Bruce W. Banfield

Insertions in the gG Gene of Pseudorabies Virus Reduce Expression of the Upstream Us3 Protein and Inhibit Cell-to-Cell Spread of Virus Infection

ABSTRACT The alphaherpesvirus Us4 gene encodes glycoprotein G (gG), which is conserved in most viruses of the alphaherpesvirus subfamily. In the swine pathogen pseudorabies virus (PRV), mutant viruses with internal deletions and insertions in the gG gene have shown no discernible phenotypes. We report that insertions in the gG locus of the attenuated PRV strain Bartha show reduced virulence in vivo and are defective in their ability to spread from cell to cell in a cell-type-specific manner. Similar insertions in the gG locus of the wild-type PRV strain Becker had no effect on the ability of virus infection to spread between cells. Insertions in the gG locus of the virulent NIA-3 strain gave results similar to those found with the Bartha strain. To examine the role of gG in cell-to-cell spread, a nonsense mutation in the gG signal sequence was constructed and crossed into the Bartha strain. This mutant, PRV157, failed to express gG yet had cell-to-cell spread properties indistinguishable from those of the parental Bartha strain. These data indicated that, while insertions in the gG locus result in decreased cell-to-cell spread, the phenotype was not due to loss of gG expression as first predicted. Analysis of gene expression upstream and downstream of gG revealed that expression of the upstream Us3 protein is reduced by insertion of lacZ or egfpat the gG locus. By contrast, expression of the gene immediately downstream of gG, Us6, which encodes glycoprotein gD, was not affected by insertions in gG. These data indicate that DNA insertions in gG have polar effects and suggest that the serine/threonine kinase encoded by the Us3 gene, and not gG, functions in the spread of viral infection between cells.

IL-27 Enhances LPS-Induced Proinflammatory Cytokine Production via Upregulation of TLR4 Expression and Signaling in Human Monocytes

IL-27, which is produced by activated APCs, bridges innate and adaptive immunity by regulating the development of Th cells. Recent evidence supports a role for IL-27 in the activation of monocytic cells in terms of inflammatory responses. Indeed, proinflammatory and anti-inflammatory activities are attributed to IL-27, and IL-27 production itself is modulated by inflammatory agents such as LPS. IL-27 primes LPS responses in monocytes; however, the molecular mechanism behind this phenomenon is not understood. In this study, we demonstrate that IL-27 priming results in enhanced LPS-induced IL-6, TNF-α, MIP-1α, and MIP-1β expression in human primary monocytes. To elucidate the molecular mechanisms responsible for IL-27 priming, we measured levels of CD14 and TLR4 required for LPS binding. We determined that IL-27 upregulates TLR4 in a STAT3- and NF-κB–dependent manner. Immunofluorescence microscopy revealed enhanced membrane expression of TLR4 and more distinct colocalization of CD14 and TLR4 upon IL-27 priming. Furthermore, IL-27 priming enhanced LPS-induced activation of NF-κB family members. To our knowledge, this study is the first to show a role for IL-27 in regulating TLR4 expression and function. This work is significant as it reveals new mechanisms by which IL-27 can enhance proinflammatory responses that can occur during bacterial infections.

Analysis of filamentous process induction and nuclear localization properties of the HSV-2 serine/threonine kinase Us3.

The Us3 serine/threonine kinase encoded by all alphaherpesviruses performs several important functions during virus multiplication. For example, expression of pseudorabies virus (PRV) Us3 causes reorganization of the actin cytoskeleton into filamentous processes (FPs) that promote cell-to-cell spread of virus infection. PRV Us3-induced FP formation requires Us3 kinase activity. To determine whether these characteristics were shared by HSV-2 Us3, expression plasmids for wild type (WT) and kinase dead (KD) Us3 variants were constructed. Expression of WT Us3 resulted in robust FP formation whereas expression of the KD Us3 variants did not. In the course of these experiments we noted that KD/K220 mutant Us3s were excluded from the nucleus in comparison to WT or KD/D305A Us3, prompting us to investigate Us3 nuclear shuttling properties. Herein we describe determinants of HSV-2 Us3-induced FP formation and present evidence for the presence of a leucine-rich nuclear export signal within HSV-2 Us3.

