HCMV-encoded NK modulators: Lessons from in vitro and in vivo genetic variation

Human cytomegalovirus (HCMV) is under constant selective pressure from the immune system in vivo. Study of HCMV genes that have been lost in the absence of, or genetically altered by, such selection can focus research toward findings of in vivo significance. We have been particularly interested in the most pronounced change in the highly passaged laboratory strains AD169 and Towne—the deletion of 13–15 kb of sequence (designated the UL/b′ region) that encodes up to 22 canonical genes, UL133-UL150. At least 5 genes have been identified in UL/b′ that inhibit NK cell function. UL135 suppresses formation of the immunological synapse (IS) by remodeling the actin cytoskeleton, thereby illustrating target cell cooperation in IS formation. UL141 inhibits expression of two activating ligands (CD155, CD112) for the activating receptor CD226 (DNAM-1), and two receptors (TRAIL-R1, R2) for the apoptosis-inducing TRAIL. UL142, ectopically expressed in isolation, and UL148A, target specific MICA allotypes that are ligands for NKG2D. UL148 impairs expression of CD58 (LFA-3), the co-stimulatory cell adhesion molecule for CD2 found on T and NK cells. Outside UL/b′, studies on natural variants have shown UL18 mutants change affinity for their inhibitory ligand LIR-1, while mutations in UL40's HLA-E binding peptide differentially drive NKG2C+ NK expansions. Research into HCMV genomic stability and its effect on NK function has provided important insights into virus:host interactions, but future studies will require consideration of genetic variability and the effect of genes expressed in the context of infection to fully understand their in vivo impact

Detection of herpes simplex virus Type-1 in patients with fibrotic lung diseases

The current study intends to investigate i) the incidence of herpes viruses including Herpes Simplex Virus type-1 (HSV-1), Cytomegalovirus (CMV) and Human Herpes Virus -6, -7, -8 (HHV6, HHV7, HHV8) in two biological samples, bronchoalveolar lavage fluid (BALF) and lung tissue biopsy, in different forms of pulmonary fibrosis, and ii) the induction of molecular pathways involved in fibrosis by herpesvirus infection in primary cell cultures. PCR was employed for the detection of CMV, HHV6-8 and HSV-1 DNA in lung specimens (4 controls and 11 IPF specimens) and BALF pellet [6 controls and 20 fibrotic Idiopathic Intestitial Pneumonias (f-IIPs) samples: 13 idiopathic pulmonary fibrosis (IPF) and 7 nonspecific idiopathic interstitial pneumonia (NSIP)] samples. Among all herpesviruses tested, HSV-1 was detected in 1/11 (9%) specimens from IPF lung tissue and in 2/20 (10%) samples of f-IIPs BALF whereas the control group was negative. Primary cell cultures from BALF of patients with IPF and healthy controls were infected in vitro with wild-type HSV-1 virus and Real Time PCR was employed for the detection of gene transcription of specific axes implicated in lung fibrosis. Primary cell cultures were permissive to HSV-1, resulting in an upregulation of the fibrotic growth factors TGFβ1 and FGF, the angiogenetic markers SDF1a, SDF1b, VEGF, FGF and the regulators of tissue wound healing MMP9 and CCR7. Downregulation was noted for the CXCR4 and MMP2 genes, while a different response has been detected in healthy donors regarding the expression of the aforementioned markers. These results implicate for the first time the HSV-1 with Fibrotic Idiopathic Interstitial Pneumonias since the virus presented similar incidence in two different biological samples

HCMV-secreted glycoprotein gpUL4 inhibits TRAIL-mediated apoptosis and NK cell activation

Human cytomegalovirus (HCMV) is a paradigm of pathogen immune evasion and sustains lifelong persistent infection in the face of exceptionally powerful host immune responses through the concerted action of multiple immune-evasins. These reduce NK cell activation by inhibiting ligands for activating receptors, expressing ligands for inhibitory receptors, or inhibiting synapse formation. However, these functions only inhibit direct interactions with the infected cell. To determine whether the virus also expresses soluble factors that could modulate NK function at a distance, we systematically screened all 170 HCMV canonical protein-coding genes. This revealed that UL4 encodes a secreted and heavily glycosylated protein (gpUL4) that is expressed with late-phase kinetics and is capable of inhibiting NK cell degranulation. Analyses of gpUL4 binding partners by mass spectrometry identified an interaction with TRAIL. gpUL4 bound TRAIL with picomolar affinity and prevented TRAIL from binding its receptor, thus acting as a TRAIL decoy receptor. TRAIL is found in both soluble and membrane-bound forms, with expression of the membrane-bound form strongly up-regulated on NK cells in response to interferon. gpUL4 inhibited apoptosis induced by soluble TRAIL, while also binding to the NK cell surface in a TRAIL-dependent manner, where it blocked NK cell degranulation and cytokine secretion. gpUL4 therefore acts as an immune-evasin by inhibiting both soluble and membrane-bound TRAIL and is a viral-encoded TRAIL decoy receptor. Interestingly, gpUL4 could also suppress NK responses to heterologous viruses, suggesting that it may act as a systemic virally encoded immunosuppressive agent

