The Equine Herpesvirus 1 UL20 Product Interacts with Glycoprotein K and Promotes Egress of Mature Particles

The Equine Herpesvirus 1 UL20 Product Interacts with Glycoprotein K and Promotes Egress of Mature Particles

The sole immediate-early (IE) gene of equine herpesvirus 1 encodes a 1,487-amino-acid (aa) regulatory phosphoprotein that independently activates expression of early viral genes. F., H. Mutations of vmw65 which abolish IE promoter transactivating activity may also be included to reduce IE gene expression generally. The genes are arranged collinearly with those in the genomes of the two previously sequenced alphaherpesviruses, varicella-zoster virus, and herpes simplex virus type-1, and comparisons of predicted amino acid sequences allowed the functions of many equine herpesvirus 1 proteins to be assigned. These abortions usually occur between the 7thand 11thmonth of gestation. Reactivation of the virus usually results in shedding of infectious virus without clinical signs. Virulence of Ab4-GFP in both mice and hamsters was weaker than that of the wild type.

Analysis of a UL20 deletion mutant of EHV-1 strain RacL11 indicated an involvement of UL20p in cell-to-cell spread, as well as in very late events in virus egress. Based on these and electron microscopic studies we suggest that the EHV-1 UL20 protein might be necessary to avoid fusion of mature virions with membranes of their transport vesicles. Alphaherpesviruses express relatively few proteins with the potential to span membranes several times. Accordingly, only four such open reading frames (ORFs) have been identified in the EHV-1 sequence, and they are homologous to the herpes simplex virus type 1 (HSV-1) ORFs UL10, UL20, UL43, and UL53 (31). A potent transcriptional activation domain lies within the first 89 aa residues of the IE protein (58), and aa 963 to 970 are necessary for nuclear localization of truncated IE polypeptides (57). Glycoproteins are crucially involved in these early stages of infection, and 11 glycoproteins in the prototype member of the Alphaherpesvirinae subfamily, Herpes simplex virus type 1 (HSV-1), have been identified. In the case of these viruses, gM and the UL49.5 product were shown to physically interact with each other by virtue of disulfide bonds that are probably formed between the first cysteine residue of the UL10 polypeptide, which is conserved throughout the UL10 homologs of all Herpesviridae sequenced to date, and one of the two equally highly conserved cysteine residues present within the UL49.5 homologs (31).

EHV-1 gK and gM share additional characteristics such as N glycosylation, early-late expression kinetics, and the fact that they both depend on a viral complex partner for complete processing (20, 25). In the present study we sought to characterize the UL20 product to further investigate the specific roles of multiply hydrophobic proteins in EHV-1 replication and to address the question whether the special structural features might be related to common functional properties. Region 5 contains a transcriptional-enhancement domain that is required for the full transactivation activity of the IE protein (5, 56). W. Such reductions in IE gene expression minimize transcription from the vast majority of the 80 or so other genes in the HSV genome. A UL20-negative PRV displayed a marked reduction in plaque sizes and replicated to decreased virus titers. Interestingly, the extent of these defects varied with the cell line used and was especially prominent on Vero cells.

The initial presenting signs include fever, malaise, lethargy, inappetance, enlarged lymph nodes, pharyngitis, nasal discharge and coughing. Similarly, HSV-1 UL20p is important for final stages of virus replication (3, 8). However, whereas the UL20 product of PRV is suggested to support intracellular transport of mature virions to the cell surface (10), the HSV-1 UL20 protein is proposed to regulate membrane fusion involved in virion assembly (8). HSV-1 UL20p is thought to either support secondary envelopment or to prevent another putative de-envelopment step, i.e., the fusion of viral envelopes with membranes of their transport vesicles or with viral envelopes of other mature virions within these vesicles. These conclusions were drawn because the number of unenveloped capsids increased within the cytoplasm of Vero cells infected with various UL20 deletion mutants, and relatively few enveloped virions were observed. Also, deleting UL20 from HSV-1 syncytial strains resulted in accumulation of unenveloped particles in vesicles surrounded by two membranes (8). TFIIB interacts with TBP and Pol II and is responsible for the recruitment of Pol II to the complex (23).