Central sympathetic innervations to visceral and subcutaneous white adipose tissue

There is a link between visceral white adipose tissue (WAT) and the metabolic syndrome in humans, with health improvements produced with small visceral WAT reduction. By contrast, subcutaneous WAT provides a site for lipid storage that is rather innocuous relative to ectopic lipid storage in muscle or liver. The sympathetic nervous system (SNS) is the principal initiator for lipolysis in WAT by mammals. Nothing is known, however, about the central origins of the SNS circuitry innervating the only true visceral WAT in rodents, mesenteric WAT (MWAT), which drains into the hepatic portal vein. We tested whether the central sympathetic circuits to subcutaneous [inguinal WAT (IWAT)] and visceral WAT (MWAT) are separate or shared and whether they possess differential sympathetic drives with food deprivation in Siberian hamsters. Using two isogenic strains of pseudorabies virus, a retrograde transneuronal viral tract tracer within the same hamsters, we found some overlap (∼20-55% doubly infected neurons) between the two circuitries across the neural axis with lesser overlap proximal to the depots (spinal cord and sympathetic chain) and with more neurons involved in the innervation of IWAT than MWAT in some brain regions. Food deprivation triggered a greater sympathetic drive to subcutaneous (IWAT) than visceral (MWAT) depots. Collectively, we demonstrated both shared and separate populations of brain, spinal cord, and sympathetic chain neurons ultimately project to a subcutaneous WAT depot (IWAT) and the only visceral WAT depot in rodents (MWAT). In addition, the lipolytic stimulus of food deprivation only increased SNS drive to subcutaneous fat (IWAT).

Hyperphosphorylation of Histone Deacetylase 2 by Alphaherpesvirus US3 Kinases

ABSTRACT A serine/threonine (S/T) kinase encoded by the US3 gene of herpes simplex virus type 1 (HSV-1) is conserved in varicella-zoster virus (VZV) and pseudorabies virus (PRV). Expression of US3 kinase in cells transformed with US3 expression plasmids or infected with each virus results in hyperphosphorylation of histone deacetylase 2 (HDAC2). Mapping studies revealed that each US3 kinase phosphorylates HDAC2 at the same unique conserved Ser residue in its C terminus. HDAC2 was also hyperphosphorylated in cells infected with PRV lacking US3 kinase, indicating that hyperphosphorylation of HDAC2 by PRV occurs in a US3-independent manner. Specific chemical inhibition of class I HDAC activity increases the plaquing efficiency of VZV and PRV lacking US3 or its enzymatic activity, whereas only minimal effects are observed with wild-type viruses, suggesting that VZV and PRV US3 kinase activities target HDACs to reduce viral genome silencing and allow efficient viral replication. However, no effect was observed for wild-type or US3 null HSV-1. Thus, we have demonstrated that while HDAC2 is a conserved target of alphaherpesvirus US3 kinases, the functional significance of these events is virus specific.

The Attenuated Pseudorabies Virus Strain Bartha Fails To Package the Tegument Proteins Us3 and VP22

ABSTRACT The Bartha strain of pseudorabies virus has several recognized mutations, including a deletion in the unique short region encompassing the glycoprotein I (gI), gE, Us9, and Us2 genes and point mutations in the gC, gM, and UL21 genes. We have determined that Bartha has mutations in the serine/threonine kinase encoded by the Us3 gene relative to the wild-type Becker strain. Our analysis revealed that Becker virions contain the Us3 protein, whereas Bartha virions do not. To test whether the mutations in the Bartha Us3 protein were responsible for this observation, we constructed a recombinant Bartha strain, PRV632, which expresses the Becker Us3 protein. PRV632 failed to package Us3 into the tegument, indicating that mutations other than those in the Us3 primary amino acid sequence were responsible for the failure of Bartha to package its Us3 protein. A recombinant Becker strain, PRV634, which expresses the Bartha Us3 protein, was constructed to test whether it was capable of being packaged into virions. The Bartha Us3 protein was not incorporated into PRV634 virions efficiently, suggesting that the primary sequence of the Bartha Us3 protein affects packaging into the tegument. To determine whether the packaging of other tegument proteins was affected in the Bartha strain, we examined VP22. Whereas Becker packaged VP22 into virions, Bartha had a severe deficiency in VP22 incorporation. Analysis of VP22 expression in Bartha-infected cells revealed that Bartha VP22 had a slower mobility on sodium dodecyl sulfate-polyacrylamide gels, indicating either primary sequence differences and/or different posttranslational modifications relative to Becker VP22. Taken together, these data indicate that, while the primary sequence of the Us3 protein does affect its incorporation into the tegument, other factors are involved. Furthermore, our data suggest that one or more of the gI, gE, Us9, or Us2 genes influences the localization of the Us3 protein in infected cells, and this effect may be important for the proper incorporation of Us3 into virions.

Localization of ERK/MAP Kinase Is Regulated by the Alphaherpesvirus Tegument Protein Us2

ABSTRACT Many different viruses activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase signaling pathway during infection and require ERK activation for the efficient execution of their replication programs. Despite these findings, no virus-encoded proteins have been identified that directly modulate ERK activities. In an effort to determine the function of a conserved alphaherpesvirus structural protein called Us2, we screened a yeast two-hybrid library derived from NIH 3T3 cells and identified ERK as a Us2-interacting protein. Our studies indicate that Us2 binds to ERK in virus-infected cells, mediates the incorporation of ERK into the virion, and inhibits the activation of ERK nuclear substrates. The association of Us2 with ERK leads to the sequestration of ERK at the plasma membrane and to a perinuclear vesicular compartment, thereby keeping ERK out of the nucleus. Us2 can bind to activated ERK, and the data suggest that Us2 does not inhibit ERK enzymatic activity. The treatment of cells with U0126, a specific inhibitor of ERK activation, resulted in a substantial delay in the release of virus from infected cells that was more pronounced with a virus deleted for Us2 than with parental and repaired strains, suggesting that both ERK and Us2 activities are required for efficient virus replication. This study highlights an additional complexity to the activation of ERK by viruses, namely, that localization of active ERK can be altered by virus-encoded proteins.

