Pseudorabies Virus Immediate-early Protein Research Articles

Motor-Coordination-Dependent Learning, More than Others, Is Impaired in Transgenic Mice Expressing Pseudorabies Virus Immediate-Early Protein IE180

The cerebellum in transgenic mice expressing pseudorabies virus immediate-early protein IE180 (TgIE96) was substantially diminished in size, and its histoarchitecture was severely disorganized, resulting in severe ataxia. TgIE96 mice can therefore be used as an experimental model to study the involvement of cerebellar circuits in different learning tasks. The performance of three-month-old TgIE96 mice was studied in various behavioral tests, including associative learning (classical eyeblink conditioning), object recognition, spatial orientation (water maze), startle response and prepulse inhibition, and passive avoidance, and compared with that of wild-type mice. Wild-type and TgIE96 mice presented similar reflexively evoked eyeblinks, and acquired classical conditioned eyelid responses with similar learning curves for both trace and delay conditioning paradigms. The two groups of mice also had similar performances during the object recognition test. However, they showed significant differences for the other three tests included in this study. Although both groups of animals were capable of swimming, TgIE96 mice failed to learn the water maze task during the allowed time. The startle response to a severe tone was similar in both control and TgIE96 mice, but the latter were unable to produce a significant prepulse inhibition. TgIE96 mice also presented evident deficits for the proper accomplishment of a passive avoidance test. These results suggest that the cerebellum is not indispensable for the performance of classical eyeblink conditioning and for object recognition tasks, but seems to be necessary for the proper performance of water maze, prepulse inhibition, and passive avoidance tests.

Impaired development of the cerebellum in transgenic mice expressing the immediate-early protein IE180 of pseudorabies virus

Pseudorabies virus (PRV) infection in animals other than its natural host almost always gives rise to fatal diseases in the central nervous system as a result of infection of peripheral neurons and subsequently to the brain. PRV immediate-early protein (IE180) activates transcription of the PRV early and late genes, and other viral and cellular genes, and represses its own transcription. To examine specific effects of IE180 in neuropathogenicity, we have generated four transgenic mouse lines expressing IE180 in a tetracycline-regulated system. In the transgenic mouse lines, cerebellar symptoms such as ataxic gait, tremor and motor discoordination were observed. Histopathology of the cerebella in the transgenic mouse lines showing severe symptoms was remarkable for a failure of layer formation and a reduction in cerebellar size. These findings suggest that IE180 affects the cascade of gene expression for development of the murine cerebellum, resulting in the impairment of the cerebellar development and differentiation.

Immunolocalization of the pseudorabies virus immediate-early protein IE180 by immunoperoxidase staining

The immediate-early (IE) gene of pseudorabies virus (PRV) expresses immediately upon infection, a phosphorylated protein (immediate-early protein, IE180) that can transactivate viral other genes and plays an essential role in regulating viral gene expression. In order to detect and localize IE180 in infected cells early on, this gene was cloned for overexpression, and the expressed products were applied to generate specific antibodies against IE180 protein. Two recombinant expression plasmids pN and pNB were constructed by cloning the IE gene onto pET 30a(+) expression vector via Nco I and BamHI sites. Plasmid pN contains the 1.8-kb NcoI- NcoI fragment of IE gene coding for the N-terminus of 616 amino acid residues, while pNB contains the 2.8-kb NcoI- BamHI fragment coding for the rest of the IE180 protein. Both pN and pNB were transformed, respectively, into E. coli cells and produced large amounts of IE protein products during induction with 1 mM IPTG. The expressed IE proteins for pN and pNB were 60 kDa and 100 kDa in size, respectively. These expression products were purified and then used as antigens to immunize mice for preparing specific antibodies against PRV IE180 protein. The specificities of the mice immune sera were confirmed by their abilities to react with IE180 protein present in the PRV infected cells in the Western immunoblotting assay. Furthermore, immunoperoxidase staining of PRV infected cells undertaken with these antisera revealed the subcellular distribution of the IE proteins in the infected cells and also demonstrated their transportation from the cytoplasm to the nucleus during infection.

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

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.

Mapping of transcriptional regulatory domains of pseudorabies virus immediate-early protein.

The 180 kilodalton immediate-early protein (IE180) of pseudorabies virus functions as a strong transactivator of several different promoters and also as a repressor of its own transcription. To map the functional domains of IE180, we prepared various truncated mutants and analyzed their transcriptional regulatory activities using the chloramphenicol acetyl transferase (CAT) assay. Analysis of mutants truncated from the carboxy-terminal end of the 1,460-amino acid polypeptide showed that a polypeptide possessing amino acids 1 to 1,081 retained significant functions of transactivation and autoregulation potential. On the other hand, removing amino acids 1 to 131 resulted in a complete loss of transactivation potential, indicating that the domain responsible for transactivation is located in the amino-terminal end of IE180. Additional amino-terminal truncation up to amino acid 453 did not affect the autoregulation activity, indicating that the region between amino acids 454 and 1081 has autoregulation potential.

