Immune- and Nonimmune-Compartment-Specific Interferon Responses Are Critical Determinants of Herpes Simplex Virus-Induced Generalized Infections and Acute Liver Failure.

Unlabelled: The interferon (IFN) response to viral pathogens is critical for host survival. In humans and mouse models, defects in IFN responses can result in lethal herpes simplex virus 1 (HSV-1) infections, usually from encephalitis. Although rare, HSV-1 can also cause fulminant hepatic failure, which is often fatal.

CCR5 limits cortical viral loads during West Nile virus infection of the central nervous system.

Background: Cell-mediated immunity is critical for clearance of central nervous system (CNS) infection with the encephalitic flavivirus, West Nile virus (WNV). Prior studies from our laboratory have shown that WNV-infected neurons express chemoattractants that mediate recruitment of antiviral leukocytes into the CNS. Although the chemokine receptor, CCR5, has been shown to play an important role in CNS host defense during WNV infection, regional effects of its activity within the infected brain have not been defined.

Immortalized human cerebral microvascular endothelial cells maintain the properties of primary cells in an in vitro model of immune migration across the blood brain barrier.

The immortalized human cerebral microvascular endothelial cell line HCMEC/D3 presents a less expensive and more logistically feasible alternative to primary human brain microvascular endothelial cells (HBMEC's) for use in constructing in vitro models of the blood brain barrier (BBB). However, the fidelity of the HCMEC/D3 cell line to primary HBMEC's in studies of immune transmigration has yet to be established. Flow cytometric analysis of primary human leukocyte migration across in vitro BBB's generated with either HCMEC/D3 or primary HBMEC's revealed that HCMEC/D3 maintains the immune barrier properties of primary HBMEC's.

Corneal replication is an interferon response-independent bottleneck for virulence of herpes simplex virus 1 in the absence of virion host shutoff.

Herpes simplex viruses lacking the virion host shutoff function (Δvhs) are avirulent and hypersensitive to type I and type II interferon (IFN). In this study, we demonstrate that even in the absence of IFN responses in AG129 (IFN-αβγR(-/-)) mice, Δvhs remains highly attenuated via corneal infection but is fully virulent via intracranial infection. The data demonstrate that the interferon-independent inherent replication defect of Δvhs has a significant impact upon peripheral replication and neuroinvasion.

Functional genomics reveals an essential and specific role for Stat1 in protection of the central nervous system following herpes simplex virus corneal infection.

Innate immune deficiencies result in a spectrum of severe clinical outcomes following infection. In particular, there is a strong association between loss of the signal transducer and activator of transcription (Stat) pathway, breach of the blood-brain barrier (BBB), and virus-induced neuropathology. The gene signatures that characterize resistance, disease, and mortality in the virus-infected nervous system have not been defined.

Bioluminescent imaging reveals divergent viral pathogenesis in two strains of Stat1-deficient mice, and in αßγ interferon receptor-deficient mice.

Pivotal components of the IFN response to virus infection include the IFN receptors (IFNR), and the downstream factor signal transducer and activator of transcription 1 (Stat1). Mice deficient for Stat1 and IFNR (Stat1(-/-) and IFNαßγR(-/-) mice) lack responsiveness to IFN and exhibit high sensitivity to various pathogens. Here we examined herpes simplex virus type 1 (HSV-1) pathogenesis in Stat1(-/-) mice and in IFNαßγR(-/-) mice following corneal infection and bioluminescent imaging.

Interferon regulatory factor 3-dependent pathways are critical for control of herpes simplex virus type 1 central nervous system infection.

The initiation of the immune response at the cellular level relies on specific recognition molecules to rapidly signal viral infection via interferon (IFN) regulatory factor 3 (IRF-3)-dependent pathways. The absence of IRF-3 would be expected to render such pathways inoperative and thereby significantly affect viral infection. Unexpectedly, a previous study found no significant change in herpes simplex virus (HSV) pathogenesis in IRF-3(-/-) mice following intravenous HSV type 1 (HSV-1) challenge (K.

Host responses to wild-type and attenuated herpes simplex virus infection in the absence of Stat1.

Humans and mice lacking the interferon signaling molecule Stat1 are sensitive to a variety of pathogens due to their presumed inability to mount a strong innate immune response. The herpes simplex virus type 1 (HSV-1) virion host shutoff (vhs) protein is a multifunctional immunomodulator that counteracts the innate immune response and viruses lacking vhs are attenuated and effective live vaccines in animal models. To investigate the interplay of viruses with an immunocompromised host, we performed functional genomics analyses on control and Stat1(-/-) mouse corneas infected with wild-type or vhs-null viruses.