The virus expressing EGFP lacks the glycoprotein gp2 gene (gene 71) and was termed HΔgp2 (49). Consistent with the absence of mature gM after infection of cells with the UL49.5-negative virus, a small-plaque phenotype and a 190-fold reduction of extracellular virus titers were observed in the absence of mature gM. Furthermore, the HSV-1 proteins have been shown to be interdependent for intracellular localization (6, 7, 9). Nevertheless, a putative physical interaction has not yet been proven, and the functional relevance of these findings is not yet understood. The PIC contains multiple components of the cellular transcriptional machinery, including RNA polymerase II (Pol II) and general transcription factors (GTFs), and can be formed on Pol II promoters in a sequential order from the individual assembly of the GTFs, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, TFIIJ, and Pol II (4, 26). Tokyo2, 117–127 (1962). The EHV-1 vmw65 homologue (gene 12) (29) has previously been shown by cotransfection experiments to be capable of transactivating HSV IE promoters (29), suggesting that the approach is possible.

Plasmids.Plasmid ptL20 contains a 2.8-kb PstI fragment of EHV-1 strain RacL11 within vector pTZ18R and encompasses UL20 encoding, as well as upstream and downstream sequences (Fig. 1B). If horses are returned to training too quickly, reactivation of the latent virus may occur. The respective primer sequences (5′-ATAGAATTCATGCCACAGGTATTAATG-3′,5′-ATATCTAGATGGGCAGAA AATGATGGG-3′, 5′-ATGGATCCAAGCTACGCTTAACGGAG-3′) included restriction enzyme recognition sites for insertion into either vector pBADgIIIB (pBAD20t; Invitrogen) or vector pcDNAI/Amp (pc20; Invitrogen). Correct amplification was confirmed by custom sequence analysis (MWG Biotech). Plasmid ptDp carries the 6.6-kb PstI-fragment of RacL11 DNA containing gene 71 and adjacent sequences (vector pTZ18R). Plasmids pcgM, pcgK, and pcgKGFP were designed to express the respective proteins and have been described elsewhere (20, 23).

The UL20-negative virus, L11Δ20, was constructed by mutagenesis of the EHV-1 BAC pRacL11 (24). J. ). A 1.6-kbp fragment on the 3′ side of the UL49.5 ORF was amplified by PCR (Table 1) and cloned as a KpnI fragment in the vector containing the 5′ fragment. Binding sites of primers used for control sequencing are given. (E) A Kyte-Doolittle hydrophilicity plot (husar; GCG Software Package, Heidelberg, Germany) of the predicted amino acid sequence of EHV-1 UL20 (31) is shown. In addition, we have mapped regions of the IE protein responsible for the IE-TFIIB interaction.

Cells and viruses.Rabbit kidney cell line Rk13 was maintained as previously described (18). HSV strains 17+/27-w and 1764/27-w were prepared by removing the lacZ insertion from the ICP27 gene in virus strains 17+/27−/pR20 and 1764/27−/pR20 (see below), respectively, by recombination with empty ICP27 flanking regions and selection of non-5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal)-staining recombinant plaques using B130/2 cells which complement ICP27 (19). UL20p expression of Geneticin-resistant cell clones was confirmed by immunofluorescence analysis using the UL20p-specific antiserum. EHV-1 strains RacL11 and RacH (11), including the recombinant viruses generated in the present study, were propagated on Rk13 or SC20 cells as indicated in the text. EHV-1 mutants deleted in gM (HΔgM-GFP), gK (HΔgK), or gp2 (L11Δgp2) have been described previously (20, 24, 29). Generation of recombinant viruses.The complete UL20 sequence was deleted in the bacterial artificial chromosome (BAC) clone of EHV-1 strain RacL11 (pRacL11 [24]) by using Red mutagenesis as described by Datsenko and Wanner (4). A linear DNA fragment was generated by PCR amplification of the kanamycin resistance (kanr) gene and flanking FRT-sites (FLP recognition target) of plasmid pKD13 (4) (Fig.
The Equine Herpesvirus 1 UL20 Product Interacts with Glycoprotein K and Promotes Egress of Mature Particles

