Regulation of pseudorabies virus gG glycoprotein gene promoter independently of pseudorabies immediate early IE180 protein

The pseudorabies virus (PRV) glycoprotein known as gG is generally regarded as an early protein, and the immediate early IE180 protein regulates its expression during infection. This study, however, provides evidence that although induction by IE180 is observed, the expression of a marker protein (EGFP), or gG itself, under the control of the gG promoter, can also occur independently of the expression of IE180. This result was demonstrated both with transient transfection assays using plasmids and with viral infections. In transient transfections, the expression under control of the gG promoter depends on the cell type and surprisingly, can be 1.3-fold higher than the expression under the control of the IE180 promoter in Hela Tet-Off cells. Recombinant PRV S3 was constructed by replacing gE in the PRV genome with a chimeric transgene, expressing EGFP under the control of the gG promoter. In PK15 cells infected with NIA-3 wild-type virus or with S3 recombinant virus, expression of gG PRV mRNA (or EGFP mRNA) under the control of the gG promoter in the presence of cycloheximide was detected by RT-PCR. This again indicates that some basal expression was produced in infected cells independently of IE180. This expression was augmented by IE180 protein in both plasmid transfections and viral infections.     

The immediate early genes of human cytomegalovirus require only proximal promoter elements to upregulate expression of interleukin-1 beta.

Human cytomegalovirus (HCMV) can infect monocytes and macrophages. The immediate early one (IE1) gene product of HCMV positively regulates its own expression, as well as the expression of the interleukin-1 beta (IL-1) gene. This study describes the IL-1 promoter proximal region required for upregulation of IL-1 gene expression by the HCMV IE1 or IE1 plus IE2 gene products. An IL-1 chloramphenicol acetyltransferase (CAT) construct containing the IL-1 genomic upstream sequence from position -1097 to +14 and four additional IL-1CAT plasmids containing progressive deletions of the -1097 to -131 sequence were used to evaluate the effect of the HCMV IE gene products on IL-1 gene expression. IL-1CAT plasmids were transfected into a monocytic cell line, THP-1, with plasmids containing either the IE promoter-regulatory region upstream of the bona fide IE1 (pIE1), IE2 (pIE2), or IE1+2 genes (pIE1+2) or a control plasmid containing the IE promoter-regulatory region alone (pLink760). In the presence of pIE1+2, there was an approximate 15-fold increase in CAT activity compared with the control, pLink760, in cells with CAT plasmids containing the -1097 to +14 IL-1 sequence. Plasmids with progressive deletions of this sequence, including the plasmid containing the shortest upstream segment (-131 to +14) also had an approximate 15-fold increase in CAT activity. The upregulation of IL-1 expression was mediated, primarily, by IE1 and not by IE2. This effect was promoter specific because an IL-1CAT plasmid with a complete deletion of the proximal promoter elements (-234 to +146) did not respond to the HCMV IE gene products.(ABSTRACT TRUNCATED AT 250 WORDS)     

A glycoprotein gX-beta-galactosidase fusion gene as insertional marker for rapid identification of pseudorabies virus mutants

We describe the isolation and characterization of infectious pseudorabies virus (PrV) mutants expressing functional beta-galactosidase. To obtain high level expression of the enzyme, sequences of the bacterial beta-galactosidase gene starting with codon 8 were inserted in frame behind the promoter and the first seven codons of the nonessential PrV glycoprotein gX-gene. Cotransfection of this construct with viral DNA yielded PrV mutants that could be easily identified after plaque staining with a chromogenic substrate. These mutants carry the gX-beta galactosidase fusion gene inserted into the authentic gX-gene leading to loss of gX-expression. The gX-beta galactosidase fusion gene could be excised as an expression cassette and placed into other non-essential PrV genomic regions, such as the thymidine kinase gene and the glycoprotein gI-gene, resulting in inactivation of the target genes. The fusion gene remains stably integrated in the viral genome at all three locations tested. It therefore appears ideal as an insertional and easily identifiable marker and greatly facilitates isolation and purification of PrV mutants.     

Mapping of a functional region conferring nuclear localization of pseudorabies virus immediate-early protein

Summary The immediate-early protein (IE180) of pseudorabies virus (PrV) is localized predominantly in the nuclei of infected cells. To define the nuclear localization signals within IE180, we prepared truncated mutants of IE180 and analyzed their localization in the transfected cells by indirect immunofluorescence. Analysis of mutants truncated from the carboxy-terminal end of the 1460-amino acid polypeptide showed that two regions including a short sequence of basic amino acid residues were associated with the nuclear localization of IE180. To assess whether these regions substantially function as signals for nuclear localization of the IE180 molecule, we then constructed two deletion mutants lacking each region. A mutant lacking amino acids 333 to 575 was detected in the nuclei of the transfected cells, whereas the other mutant lacking amino acids 900 to 950 was detected mainly in the cytoplasm. These results suggest that the region of amino acids 900 to 950 is responsible for nuclear localization of IE180.     