Μελέτη της αντιικής δράσης του CD40L κατά τη διάρκεια λυτικής λοίμωξης από τον ιό του απλού έρπητα τύπου 1 (HSV-1)

Ο ιός του απλού έρπητα τύπου 1 (HSV-1) είναι ένας νευροτροπικός, άλφα ερπητοϊός της οικογένειας Herpesviridae. Όπως και άλλοι ιοί, χρησιμοποιεί τους μηχανισμούς του κυττάρου ξενιστή ώστε να ενορχηστρώσει τον κύκλο της ζωής του. Γι το σκοπό αυτό, ακολουθεί ένα σαφώς οργανωμένο πρότυπο έκφρασης των ιϊκών γονιδίων που προσαρμόζεται ανάλογα με τις συνθήκες του περιβάλλοντος του ιού. Με αυτόν τον τρόπο ο ιός μπορεί να αποφεύγει την ανοσολογική απόκριση, να αναστέλλει την απόπτωση και επίσης να ρυθμίζει την τροποποίηση των ιστονών ώστε να μπορεί να μεταβεί από κατάσταση λυτικού κύκλου σε λανθάνουσα κατάσταση και αντίστροφα. Διάφορες προσεγγίσεις έχουν πραγματοποιηθεί με στόχο την αποσαφήνιση της βιολογίας του απλού έρπητα και την ταυτοποίηση νέων στόχων για την θεραπεία του. Στα πλαίσια αυτά, έχουν μελετηθεί ο υποδοχέας CD40 καθώς και ο συνδέτης του CD40L αφού η σηματοδότησή τους είναι μείζονος σημασίας για το ανοσοποιητικό σύστημα. Πειράματα σε ποντίκια έχουν δείξει ότι απουσία του CD40L οδηγεί σε σοβαρή αποσταθεροποίηση της χυμικής ανοσίας. Παράλληλα, έχει δειχθεί ότι ο CD40L έχει και άμεση αντιϊκή δράση σε κύτταρα που εκφράζουν τον υποδοχέα του. Προς αυτή την κατεύθυνση επιχειρήσαμε να διερευνήσουμε την επίδραση της σηματοδότησης του CD40 στην μόλυνση από τον ιό HSV-1 και πώς αυτή επηρεάζει την έκφραση των ιϊκών πρωτεϊνών κατά τη διάρκεια του λυτικού κύκλου. Πράγματι, παρατηρήθηκε μείωση του ιϊκού φορτίου έπειτα από επίδραση με CD40L σε κύτταρα πουέφεραν τονυποδοχέα καθώς και κάποια μείωση της έκφρασης της πρώιμης πρωτεΐνης του ιού, ICP8.Herpes simplex virus type 1 (HSV-1) is a neurotropic, alpha herpesvirus that belongs to the Herpesviridae family. Similar to other viruses, it utilizes the host cell machinery in order to orchestrate its life cycle. In order to do so, it follows a well organized pattern of viral gene expression which is modulated according to the environmental conditions. Thereby, HSV-1 can evade immune responses, apoptosis and also modulate histone modifications in order to switch from an active to a latent state and vice versa. Several approaches have been made towards the elucidation of HSV-1 biology and to identification of targets for its treatment. In this context, CD40 and CD40 ligand have also been studied since their signaling is of major importance in the immune system. Experiments in mice have revealed severely compromised humoral immune responses in the absence of CD40L while a direct antiviral effect has been demonstrated in cells expressing CD40. To that direction, we attempted to investigate the effect of CD40-CD40L signaling to HSV-1 infection and how this affects viral protein expression during lytic infection. We indeed, observed a reduction in viral yield upon treatment with CD40L as well as some decrease in the protein expression of the virus early protein ICP8

Μελέτη της αντιικής δράσης του CD40L κατά τη διάρκεια λυτικής λοίμωξης από τον ιό του απλού έρπητα τύπου 1 (HSV-1)