Enhanced pathogenesis of an attenuated herpes simplex virus for mice lacking Stat1.

Mice lacking the Stat1 interferon signaling gene were infected with herpes simplex virus type 1 (HSV-1) or an attenuated recombinant lacking virion host shutoff (Delta vhs). Delta vhs virus-infected Stat1(-/-) mice showed levels of replication equivalent to that of the wild-type virus-infected control mice but reduced relative to wild-type virus-infected Stat1(-/-) mice. Stat1 deficiency relieves the immunomodulatory deficiency of Delta vhs virus, but not its inherent growth defect.

Herpes simplex virus virion host shutoff attenuates establishment of the antiviral state.

Herpes simplex virus mutants lacking the vhs protein are severely attenuated in animal models of pathogenesis and exhibit reduced growth in primary cell culture. As a result of these properties, viruses with vhs deleted have been proposed as live-attenuated vaccines. Despite these findings and their implications for vaccines, the mechanisms by which vhs promotes infection in cell culture and in vivo are not understood.

Functional genomic analysis of herpes simplex virus type 1 counteraction of the host innate response.

Herpes simplex virus type 1 (HSV-1) mutants lacking the ICP34.5 gene are severely attenuated in mouse models and have a significant growth defect in confluent mouse embryo fibroblasts. Previously, ICP34.

Incorporation of three endocytosed varicella-zoster virus glycoproteins, gE, gH, and gB, into the virion envelope.

The cytoplasmic tails of all three major varicella-zoster virus (VZV) glycoproteins, gE, gH, and gB, harbor functional tyrosine-based endocytosis motifs that mediate internalization. The aim of the present study was to examine whether endocytosis from the plasma membrane is a cellular route by which VZV glycoproteins are delivered to the final envelopment compartment. In this study, we demonstrated that internalization of the glycoproteins occurred in the first 24 h postinfection but was reduced later in infection.

Regulation of varicella-zoster virus-induced cell-to-cell fusion by the endocytosis-competent glycoproteins gH and gE.

The gH glycoprotein of varicella-zoster virus (VZV) is a major fusogen. The realigned short cytoplasmic tail of gH (18 amino acids) harbors a functional endocytosis motif (YNKI) that mediates internalization in both VZV-infected and transfected cells (T. J.

Viral induced fusion and syncytium formation: measurement by the Kolmogorov-Smirnov statistical test.

Fusion and syncytium formation induced by the expression of fusogenic viral glycoproteins are common cytopathic effects. Numerous laboratories have studied mechanisms of fusion, generally with each laboratory utilizing a different method to measure polykaryote formation. There is little consensus as to which methods are preferred for measuring and comparing fusion.

A functional YNKI motif in the short cytoplasmic tail of varicella-zoster virus glycoprotein gH mediates clathrin-dependent and antibody-independent endocytosis.

The trafficking of varicella-zoster virus (VZV) gH was investigated under both infection and transfection conditions. In initial endocytosis assays performed in infected cells, the three glycoproteins gE, gI, and gB served as positive controls for internalization from the plasma membrane. Subsequently, we discovered that gH in VZV-infected cells was also internalized and followed a similar trafficking pattern.

Identification of the authentic varicella-zoster virus gB (gene 31) initiating methionine overlapping the 3' end of gene 30.

The varicella-zoster virus (VZV) gB sequence was re-examined in light of recent knowledge about unusually long gB signal peptides in other herpesviral gB homologs. Through mutational analysis, the discovery was made that the authentic initiating methionine for VZV gB is a codon beginning at genome nucleotide 56,819. The total length for the VZV gB primary translation product was 931 amino acids (aa) with a 71-aa signal sequence.

Varicella-zoster Virus gB and gE coexpression, but not gB or gE alone, leads to abundant fusion and syncytium formation equivalent to those from gH and gL coexpression.

Varicella-zoster virus (VZV) is distinguished from herpes simplex virus type 1 (HSV-1) by the fact that cell-to-cell fusion and syncytium formation require only gH and gL within a transient-expression system. In the HSV system, four glycoproteins, namely, gH, gL, gB, and gD, are required to induce a similar fusogenic event. VZV lacks a gD homologous protein.

Multimeric humanized varicella-zoster virus antibody fragments to gH neutralize virus while monomeric fragments do not.

Murine monoclonal antibody 206 (MAb mu206) binds to gH, the varicella-zoster virus (VZV) fusogen, neutralizing the virus in vitro in the absence of complement and inhibiting cell-to-cell spread and egress of VZV in cultured cells. We have humanized this antibody to generate MAb hu206 by complementarity determining region grafting. MAb hu206 retained binding and in vitro neutralizing activity, as well as cross-reactivity with ten different VZV strains.