1B). Besides kanr recognition sequences, the respective primers included sequences complementary to those up- and downstream of UL20 (primer 1, 5′-GAC-TAG-TCC-ATA-CCC-TGC-ACC-GCT-CGC-AGG-CTG-CCA-GAA-ATA-TTT-CTC-TCC-GAA-TTT-TTG-AGG-GTT-GGA- GGT-GTA-GGC-TGG-AGC-TGC-TTC-3′; primer 2, 5′-TCT-CTT-CTC-ACG-TTT-TAT-ACT-GTC-AGT-CGG-CCG-CTA-CCC-CAG-TGG-TGC-CTC- CCA-CGG-AGG-CAC-CCC-TAA-TTC-CGG-GGA-TCC-GTC-GAC-CT-3′). After transformation of the PCR product into E. Plasmid pGEX-IE was digested with EagI and self-ligated to generate the pGEX-IEΔ644/824. Samples were adjusted to 1 mg/ml protein (BCA protein assay; Pierce) and mixed with buffer containing 5% 2-mercaptoethanol (38). For each combination of virus and cells, 150 plaques were measured and the average plaque diameters were determined. The final UL20 negative virus, L11Δ20, was plaque purified after cotransfection into SC20 cells DNA of pRacL11Δ20 and of plasmid ptDp, which contains the RacL11 sequences necessary to replace the mini F plasmid pHA2 and to repair gene 71 (24).

The UL20-negative but gp2-positive (gene 71 repaired) L11Δ20 virus was used for generating a UL20 revertant virus, L11Δ20R, by homologous recombination of L11Δ20 with plasmid ptL20 in Rk13 cells. (i) Human TFIIB expression plasmids.The full-length TFIIB gene from pM270 was amplified by PCR, using the 5′ primer hIIB#F1 and the 3′ primer hIIB#R1, which contained SmaI andBamHI restriction sites, respectively, and was cloned in-frame into the same sites of pGBKT7 yeast two-hybrid vector (Clontech) to obtain pGBKhIIB. Western blotting.Cells were infected at a multiplicity of infection (MOI) of 2 (unless stated otherwise) with the respective viruses and lysed at the indicated times postinfection (p.i.). For mouse mammary tumor virus (MMTV) promoter control, the neomycin resistance gene (excised as a BamHI fragment) in plasmid pMAMneo (Invitrogen) was replaced with the phleomycin resistance gene as described above, again as a BamHI fragment. Proteins were separated by SDS-12% polyacrylamide gel electrophoresis (PAGE) and detected by Western blotting as previously described (22). Apparent Mrs were exactly calculated in relation to a precision protein standard (Bio-Rad), but in most experiments another protein marker (Biolabs) was used running slightly different. Extracellular virions were purified from supernatants of infected Rk13 cells (MOI = 2; 16 h p.i.) by repeated centrifugation through a 30% sucrose cushion (22).

Purified virions were split into two fractions by incubation at 45°C in buffer containing Triton X-100 (1%; 20 min) and pelleting of capsids and attached proteins at 45,000 × g for 30 min. Supernatants that contained envelopes and solubilized tegument proteins were carefully separated from pellets. After another washing step, the pellet was resuspended in lysis buffer. To remove N-linked carbohydrates, virion preparations (5 μg) were incubated in the presence or absence of 1 U of PNGase-F (16 h, 37°C; Roche Molecular Biochemicals) in deglycosylation buffer (100 μl [13]). O-linked carbohydrates were digested after removal of sialic acid side chains. To generate pSVEICP0(1-164), pSVICP0K was digested with SstI and then self-ligated. Seven days after the third immunization, spleens were recovered.

or after transfection, and IIF and confocal laser scanning microscopy using a Zeiss LSM510 confocal microscope were done exactly as described previously (19, 23, 27). At the stated times postinfection or transfection cells were fixed with 2% paraformaldehyde (15 min), permeabilized with Triton X-100 (0.1%; 10 min) unless stated otherwise, and processed by standard procedures (22). Concanavalin A (ConA) directly labeled with Alexa Fluor 594 was used to track the endoplasmic reticulum (2 μg/ml, 30 min). First, an 8-bp NcoI linker (New England Biolabs) was inserted into the blunt-ended RsrII site of the 7.09-kbp fragment derived from RsrII-digested pG3IE (1–1487) to obtain the pG3IENΔ621–757 intermediate. Pictures were adjusted in Adobe Photoshop 7.0, and figures were assembled by using CorelDraw 7. Percent conversion of the substrate to the acetylated form is shown. coli as a mycHis fusion protein using the pBADgIII expression system (Invitrogen) and purified by affinity chromatography according to the manufacturer’s instructions.

