Inhibition of Phosphorylated-STAT1 Nuclear Translocation and Antiviral Protein Expression in Human Brain Vascular Adventitial Fibroblasts Infected with Varicella-Zoster Virus

Abstract

In varicella-zoster virus (VZV)-infected primary human brain vascular adventitial fibroblasts (BRAFs), levels of beta interferon (IFN-␤V aricella-zoster virus (VZV) vasculopathy is often protracted, perhaps due in part to persistent infection of arterial adventitial fibroblasts (1). A potential mechanism of virus persistence is evasion of the antiviral type I interferon (interferon alpha [IFN-␣] and IFN-␤) response (2) which induces antiviral interferon-inducible genes such as Mx1. Mx1 belongs to the dynamin superfamily of large GTPases (3,4), is present in all vertebrates in one to three copies, and is strictly controlled by type I and type III interferons. Mx1 encodes the human MxA protein, which accumulates in the cytoplasm, recognizes viral nucleocapsids, and blocks viral replication. In human lung embryonic fibroblasts, VZV immediateearly protein (IE) 62 blocks phosphorylation of interferon regulatory factor 3 (IRF3) and subsequent induction of IFN-␤ (5). Similarly, in HEK 293T and MeWo cells, VZV IE61 degrades activated IRF3 (6), and in VZV-infected epidermal cells, IFN-␣ is downregulated and IFN-␣-induced phosphorylated STAT1 (pSTAT1) is absent (7). Thus, we examined interference with type 1 IFN signaling in VZVinfected cerebrovascular adventitial fibroblasts as a potential mechanism for virus persistence.Primary human brain vascular adventitial fibroblasts (BRAFs) (ScienCell, Carlsbad, CA) were seeded at 5,000 cells/cm 2 in basal fibroblast medium supplemented with 2% fetal bovine serum (FBS), 1% fibroblast growth serum, and 1% 100ϫ penicillinstreptomycin (ScienCell). After 24 h, medium was changed to basal fibroblast medium supplemented with 0.1% FBS and 1% 100ϫ penicillin-streptomycin and replenished every 48 h for 1 week to establish quiescence. Quiescent BRAFs were cocultivated with VZV-infected or uninfected BRAFs or treated for 24 h with 1,000 U of IFN-␣ (PBL Interferon Source, Piscataway, NJ). VZVinfected BRAFs were analyzed at the height of the cytopathic effect (CPE) 3 days later and positive controls 24 h after treatment in 4 independent experiments. mRNA was analyzed by reverse-transcription PCR (RT-PCR) using SYBR green (8) and primers for IFN-␣, IFN-␤, STAT1, STAT2, Mx1, and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Fig. 1A). Primer efficiencies were 104%, 93%, 92%, 89%, 107%, and 102%, respectively. Data were normalized to GAPDH and analyzed using the delta delta threshold cycle (C T ) method (9).BRAFS were propagated on coverslips, fixed, and permeabilized (10). Mouse anti-VZV gE (Santa Cruz Biotechnology, Santa Cruz, CA) (1:500) was added in addition to another primary antibody: rabbit anti-STAT1 (Epitomics, Burlingame, CA) (1:200); rabbit anti-pSTAT1 (Epitomics) (1:300); or rabbit anti-Mx1 (Abcam, Cambridge, MA) (1:100). Secondary antibodies were 1:1,000 dilutions of both Alexa Fluor 594 donkey anti-mouse IgG and Alexa Fluor 488 donkey anti-rabbit IgG 488 (Life Technologies, Grand Island, NY). Coverslips were mounted with Vectashield containing DAPI (4=,6-]diamidino-2-phenylindo...

Simian varicella virus inhibits the interferon gamma signalling pathway

“…Although HCMV and vaccinia virus infection block phosphorylation of STAT1 via the induction of cellular or viral tyrosine phosphatase expression [24,25], we showed that SVVinduced inhibition of IFNg-induced STAT1 phosphorylation did not involve active dephosphorylation by phosphatases. Our data suggest that SVV infection does not induce degradation of STAT1 protein, suggesting that the virus may reduce STAT1 mRNA expression levels, as was recently demonstrated for VZV [29]. Moreover, we demonstrate that SVV utilizes distinct mechanisms to reduce STAT1 protein levels and block IFNg-induced phosphorylation of STAT1.…”

“…STAT1 is shared between the IFN-a/b and IFNg signalling pathways, thereby providing an ideal target for immune evasion [12]. Similarly, previous studies reported that VZVthe human homologue of SVVreduced STAT1 protein levels in infected human foreskin fibroblasts and brain-derived vascular adventitial fibroblasts [18,29]. Interestingly, others did not detect SVV-or VZV-induced reduction of STAT1 protein level in rhesus macaque and human lung fibroblasts [27], possibly due to a cell-type specific effect.…”

Differential regulation of matrix metalloproteinases in varicella zoster virus-infected human brain vascular adventitial fibroblasts

“…At the time of CPE, conditioned supernatant and cell lysates were prepared as described [8] from VZV- and mock-infected BRAFs, and analyzed for levels of MMP-1, -2, -3 and -9 protein by a multiple immunoassay (Meso Scale Discovery, Rockville, MD) according to the manufacturer’s directions. Each MMP was quantitated (ng/mL) using a standard curve, and the fold change determined for VZV-infected and mock-infected conditioned supernatant.…”

Attack, parry and riposte: molecular fencing between the innate immune system and human herpesviruses

“…Almost all conceivable mechanisms of inhibition have been realized by herpesviruses (see Table ): (I) loss of IFN receptor or their signaling capability [both shown for KSHV ], (II) loss of janus kinases or their enzymatic activity [shown for EBV ], (III) loss of certain STAT molecules , their interaction partner IRF‐9 [shown for HCMV ], their nuclear translocation [shown for HSV and VZV ] or their transcriptional activity , (IV) inhibition of signaling by the expression of viral IRF homologues [shown for KSHV ], (V) block of ISG induction by a global host‐shut‐off [as shown for α‐herpesviruses ] and (VI) upregulation of cell‐intrinsic negative feed‐back regulatory proteins [e.g. PIAS or SOCS proteins – as shown for EBV ].…”