Patrick K. Lai

Characterization of the P Protein-Binding Domain on the 10-Kilodalton Protein of Borna Disease Virus

ABSTRACT The Borna disease virus (BDV) is the prototype member of the Bornaviridae, and it replicates in the cell nucleus. The BDV p24P and p40N proteins carry nuclear localization signals (NLS) and are found in the nuclei of infected cells. The BDV p10 protein does not have an NLS, but it binds with P and/or N and is translocated to the nucleus. Hence, p10 may play a role in the replication of BDV in the cell nucleus. Here, we show that the P-binding domain is located in the N terminus of p10 and that S3 and L16 are important for the interaction.

Interaction of Epstein-Barr viral (EBV) origin of replication (oriP) with EBNA-1 and cellular anti-EBNA-1 proteins

We have previously shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) which activates expression of the latent genome of the Epstein-Barr virus (EBV) in Burkitt lymphoma cells induces the synthesis of two cellular anti-EBNA-1 competitor proteins, anti-EBNA-1.1 and anti-EBNA-1.2. Both anti-EBNA-1 proteins can uncouple the specific binding of the EBNA-1 to the region required for EBV plasmid maintenance (oriP). Here, we show by DNase I footprinting that the binding sites on oriP for the EBNA-1 and the anti-EBNA-1 proteins were indistinguishable. The proteins bound to the 30-bp tandem repeats of the oriP. Glycerol-gradient centrifugation and gel retardation assay revealed that a 60-kDa protein formed the anti-EBNA-1.1-DNA complex and a 40-kDa protein formed the anti-EBNA-1.2-DNA complex.

Multiple immunological functions of extracts from the cone of Japanese white pine, Pinus parviflora Sieb. et Zucc.

Japanese folklore suggests a therapeutic capacity of the extract of the pine cone of Pinus parviflora Sieb, et Zucc. for gastric cancer. We partially purified the antitumor substances (Fr. VI and Fr. VII) by acid- and ethanol- precipitation from the NaOH extract (1). It was unexpectedly found that the replication of human immunodeficiency virus (HIV) was potently inhibited by these fractions (2, 3). We report here their structural analysis and ability to induce several kinds of interesting biological activities.

Amplification of a Full-Length Borna Disease Virus (BDV) cDNA from Total RNA of Cells Persistently Infected with BDV

We have developed a novel reverse transcriptase‐polymerase chain reaction (RT‐PCR) to amplify the full‐length 8.9 kilobase (kbp) cDNA of the Borna disease virus (BDV) RNA genome from the total cellular RNA of MDCK cells persistently infected with BDV (MDCK/BDV). Antigenomic BDV cDNA was reverse transcribed using a 53‐mer oligonucleotide primer, corresponding to the 5′‐terminus of a putative 3′‐leader sequence of the BDV RNA genome, for 2 hr at 42 C followed by 30 min at 55 C. PCR was performed in the presence of this 53‐mer antigenomic primer and a 25‐mer primer, corresponding to the 3′‐terminus of the BDV antigenomic cDNA, by use of an rTth DNA polymerase with proof‐reading activity. The amplified full‐length BDV cDNA was detected in as little as 20 ng of total cellular RNA of MDCK/BDV. This RT‐PCR method should be a useful technique to study the molecular quasispecies of BDV.

Two Major Histocompatibility Complex Class I-Restricted Epitopes of the Borna Disease Virus p10 Protein Identified by Cytotoxic T Lymphocytes Induced by DNA-Based Immunization

ABSTRACT Borna disease virus (BDV) infection of Lewis rats is the most studied animal model of Borna disease, an often fatal encephalomyelitis. In this experimental model, BDV-specific CD8+ cytotoxic T lymphocytes (CTLs) play a prominent role in the immunopathogenesis of infection by the noncytolytic, persistent BDV. Of the six open reading frames of BDV, CTLs to BDV X (p10) and the l-polymerase have never been studied. In this study, we used plasmid immunization to investigate the CTL response to BDV X and N. Plasmid-based immunization was a potent CTL inducer in Lewis rats. Anti-X CTLs were primed by a single injection of the p10 cDNA. Two codominant p10 epitopes, M1SSDLRLTLL10 and T8LLELVRRL16, associated with the RT1.Al major histocompatibility complex class I molecules of the Lewis rats, were identified. In addition, immunization with a BDV p40-expressing plasmid confirmed the previously reported RT1.Al-restricted A230SYAQMTTY238 peptide as the CTL target for BDV N. In contrast to the CTL responses, plasmid vaccination was a poor inducer of an antibody response to p10. Three injections of a recombinant eukaryotic expression plasmid of BDV p10 were needed to generate a weak anti-p10 immunoglobulin M response. However, the antibody response could be optimized by a protein boost after priming with cDNA.

Cytokine regulation of the human immunodeficiency virus (HIV)

The remarkable ability of HIV to insinuate itself into the working of the immune system is the key of its success as an infectious agent. Given that the cytokine network regulates the immune responses, it is not surprising that cytokines can modulate HIV infection. GM-CSF, IL6 and TNF-alpha enhance HIV, but TGF-beta and HIF inhibits the virus. However, the anti-HIV activity of TGF-beta is restricted to myeloid cells, while HIF inhibits HIV in myeloid cells and in T-lymphocytes. HIF is produced by CEM cells after induction by an extract from pine cones. It is not an interferon and is likely a novel cytokine. It is pepsin-sensitive but trypsin-resistant and has an apparent molecular weight of 7-12 KDa. Apart from having anti-HIV activity, crude preparations of HIF also inhibit HTLV-1 virus but not HSV virus replication.

Overview: Proteins and Peptides that have Activity Against the Human Immunodeficiency Virus (HIV)

SummaryThis review is comprized of two sections. The first discusses the life-cycle of HIV and potential target sites for anti-HIV drugs. The second reviews anti-HIV agents, which are limited to proteins and peptides in this article. Agents that interfere with events early in the HIV life-cycle are divided into two categories, those that interfere with events prior to viral entry and those that are active after viral entry but prior to proviral integration. These agents can abort virus infection if appropriately used. In contrast, agents interfering with events late in the HIV life-cycle do not abort HIV infection, rather, they suppress events during viral replication such as gene transcription, translation and/or virus assembly and maturation. It is likely that combination therapy with agents inhibiting an event early and another event late in the HIV life-cycle will be most efficacious.Soluble CD4, chimaeric molecules of CD4 and IgG, neutralizing antibodies and N-carbomethoxy-carbonyl-prolyl-phenylalany...