Two rabbits were immunized five times in 3-week intervals with 50 to 100 μg of the purified protein mixed with Freund adjuvant. The monoclonal anti-major capsid protein antibody ZB4 (kindly provided by G. B. Caughman), anti-gB monoclonal antibody (MAb) 3F6 (1), anti-gB MAb 4B6 (17), anti-gM MAb F6 (5), and anti-gp2 MAb 3B12 (17), as well as rabbit polyclonal antisera directed against gM (28), UL34p (19), and UL45p (22), were used in the present study. Antibodies directed against green fluorescent protein (GFP; rabbit serum) and Alexa Fluor 546- or 488-conjugated anti-rabbit or anti-mouse immunoglobulin G (IgG) secondary antibodies were purchased from Molecular Probes; antibodies to γ-adaptin (clone 100/3) and peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies were from Sigma. Virus growth kinetics, penetration assays, and plaque size measurements.Rk13 or SC20 cells in six-well plates were infected with the indicated viruses (MOI = 5). After a 60-min period of adsorption at 4°C, inocula were removed, and attached viruses were allowed to penetrate for another 60 min at 37°C, followed by inactivation of remaining extracellular virus with a citrate buffer (40 mM citric acid, 10 mM KCl, 135 mM NaCl; pH 3.0).

(iv) Human GST-TFIIB deletion and GST-TBP mutants.To facilitate the mapping of the domain of TFIIB that mediates its interaction with the EICP0 protein, a panel of GST-TFIIB deletion mutants was constructed (Fig. Controls were equine or murine sera containing anti-EHV-1 antibodies (42). After 16 h of incubation at 37°C, lysates were separated by SDS-10% PAGE, transferred to nitrocellulose, and examined by Western blotting. The efficiency of penetration was determined by the number of plaques present after citrate buffer treatment relative to the number of plaques present after control treatment (phosphate-buffered saline) at various times after the temperature was shifted from 4°C (adsorption) to 37°C. Sizes of the virus plaques were examined on Rk13 or SC20 cells (50 PFU/well) at day 3 under a methocellulose overlay. The resultant plasmid, pTriExIE (1–1487), encodes a tagged protein in the mammalian L-M cells and was used in this study. Representative plaques were immunofluorescently labeled for gB and digitally documented by using a Zeiss Axioskop.

Next, cell lines were cloned out after transfection with only the ICP27 gene-containing plasmid or the ICP27 gene-containing plasmid together with the EHV-1 gene 12-containing plasmid. as previously described (19). Briefly, cells were fixed in 5% glutaraldehyde and 4% formaldehyde (pH 7.4, 2 h) and then washed with 0.1 M sodium phosphate buffer (Ph 7.4) before they were postfixed with 1% OsO4-0.8% K3Fe(CN)6 in 0.1 M sodium phosphate buffer (2 h). All samples were stained with 2% aqueous uranyl acetate for 90 min, dehydrated in graded ethanol, and finally embedded in ERL 4206. Thin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope EM 10C/CR (Zeiss, Oberkochen, Germany) at 60 kV. Pictures were digitally scanned and assembled by using Adobe Photoshop 7.0. Construction of an EHV-1 UL20 deletion mutant.To investigate the function of the UL20 product in EHV-1 replication, a UL20-deleted RacL11 was generated by using BAC-mutagenesis.

In a first step, the complete ORF was removed from the pRacL11 BAC clone in E. coli (24), resulting in recombinant clone pRacL11Δ20 (Fig. Plasmid pGSTKG-hIIBaa174N was subsequently digested with NcoI and religated to generate pGST-TFIIBΔ125-174. ). Western blot analyses of RK13, RK49.5, and RKgM cells transfected with pc49.5, pcgM, or pcgB. To, in a second step, reconstitute in eukaryotic cells a virus carrying the UL20-deletion only, UL20p-expressing SC20 cells were cotransfected with DNA of pRacL11Δ20 and of plasmid ptDp. GFP-negative progeny virus was purified to homogeneity, and gp2 expression of the isolated L11Δ20 was confirmed by immunofluorescence analysis with a gp2-specific antibody (17; data not shown).