氯霉素乙酰基转移酶双抗体夹心ELISA检测方法的建立

目的 建立氯霉素乙酰基转移酶（chloramphenicol acetyltransferase,CAT)双抗体夹心ELISA的实验室检测方法。方法 从质粒pBLCAT6经PCR扩增CAT基因序列，插入到原核表达质粒pGEX-2T中，在大肠埃希菌DH5α中诱导表达；以纯化的融合蛋白GST-CAT为抗原免疫实验用兔，得到的CAT抗血清进行生物素标记，并在此基础上利用生物素链和亲和素放大系统建立双抗体夹心ELISA法，构建不同YY1结合位点突变的HPV16LCRCAT报道质粒，体外瞬时转染真核细胞，提取胞质蛋白，以建立的方法进行CAT表达检测。结果 SDS-PAGE显示表达的GST-CAT融合蛋白相对分子质量约为54000；制备的CAT抗血清可有效地识别原核及哺乳动物细胞表达的CAT蛋白；在不同启动子及调节序列的控制下，利用建立的CAT检测技术证明在瞬时转染的细胞中可诱导2-8倍的CAT表达增强。结论 建立的双抗体夹心ELISA方法可敏感地反映上游序列的启动子活性，以及启动子调节序列变化对启动子活性的影响，使CAT作为报道基因的研究更为简单化。     

Inhibition of TAP by pseudorabies virus is independent of its vhs activity

Previously we have shown that pseudorabies virus (PrV) down-regulates the expression of porcine MHC class I molecules by interfering with the transporter associated with antigen processing (TAP). During lytic PrV infection, the half-lives of both host and viral mRNA are regulated by the product of virion host shut-off (vhs) gene, UL41. PrV vhs protein induces degradation of cellular mRNA including those encoding class I and TAP. Therefore, further elucidation of specific mechanisms of down-regulation of class I molecules by PrV necessitates construction of a vhs deletion mutant. Two such mutants (vhsDelta1 and vhsDelta2) were generated by homologous recombination between the wild type (wt) PrV Indiana-F strain, and plasmids containing truncated UL41 gene of PrV into which the enhanced green fluorescent protein (EGFP) cassette was inserted. Compared with the wt virus, both the vhs mutants exhibited slower in vitro growth kinetics. The mutants, like the wt virus, inhibited the peptide transport activity of TAP and down-regulated cell surface expression of class I molecules. These findings suggest that, inhibition of TAP activity in PrV-infected cells is due to mechanism(s) specifically directed at class I pathway and not due to the non-specific vhs activity of the virus.     

Use of an N-terminal half truncated IE1 as an antagonist of IE1, an essential regulatory protein in baculovirus

An immediate-early gene product of baculovirus, IE1, is essential for viral gene expression and for viral DNA replication. It has been demonstrated for Autographa californica nuclear polyhedrosis virus (AcNPV) that the C-terminal region of IE1 is required for dimerization. And the acidic N-terminal region of IE1 has been identified as the activation domain. We constructed an N-terminal 267 amino acid (a.a.) truncated mutant of Bombyx mori nuclear polyhedrosis virus (BmNPV) IE1, which was defective as a transactivator of a viral early gene (p35) promoter. We then examined possible IE1 antagonistic functions of this defective IE1, IE1TN, in BmNPV-infected cells. A transient expression experiment demonstrated that IE1TN strongly repressed the activation of the hr5-dependent p35 promoter derived from BmNPV infection. In addition, DpnI assay elucidated an inhibitory effect of IE1TN on the hr5-dependent replication of plasmid in BmN cells induced by NPV infection. A marked reduction in the production of virus was observed when the BmN cells were infected with BmNPV after transfection with IE1TN-expression plasmids. These results suggested that IE1TN could act as an IE1 antagonist in silkworm cells infected with BmNPV. We then analyzed the ability of IE1TN to inhibit the multiplication of BmNPV using transgenic silkworms. The BmNPV-resistance of the transgenic silkworms was very weak, suggesting insufficient expression of the transgene product, IE1TN.     

Role of the cytoplasmic tail of gE in antibody-induced redistribution of viral glycoproteins expressed on pseudorabies-virus-infected cells.

Pseudorabies virus (PrV) glycoprotein gE is a nonessential glycoprotein involved in virulence and spread of the virus. It also has an important, yet unknown, function during antibody-induced capping of viral glycoproteins on the plasma membrane of PrV-infected swine kidney cells. In the present study, it was shown, by the use of a PrV strain expressing a truncated gE glycoprotein, that the cytoplasmic tail of gE is of significant importance for viral glycoprotein capping to occur. In addition, using PrV strains carrying point mutations in the cytoplasmic tail of gE, it was demonstrated that two tyrosine-based motifs are very important for correct functioning of gE during viral glycoprotein capping. Furthermore it was shown that genistein and tyrphostin, two tyrosine kinase activity inhibitors, inhibit viral glycoprotein capping in a concentration-dependent manner. In conclusion, it can be stated that efficient antibody-induced viral glycoprotein capping requires the presence of two YxxL sequences in the cytoplasmic tail of glycoprotein gE, as well as the activation of a tyrosine phosphorylation signal transduction pathway.     

Optimization of gene transfer in cultured insect cells

Summary The use of polybrene for the efficient transfection of cultured mosquito cells is described. Cells are transfected with purified plasmid DNA coding for a bacterial chloramphenicol acetyltransferase (CAT) gene regulated by sequences from aDrosophila heat shock protein promoter. The DNA is added to the cells in serumfree medium containing polybrene, which facilitates adsorption of DNA to the cell surface. Expression of the transfected gene is induced by subjecting recipient cells to heat shock conditions. CAT activity in lysates from transfected cells is assayed using a simple thin layer chromatographic procedure. Adaptation of the transfection protocol to other insect cell types is discussed.