCV, HBV). The outcome of a viral infection depends on the antiviral immune responses of the host.

Deregulated host innate immune responses fail to eliminate the virus during the acute phase, thereby facilitating the establishment of viral chronicity. Innate immune mechanisms, including the activation of TLR-mediated antiviral pathways are deployed in the presence of viral nucleic acids, leading to IFN type- and cell-specific transcriptional gene regulation. Infected-cell killing by NKCs and viral antigen-presenting by DCs are both instrumental for the early innate immune responses. Monocytes, infiltrating macrophages from the periphery and tissue-resident macrophages also hold key roles in viral infection, including viral antigen presentation, production of pro- and anti-inflammatory cytokines/chemokines and induction of T-cell activity.

Chronic persistent infections are typically associated with compromised adaptive immune responses. CD4+ T-cells, and CD8+ T-cells play a significant antiviral role by balancing the combat against viral pathogens and the risk of developing autoimmunity or overwhelming inflammation. Tregs have also been shown to play an important role in the attenuation of antiviral T-cell responses and immune-mediated host injury and inflammation, through CD8+ T-cell inhibition in both the acute and chronic phases.

Recent findings suggest that immune responses may be modulated by changes in cellular metabolic pathways that can effectively alter the function of immune cells of both adaptive and innate immunity.

3.1 Exosome-mediated Immunomodulation in Viral Hepatitis

Exosomes are extracellular vesicles secreted by various cells to participate in intercellular communication in health and disease. Viruses exploit exosomes to establish persistent infection and evade the immune response.

Exosomal cargo promotes disease progression possibly by “dampening down” host immune responses. Direct-acting antivirals (DAAs) have been recently used for successful eradication of HCV, but host immunity fails to fully recover. We have discovered specific anti-inflammatory and immunosuppressive molecules within exosomes that may contribute to the observed post-cure skewed immune responses, depending on the depth of HCV-induced liver damage before treatment. Furthermore, we observed that specific T-cell populations, such as Tregs, isolated by chronic HCV patients following DAA treatment, could be correlated with the level of liver fibrosis, ie. the degree of hepatic damage in cured patients (Karamichali et al., 27th International Symposium on Hepatitis C Virus and Related Viruses July 6 – 9, 2021).

Laboratory members involved: E. Karamichali (co-PI), P. Foka, U. Georgopoulou (PI)

3.2 Manipulation of Host Iron Homeostasis by the Virus Blunts Innate Immune Responses in HCV Infection

Functional systemic iron homeostasis is critical for proper immune responses against bacterial and viral pathogens. We have built a novel triple-cell co-culture model using liver and intestinal cells co-cultured with macrophages to simulate “more physiological aspects” of systemic iron homeostasis.

We have studied the relationships among key iron-related genes, like the iron regulatory hormone hepcidin and the iron storage protein ferritin, to validate our system. We propose that HCV-modulated changes in iron homeostasis elicit immunotolerant M2 macrophage phenotypes that promote productive infection and persistence

(Foka et al., Cells, 2021).

Laboratory members involved: U. Georgopoulou, E. Karamichali, P. Foka (PI)

This project is in collaboration with Dr A. Mamalaki (Laboratory of Molecular Biology and Immunobiotechnology, HPI).

3.3. SARS-CoV-2 and host immune responses

Emerging SARS-CoV-2 causes a “cytokine storm” with high-levels of pro-inflammatory cytokines and chemokines upon infection. NK- and total T-cells counts are low, but circulatory CD8+ T-cells become highly granulated and infiltrate the lung interstitium, causing extensive injury. Such events may be promoted by viral capsid proteins on entry in collaboration with cell immune mechanisms. Ensuing signalling regulates gene transcription of cellular proliferation, differentiation, degranulation, phagocytosis and expression of inflammatory factors/cytokines. We have been constructing SARS-CoV-2 pseudocapsids in order to delineate the interactions between viral proteins and host immune determinants during viral entry and endocytosis.

Laboratory Members involved: E. Karamichali, U. Georgopoulou, P. Foka (PI)

4. Development of a toe-hold switch-based diagnostic method for the detection of MERS-CoV

 

MERS-CoV is a virus with 35% mortality rate, considered to be one of the most likely to cause major epidemics. Attempting to strengthen the response provisions in case of an outbreak, this project aimed to develop an easy-to-use, reliable and safe, rapid test for MERS-CoV diagnosis. The engineered biosensor was based on the toehold switch mechanism. Toehold switches are mRNA molecules with riboswitch functionality. In this case, the switches under study were designed to regulate the expression of the reporter gene trehalase, which codes for an enzyme that hydrolyses the disaccharide trehalose to glucose. Trehalase expression is then allowed only when the virus is present in the sample. Part of the project were presented in the International iGEM 2018 Competition in the “Genomers” project.

Laboratory Members Involved: E. Karamichali, U. Georgopoulou, P. Foka (PI)

This project was a collaboration with Dr P. Eliadis (Laboratory of Molecular Biology and Immunobiotechnology, HPI) and Dr E. Topakas (School of Chemical Engineering, NTUA)