The Pseudorabies Virus Us2 Protein, a Virion Tegument Component, Is Prenylated in Infected Cells

ABSTRACT The Us2 gene is conserved among alphaherpesviruses, but its function is not known. We demonstrate here that the pseudorabies virus (PRV) Us2 protein is synthesized early after infection and localizes to cytoplasmic vesicles and to the plasma membrane, despite the lack of a recognizable signal sequence or membrane-spanning domain. Us2 protein is also packaged as part of the tegument of mature virions. The Us2 carboxy-terminal four amino acids comprise a CAAX motif, a well-characterized signal for protein prenylation. Treatment of infected cells with lovastatin, a drug that disrupts protein prenylation, changed the relative electrophoretic mobility of Us2 in sodium dodecyl sulfate-polyacrylamide gels. In addition, lovastatin treatment caused a dramatic relocalization of Us2 to cytoplasmic punctate structures associated with microtubules, which appeared to concentrate over the microtubule organizing center. When the CAAX motif was changed to GAAX and the mutant protein was synthesized from an expression plasmid, it concentrated in punctate cytoplasmic structures reminiscent of Us2 localization in infected cells treated with lovastatin. We suggest that prenylation of PRV Us2 protein is required for proper membrane association. Curiously, the Us2 protein isolated from purified virions does not appear to be prenylated. This is the first report to describe the prenylation of an alphaherpesvirus protein.

The Herpes Simplex Virus 2 UL21 Protein Is Essential for Virus Propagation

ABSTRACT Herpes simplex virus 2 (HSV-2) is an important human pathogen that is the major cause of genital herpes infections and a significant contributor to the epidemic spread of human immunodeficiency virus infections. The UL21 gene is conserved throughout the Alphaherpesvirinae subfamily and encodes a tegument protein that is dispensable for HSV-1 and pseudorabies virus replication in cultured cells; however, its precise functions have not been determined. To investigate the role of UL21 in the HSV-2 replicative cycle, we constructed a UL21 deletion virus (HSV-2 ΔUL21) using an HSV-2 bacterial artificial chromosome, pYEbac373. HSV-2 ΔUL21 was unable to direct the production of infectious virus in noncomplementing cells, whereas the repaired HSV-2 ΔUL21 strain grew to wild-type (WT) titers, indicating that UL21 is essential for virus propagation. Cells infected with HSV-2 ΔUL21 demonstrated a 2-h delay in the kinetics of immediate early viral gene expression. However, this delay in gene expression was not responsible for the inability of cells infected with HSV-2 ΔUL21 to produce virus insofar as late viral gene products accumulated to WT levels by 24 h postinfection (hpi). Electron and fluorescence microscopy studies indicated that DNA-containing capsids formed in the nuclei of ΔUL21-infected cells, while significantly reduced numbers of capsids were located in the cytoplasm late in infection. Taken together, these data indicate that HSV-2 UL21 has an early function that facilitates viral gene expression as well as a late essential function that promotes the egress of capsids from the nucleus.

Herpes Simplex Virus 2 Infection Impacts Stress Granule Accumulation

ABSTRACT Interference with stress granule (SG) accumulation is gaining increased appreciation as a common strategy used by diverse viruses to facilitate their replication and to cope with translational arrest. Here, we examined the impact of infection by herpes simplex virus 2 (HSV-2) on SG accumulation by monitoring the localization of the SG components T cell internal antigen 1 (TIA-1), Ras-GTPase-activating SH3-domain-binding protein (G3BP), and poly(A)-binding protein (PABP). Our results indicate that SGs do not accumulate in HSV-2-infected cells and that HSV-2 can interfere with arsenite-induced SG accumulation early after infection. Surprisingly, SG accumulation was inhibited despite increased phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), implying that HSV-2 encodes previously unrecognized activities designed to maintain translation initiation downstream of eIF2α. SG accumulation was not inhibited in HSV-2-infected cells treated with pateamine A, an inducer that works independently of eIF2α phosphorylation. The SGs that accumulated following pateamine A treatment of infected cells contained G3BP and PABP but were largely devoid of TIA-1. We also identified novel nuclear structures containing TIA-1 that form late in infection. These structures contain the RNA binding protein 68-kDa Src-associated in mitosis (Sam68) and were noticeably absent in infected cells treated with inhibitors of viral DNA replication, suggesting that they arise as a result of late events in the virus replicative cycle.