Experiments involving the in vitro-synthesized proteins were performed in parallel in the presence or absence (competition assays) of 35S-labeled proteins. The generated recombinant viruses were genetically characterized as explained in Materials and Methods to confirm their genotypes and to exclude spurious modifications (data not shown) before they were functionally assessed in the course of the present study.

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The Equine Herpesvirus 1 UL20 Product Interacts with Glycoprotein K and Promotes Egress of Mature Particles

The Equine Herpesvirus 1 UL20 Product Interacts with Glycoprotein K and Promotes Egress of Mature Particles

Bovine herpesvirus type 5 (BHV-5) is an alphaherpesvirus associated with meningoencephalitis, a disease highly prevalent in South America. Particular emphasis is given to the epidemiology, prevention and control of infectious diseases. An LR mutant virus with stop codons at the amino terminus of ORF2 does not reactivate from latency after treatment with the synthetic corticosteroid dexamethasone, in part because it induces higher levels of apoptosis during the establishment of latency. The UL7 open reading frame encodes a protein of 300 amino acids with a calculated molecular mass of 32 kDa. Out of 32 flocks investigated 10 were found positive for ILTV infection by PCR. Neuronal cell cycle progression or inappropriate expression of cyclin A leads to apoptosis, suggesting that a viral factor inhibits the deleterious effects of cyclin A expression. Additionally, the virus was detected in a single prematurely born pup, suggesting the possibility of perinatal transmission.

Caughman, D. Careful analyses of these recombinants has allowed a better understanding of the molecular mechanisms underlying recombination (49). Alphaherpesviruses express relatively few proteins with the potential to span membranes several times. Genotyping identified 3 unique sequences clustered in the A clade. They were euthanized, respectively, at 4 (goat 2) and 6 (goats 3 and 4) days following the DMS treatment, and their ganglia were immediately collected. The simplest is the use of KHSV-infected saliva for hygiene, particularly in water-shortage regions4 or for soothing insect bites.14 The second is the alteration of cellular immunity, as induced by insect bites or particularly by parasites,9 that may increase susceptible target cells, hamper effective responses, or increase KSHV shedding in close contacts. Prior to DNA extraction, PBMC or BCP-1 cells were washed with phosphate-buffered saline and stored at −70°C as a dry pellet of 3 × 106 cells.

EHV-1 gK and gM share additional characteristics such as N glycosylation, early-late expression kinetics, and the fact that they both depend on a viral complex partner for complete processing (20, 25). In the present study we sought to characterize the UL20 product to further investigate the specific roles of multiply hydrophobic proteins in EHV-1 replication and to address the question whether the special structural features might be related to common functional properties. In contrast to the EHV-1 UL20 protein, which has not been studied before, the respective homologs of HSV-1 and pseudorabies virus (PRV) are well characterized. The structure of the PRV UL20p is still unknown, since no specific antibodies are available, but the HSV-1 protein is an unglycosylated, early-late structural protein which localizes to nuclear membranes and the Golgi apparatus (9, 32). A recombinant virus, PRV-GS443, carrying the fusion allele in place of the wild-type UL35 gene, was made by using the pBecker3 infectious Escherichia coli clone (5). A UL20-negative PRV displayed a marked reduction in plaque sizes and replicated to decreased virus titers. Interestingly, the extent of these defects varied with the cell line used and was especially prominent on Vero cells.

Electron microscopy of again Vero cells infected in the absence of UL20p revealed the accumulation of complete, enveloped particles in cytoplasmic vesicles, whereas all other stages of replication appeared normal (10). Similarly, HSV-1 UL20p is important for final stages of virus replication (3, 8). However, whereas the UL20 product of PRV is suggested to support intracellular transport of mature virions to the cell surface (10), the HSV-1 UL20 protein is proposed to regulate membrane fusion involved in virion assembly (8). HSV-1 UL20p is thought to either support secondary envelopment or to prevent another putative de-envelopment step, i.e., the fusion of viral envelopes with membranes of their transport vesicles or with viral envelopes of other mature virions within these vesicles. These conclusions were drawn because the number of unenveloped capsids increased within the cytoplasm of Vero cells infected with various UL20 deletion mutants, and relatively few enveloped virions were observed. Also, deleting UL20 from HSV-1 syncytial strains resulted in accumulation of unenveloped particles in vesicles surrounded by two membranes (8). Taken together, both the PRV and HSV UL20 homologs function in a late step of virus egress in Vero cells, but the precise mechanisms seem to differ.

