Nuclear IE2 structures are related to viral DNA replication sites during baculovirus infection

The ie2 gene of Autographa californica multicapsid nuclear polyhedrosis virus is I of the 10 baculovirus genes that have been identified as factors involved in viral DNA replication. IE2 is detectable in the nucleus as one of the major early- expressed proteins and exhibits a dynamic localization pattern during the infection cycle (D. Murges, 1. Quadt, J. Schroer, and D. Knebel-Morsdorf, Exp. Cell Res. 264:219-232, 2001). Here, we investigated whether IE2 localized to regions of viral DNA replication. After viral DNA was labeled with bromode- oxyuridine (BrdU), confocal imaging indicated that defined IE2 domains colocalized with viral DNA replication centers as soon as viral DNA replication was detectable. In addition, a subpopulation of IE2 structures colocalized with two further virus-encoded replication factors, late expression factor 3 (LEF-3) and the DNA binding protein (DBP). While DBP and LEF-3 structures always colocalized and enlarged simultaneously with viral DNA replication sites, only those IE2 structures that colocalized with replication sites also colocalized with DBP. Replication and transcription of DNA viruses in association with promyelocytic leukemia protein (PML) oncogenic domains have been observed. By confocal imaging we demonstrated that the human PML colocalized with IE2. Triple staining revealed PML/IE2 domains in the vicinity of viral DNA replication centers, while IE2 alone colocalized with early replication sites, demonstrating that PML structures do not form common domains with viral DNA replication centers. Thus, we conclude that IE2 colocalizes alternately with PML and the sites of viral DNA replication. Small ubiquitin-like modifier SUMO-1 has been implicated in the nuclear distribution of PML. Similar to what was found for mammalian cells, small ubiquitin-like modifiers were recruited to PML domains in infected insect cells, which suggests that IE2 and PML colocalize in conserved cellular domains. In summary, our results support a model for IE2 as part of various functional sites in the nucleus that are connected with viral DNA replication

<em>Ex Vivo</em> Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice

Expression of Baculovirus Late and Very Late Genes Depends on LEF-4, a Component of the Viral RNA Polymerase Whose Guanyltransferase Function Is Essential

Baculovirus lef-4 encodes one subunit of the viral RNA polymerase. Here, we demonstrate the essential nature of LEF-4 by RNA interference and bacmid knockout technology. Silencing of LEF-4 in wild-type virus-infected cells suppressed expression of structural genes, while early expression was unaffected, demonstrating its essential role in late gene expression. After transfection of insect cells with lef-4 mutant bacmid, no viral progeny was produced, further defining its central role in infection. Cotransfection with wild-type lef-4 plasmid restored normal replication, but plasmid encoding a guanyltransferase-deficient version failed to rescue. These results emphasize the importance of the mRNA capping function of LEF-4

HSV-1 infection of murine epidermis pretreated with dynasore.

<p>Epidermal sheets prepared from newborn mouse skin were separated from dermis by dispase II treatment, followed by incubation on 40 or 120 µM dynasore-containing medium or DMSO-containing medium. After 1 h at 37°C HSV-1 was added at 100 PFU/cell. At 3 h p.i. epidermal whole mounts showing the basal keratinocyte layer and developing hair follicles were costained with mouse anti-ICP0 (red) and rabbit anti-keratin 14 (green) visualized with AF555-conjugated anti-mouse (Molecular Probes) and AF488-conjugated anti-rabbit (Molecular Probes), respectively. Single immunofluorescence analyses are shown. Bar, 80 µm.</p

Uptake of HSV-1 into human keratinocytes.

<p>(A) HaCaT cells or (B) primary human keratinocytes (phK) were incubated with HSV-1 at 200 PFU/cell (A, a, b), or 1500 PFU/cell (A, c, d; B, a–d) for 1 h at 4°C to allow attachment followed by incubation at 37°C to allow uptake. Cells were fixed and prepared for electron microscopy at 2, 10, or 30 min p.i. (A, B) Particles on the cell surface (a), free cytoplasmic capsids (b, c) and enveloped particles in vesicles (d) are shown at 2 and 10 min p.i. Bar, 0.2 µm. (C) In two independent experiments 133, 138, and 109 particles in total were evaluated at 2, 10, and 30 min, respectively, in HaCaT cells, and 159 (at 2 min) and 254 (at 10 min) were analyzed in primary human keratinocytes. The percentages of particles on the surface, cytoplasmic capsids and enveloped particles in vesicles are given at 2, 10, and 30 min p.i. Results are mean ± standard deviation values. In primary keratinocytes 58 particles in total were analyzed in one experiment at 30 min p.i.</p