Ο ιός του απλού έρπητα τύπου 1 (HSV-1) είναι ένας νευροτροπικός, άλφα ερπητοϊός της οικογένειας Herpesviridae. Όπως και άλλοι ιοί, χρησιμοποιεί τους μηχανισμούς του κυττάρου ξενιστή ώστε να ενορχηστρώσει τον κύκλο της ζωής του. Γι το σκοπό αυτό, ακολουθεί ένα σαφώς οργανωμένο πρότυπο έκφρασης των ιϊκών γονιδίων που προσαρμόζεται ανάλογα με τις συνθήκες του περιβάλλοντος του ιού. Με αυτόν τον τρόπο ο ιός μπορεί να αποφεύγει την ανοσολογική απόκριση, να αναστέλλει την απόπτωση και επίσης να ρυθμίζει την τροποποίηση των ιστονών ώστε να μπορεί να μεταβεί από κατάσταση λυτικού κύκλου σε λανθάνουσα κατάσταση και αντίστροφα. Διάφορες προσεγγίσεις έχουν πραγματοποιηθεί με στόχο την αποσαφήνιση της βιολογίας του απλού έρπητα και την ταυτοποίηση νέων στόχων για την θεραπεία του. Στα πλαίσια αυτά, έχουν μελετηθεί ο υποδοχέας CD40 καθώς και ο συνδέτης του CD40L αφού η σηματοδότησή τους είναι μείζονος σημασίας για το ανοσοποιητικό σύστημα. Πειράματα σε ποντίκια έχουν δείξει ότι απουσία του CD40L οδηγεί σε σοβαρή αποσταθεροποίηση της χυμικής ανοσίας. Παράλληλα, έχει δειχθεί ότι ο CD40L έχει και άμεση αντιϊκή δράση σε κύτταρα που εκφράζουν τον υποδοχέα του. Προς αυτή την κατεύθυνση επιχειρήσαμε να διερευνήσουμε την επίδραση της σηματοδότησης του CD40 στην μόλυνση από τον ιό HSV-1 και πώς αυτή επηρεάζει την έκφραση των ιϊκών πρωτεϊνών κατά τη διάρκεια του λυτικού κύκλου. Πράγματι, παρατηρήθηκε μείωση του ιϊκού φορτίου έπειτα από επίδραση με CD40L σε κύτταρα πουέφεραν τονυποδοχέα καθώς και κάποια μείωση της έκφρασης της πρώιμης πρωτεΐνης του ιού, ICP8.Herpes simplex virus type 1 (HSV-1) is a neurotropic, alpha herpesvirus that belongs to the Herpesviridae family. Similar to other viruses, it utilizes the host cell machinery in order to orchestrate its life cycle. In order to do so, it follows a well organized pattern of viral gene expression which is modulated according to the environmental conditions. Thereby, HSV-1 can evade immune responses, apoptosis and also modulate histone modifications in order to switch from an active to a latent state and vice versa. Several approaches have been made towards the elucidation of HSV-1 biology and to identification of targets for its treatment. In this context, CD40 and CD40 ligand have also been studied since their signaling is of major importance in the immune system. Experiments in mice have revealed severely compromised humoral immune responses in the absence of CD40L while a direct antiviral effect has been demonstrated in cells expressing CD40. To that direction, we attempted to investigate the effect of CD40-CD40L signaling to HSV-1 infection and how this affects viral protein expression during lytic infection. We indeed, observed a reduction in viral yield upon treatment with CD40L as well as some decrease in the protein expression of the virus early protein ICP8

CD40 ligand exhibits a direct antiviral effect on Herpes Simplex Virus type-1 infection via a PI3K-dependent, autophagy-independent mechanism

The interaction between CD40 and its ligand, CD40L/CD154, is crucial for the efficient initiation and regulation of immune responses against viruses. Herpes Simplex Virus type-1 (HSV-1) is a neurotropic virus capable of manipulating host responses and exploiting host proteins to establish productive infection. Herein we have examined the impact of CD40L-mediated CD40 activation on HSV-1 replication in U2OS cells stably expressing the CD40 receptor. Treatment of these cells with CD40L significantly reduced the HSV-1 progeny virus compared to non-treated cells. The activation of CD40 signaling did not affect the binding of HSV-1 virions on the cell surface but rather delayed the translocation of VP16 to the nucleus, affecting all stages of viral life cycle. Using pharmacological inhibitors and RNAi we show that inhibition of PI3 kinase but not autophagy reverses the effects of CD40L on HSV-1 replication. Collectively, these data demonstrate that CD40 activation exerts a direct inhibitory effect on HSV-1, initiating from the very early stages of the infection by exploiting PI3 kinase-dependent but autophagy-independent mechanisms