The primary aim of the present study was to assess whether UL20p might play a role in EHV-1 egress and whether a putative overlap in function with other multiply hydrophobic proteins like gK or gM would be detectable. An interaction of UL20p with gK was likely because (i) processing and localization of EHV-1 gK has been previously demonstrated to depend on the presence of (an)other as-yet-unidentified viral component(s) (20) and (ii) expression of the respective UL20 proteins is necessary for complete processing of gK in HSV-1 and PRV. Furthermore, the HSV-1 proteins have been shown to be interdependent for intracellular localization (6, 7, 9). Nevertheless, a putative physical interaction has not yet been proven, and the functional relevance of these findings is not yet understood. To determine the precise function of the UL20 homolog in EHV-1 replication in Rk13 cells, an antiserum was raised allowing direct identification and in-depth analysis of the UL20 gene product. Physical and functional interactions were investigated by Western blot analysis and confocal laser scanning microscopy, and the presence of a stable complex dependent on gK and UL20p expression for formation and positive for UL20p and gK components was demonstrated. Interestingly, the defect of UL20 negative EHV-1 seems to affect even a later step in virus replication than that of the respective gK deleted mutant, suggesting that UL20p functions not only in complex with gK but also independently of the glycoprotein.

Plasmid ptL20 contains a 2.8-kb PstI fragment of EHV-1 strain RacL11 within vector pTZ18R and encompasses UL20 encoding, as well as upstream and downstream sequences (Fig. ). To either express a truncated UL20-mycHIS fusion protein in Escherichia coli or the full-length UL20 polypeptide in eukaryotic cells, either bp 1 to 208 or the complete 719-bp UL20 ORF (gene 41 [31]) was amplified by PCR. The respective primer sequences (5′-ATAGAATTCATGCCACAGGTATTAATG-3′,5′-ATATCTAGATGGGCAGAA AATGATGGG-3′, 5′-ATGGATCCAAGCTACGCTTAACGGAG-3′) included restriction enzyme recognition sites for insertion into either vector pBADgIIIB (pBAD20t; Invitrogen) or vector pcDNAI/Amp (pc20; Invitrogen). Correct amplification was confirmed by custom sequence analysis (MWG Biotech). Plasmid ptDp carries the 6.6-kb PstI-fragment of RacL11 DNA containing gene 71 and adjacent sequences (vector pTZ18R). Plasmids pcgM, pcgK, and pcgKGFP were designed to express the respective proteins and have been described elsewhere (20, 23).

The UL20-negative virus, L11Δ20, was constructed by mutagenesis of the EHV-1 BAC pRacL11 (24). (A) The BamHI map of pRacL11 is illustrated, and sequences of mini F plasmid pHA2 are highlighted as a black box. (B) The position of the UL20 ORF (gene … Rabbit kidney cell line Rk13 was maintained as previously described (18). E. UL20p expression of Geneticin-resistant cell clones was confirmed by immunofluorescence analysis using the UL20p-specific antiserum. EHV-1 strains RacL11 and RacH (11), including the recombinant viruses generated in the present study, were propagated on Rk13 or SC20 cells as indicated in the text.

The Equine Herpesvirus 1 UL20 Product Interacts with Glycoprotein K and Promotes Egress of Mature Particles
EHV-1 mutants deleted in gM (HΔgM-GFP), gK (HΔgK), or gp2 (L11Δgp2) have been described previously (20, 24, 29). Donors had been examined and excluded if they had a predisposition to certain inheritable diseases (http://dk.cryosinternational.com/clinics/screening.aspx), previous known genital infections, current infection of C. The spectrophotometer absorbance at 570 nm was determined. At the indicated times, RNA was prepared and analyzed by probing a Northern blot with a single-stranded oligonucleotide complementary to the 3.6-kb ORF50 mRNA. Here we describe the establishment and characterization of gD:R222N/F223I and gD:A3C/Y38C mutant viruses. After transformation of the PCR product into E. coli cells harboring pRacL11, colonies containing an insertion of the kanr gene were selected on kanamycin-containing agar plates.