Nucleotide sequence of the pseudorabies virus immediate early gene, encoding a strong transactivator protein

We report the complete DNA sequence of teh pseudorabies virus (PRV) immediate early (IE) gene and its flanking nucleotide sequences, together comprising 5091 base pairs. An open reading frame starts with an ATG codon in position 263 from the transcription-initiation site and ends with a TGA codon in position 4601, thus encoding a predicted protein of 1446 amino acids (150 kD). The PRV IE protein exhibits significant homology with the functionally related transactivator proteins, ICP4 of herpes simplex virus-1 (HSV-1) and p140 of varicella zoster virus (VZV). The extent of homology varies widely along the three sequences: Two regions of the PRV IE protein extending from amino acids 482 to 659 and 959 to 1350 exhibit 50% to 60% identity with the cognate sequences, whereas the remaining sequence reveals little homology apart from a common polyserine stretch. The base composition of the PRV IE coding region is 80% G + C, compared with 81.5% for HSV-1 and 64.1% for VZV. Yet the PRV IE protein appears to be as closely related to VZV p140 as to HSV-1 ICP4. The regions of strong homology are also apparent in plots predicting secondary structure.

Transcriptionally active immediate-early protein of pseudorabies virus binds to specific sites on class II gene promoters

In the presence of partially purified pseudorabies virus immediate-early protein, multiple sites of DNase I protection were observed on the adenovirus major late and human hsp 70 promoters. Southwestern (DNA-protein blot) analysis demonstrated that the immediate-early protein bound directly to the sequences contained in these sites. These sequences share only limited homology, differ in their affinities for the immediate-early protein, and are located at different positions on these two promoters. In addition, the site-specific binding of a temperature-sensitive immediate-early protein was eliminated by the same heat treatment which eliminates its transcriptional activating function, whereas the binding of the wild-type protein was unaffected by heat treatment. Thus, site-specific binding requires a functionally active immediate-early protein. Furthermore, immediate-early-protein-dependent in vitro transcription from the major late promoter was preferentially inhibited by oligonucleotides which are homologous to the high-affinity binding sites on the major late or hsp 70 promoters. These observations suggest that transcriptional stimulation by the immediate-early protein involves binding to cis-acting elements.

Binding of the pseudorabies virus immediate-early protein to single-stranded DNA.

In an attempt to correlate the ability to activate transcription with affinity for single-stranded DNA, both wild-type and temperature-sensitive pseudorabies virus immediate-early proteins were tested for the ability to bind to single-stranded DNA columns. Wild-type and temperature-sensitive immediate-early proteins bound to nonspecific single-stranded DNA columns with similar affinities at both 0 and 40 degrees C. There did not seem to be a direct correlation between the ability to activate transcription and the ability to bind to single-stranded DNA. To study further the interactions that are involved in binding to single-stranded DNA, we expressed the immediate-early protein in an Escherichia coli expression vector. In this system the expressed immediate-early protein was not phosphorylated, nor could it be complexed with mammalian cell factors. The first trp construct did not express a soluble form of the immediate-early protein, presumably due to the insoluble nature of the trp leader. We deleted a large segment of the trpE gene and found that the immediate-early fusion protein was soluble. We tested this protein for its affinity for single-stranded DNA by passage over single-stranded DNA cellulose columns. The bacterially expressed immediate-early protein bound single-stranded DNA at least as well as did the wild-type protein. Affinity for single-stranded DNA did not appear to be dependent on the phosphorylation state nor on the presence of mammalian cell factors.

Effects of a temperature-sensitive mutation in the immediate-early gene of pseudorabies virus on class II and class III gene transcription.

The pseudorabies virus immediate-early protein activates transcription of both class II and class III genes. At present it is not known whether the activation of class II genes occurs through an activation of cellular factors or by a direct interaction of the immediate-early protein with factors or DNA. It is also not known whether the activation of class II and class III genes occurs by a similar or different mechanism. We utilized tsG, a temperature-sensitive mutation in the immediate-early gene of pseudorabies virus, to study the activation of viral genes transcribed by RNA polymerases II and III. Previous studies have shown that tsG inhibits wild-type adenovirus early gene transcription in coinfections at the nonpermissive temperature (L. T. Feldman and S. E. Ahlers, J. Virol. 57:13-17, 1986). Using this system of mixed infections as an assay, we obtained several results which allowed us to draw certain conclusions about the mode of action of the pseudorabies virus immediate-early protein (IEP). First, the tsG mutation inhibits the formation of new transcription complexes on class II genes, but does not affect transcription from preestablished transcription complexes formed in the presence or absence of the adenovirus E1A protein. Second, tsG does not inhibit transcription from class III genes, suggesting that the activation by IEP of class II and class III genes occurs by different mechanisms. Third, activation of transcription by the adenovirus E1A protein is not dominant to inhibition by tsG, suggesting that the temperature-sensitive IEP is involved in some physical interaction which actively inhibits transcription of viral genes.

In vitro stimulation of specific RNA polymerase II-mediated transcription by the pseudorabies virus immediate early protein.

Nuclear extracts from human cells infected with pseudorabies virus (PRV) exhibited higher levels of accurate transcription of RNA polymerase II genes than did control extracts from mock-infected cells. Stimulation was maximal at low DNA concentrations and was not gene-specific. It was heat sensitive in extracts from cells infected with a virus containing a temperature sensitive mutation in the immediate early (IE) gene. The stimulatory activity copurified from the IE protein and was also heat sensitive when purified with cells infected with tsG, further indicating that the IE protein was responsible for this stimulation. These results thus demonstrate an in vitro system that mimics, at least in part, the in vivo stimulatory action of the PRV IE protein. They further imply that the IE protein acts not by increasing the amounts of cellular transcription factors, but rather by directly or indirectly altering their activities.