The inserted kanr sequences were then removed by conditionally expressing the FLP recombinase in these cells, and a UL20-deleted pRacL11, pRacL11Δ20, resulted that carried an insertion of 82 bp only (Fig. Shellfish Aquaculture in the Irish Sea – detection and prevention of diseases in Crassostrea gigas. The final UL20 negative virus, L11Δ20, was plaque purified after cotransfection into SC20 cells DNA of pRacL11Δ20 and of plasmid ptDp, which contains the RacL11 sequences necessary to replace the mini F plasmid pHA2 and to repair gene 71 (24). The UL20-negative but gp2-positive (gene 71 repaired) L11Δ20 virus was used for generating a UL20 revertant virus, L11Δ20R, by homologous recombination of L11Δ20 with plasmid ptL20 in Rk13 cells. The inoculated and control groups were kept in separated areas. Cells were infected at a multiplicity of infection (MOI) of 2 (unless stated otherwise) with the respective viruses and lysed at the indicated times postinfection (p.i.). Forty-eight hours after transfection, cells were collected, washed once with phosphate-buffered saline (PBS), and suspended in lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Triton X-100) with protease inhibitors (Roche).

Proteins were separated by SDS-12% polyacrylamide gel electrophoresis (PAGE) and detected by Western blotting as previously described (22). The percent identity matrix calculated for Indian ILT viruses shared 99.8 % homology with chicken embryo origin (CEO) vaccine strains of Italy, USA and China, and 98.4 % with Brazilian strain. Extracellular virions were purified from supernatants of infected Rk13 cells (MOI = 2; 16 h p.i.) by repeated centrifugation through a 30% sucrose cushion (22). Purified virions were split into two fractions by incubation at 45°C in buffer containing Triton X-100 (1%; 20 min) and pelleting of capsids and attached proteins at 45,000 × g for 30 min. Supernatants that contained envelopes and solubilized tegument proteins were carefully separated from pellets. MAb 3402 is a mouse IgG2a, used at a dilution of 1/10,000, and was detected by using FITC-conjugated goat anti-mouse IgG2a antibodies (FITC-GAM-IgG2a; Imtech). To remove N-linked carbohydrates, virion preparations (5 μg) were incubated in the presence or absence of 1 U of PNGase-F (16 h, 37°C; Roche Molecular Biochemicals) in deglycosylation buffer (100 μl [13]).

After incubation at 37°C for 2 h, the plates were washed again five times, and a substrate solution containing 1 mg/ml of para-nitrophenylphosphate in 10% diethanolamine, pH 9.8, at 50 μl per well was added. To do this, virions (5 μg) were first incubated with 1 U of neuraminidase (1 h, 37°C; Roche Molecular Biochemicals) in a buffer (100 μl) containing sodium acetate (pH 5.2, 50 mM) and calcium chloride (4 mM) and then pelleted and suspended in a Tris-phosphate buffer (20 mM, pH 7.4, 100 μl). These preparations were then incubated in the presence or absence of O-glycosidase (2 U, 16 h, 37°C; Roche Molecular Biochemicals). For a single PBMC specimen, fluorescence exceeded background in cycle 37 of PCR amplification, but the value of CT corresponded to a KSHV DNA level of 0.2 copies/2 μg of PBMC DNA (a value below the theoretical minimum level of detection of 1 copy/2 μg). At the stated times postinfection or transfection cells were fixed with 2% paraformaldehyde (15 min), permeabilized with Triton X-100 (0.1%; 10 min) unless stated otherwise, and processed by standard procedures (22). Concanavalin A (ConA) directly labeled with Alexa Fluor 594 was used to track the endoplasmic reticulum (2 μg/ml, 30 min). Fluorescence of samples was digitally documented by using either conventional fluorescence microscopy (Zeiss Axioskop; Spot Advanced Software, Diagnostic Instruments) or an LSM510 laser scanning microscope (LSM510 Software; Zeiss).

Pictures were adjusted in Adobe Photoshop 7.0, and figures were assembled by using CorelDraw 7. Capsids moving in the retrograde direction were less common than those moving in the anterograde direction, with a ratio of anterograde-to-retrograde-moving capsids of approximately 7:1. coli as a mycHis fusion protein using the pBADgIII expression system (Invitrogen) and purified by affinity chromatography according to the manufacturer’s instructions. Two rabbits were immunized five times in 3-week intervals with 50 to 100 μg of the purified protein mixed with Freund adjuvant. The monoclonal anti-major capsid protein antibody ZB4 (kindly provided by G. B. Caughman), anti-gB monoclonal antibody (MAb) 3F6 (1), anti-gB MAb 4B6 (17), anti-gM MAb F6 (5), and anti-gp2 MAb 3B12 (17), as well as rabbit polyclonal antisera directed against gM (28), UL34p (19), and UL45p (22), were used in the present study.

Antibodies directed against green fluorescent protein (GFP; rabbit serum) and Alexa Fluor 546- or 488-conjugated anti-rabbit or anti-mouse immunoglobulin G (IgG) secondary antibodies were purchased from Molecular Probes; antibodies to γ-adaptin (clone 100/3) and peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies were from Sigma. Rk13 or SC20 cells in six-well plates were infected with the indicated viruses (MOI = 5). After a 60-min period of adsorption at 4°C, inocula were removed, and attached viruses were allowed to penetrate for another 60 min at 37°C, followed by inactivation of remaining extracellular virus with a citrate buffer (40 mM citric acid, 10 mM KCl, 135 mM NaCl; pH 3.0). At the indicated time points p.i., supernatants and infected cells, which were again treated with low pH to achieve inactivation of contaminating extracellular infectivity, were collected separately. Virus titers were determined independently by plaque titration on Rk13 or SC20 cells. Penetration assays were performed as previously described (18). The efficiency of penetration was determined by the number of plaques present after citrate buffer treatment relative to the number of plaques present after control treatment (phosphate-buffered saline) at various times after the temperature was shifted from 4°C (adsorption) to 37°C.

Sizes of the virus plaques were examined on Rk13 or SC20 cells (50 PFU/well) at day 3 under a methocellulose overlay. Plaque areas were measured by using the SPOTadvanced software (Diagnostic instruments), and mean areas of respective plaques were set in relation to mean areas of parental RacL11 plaques (100%). Representative plaques were immunofluorescently labeled for gB and digitally documented by using a Zeiss Axioskop. Rk13 cells were infected with the indicated viruses (MOI of 2) and prepared for electron microscopic investigations at 16 h p.i. as previously described (19). Briefly, cells were fixed in 5% glutaraldehyde and 4% formaldehyde (pH 7.4, 2 h) and then washed with 0.1 M sodium phosphate buffer (Ph 7.4) before they were postfixed with 1% OsO4-0.8% K3Fe(CN)6 in 0.1 M sodium phosphate buffer (2 h). All samples were stained with 2% aqueous uranyl acetate for 90 min, dehydrated in graded ethanol, and finally embedded in ERL 4206.

Thin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope EM 10C/CR (Zeiss, Oberkochen, Germany) at 60 kV. Pictures were digitally scanned and assembled by using Adobe Photoshop 7.0. To investigate the function of the UL20 product in EHV-1 replication, a UL20-deleted RacL11 was generated by using BAC-mutagenesis. In a first step, the complete ORF was removed from the pRacL11 BAC clone in E. coli (24), resulting in recombinant clone pRacL11Δ20 (Fig. ). In pRacL11, the mini F plasmid pHA2, which contains a GFP cassette, replaces gene 71 encoding for gp2 (Fig.

) (24). The blots were incubated overnight in 5% skim milk in PBS containing 0.05% Tween 20 (PBST) and reacted with primary antibodies for 2 h. GFP-negative progeny virus was purified to homogeneity, and gp2 expression of the isolated L11Δ20 was confirmed by immunofluorescence analysis with a gp2-specific antibody (17; data not shown). In addition, L11Δ20R, the respective UL20 repair virus, was isolated in Rk13 cells. The generated recombinant viruses were genetically characterized as explained in Materials and Methods to confirm their genotypes and to exclude spurious modifications (data not shown) before they were functionally assessed in the course of the present study.

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