Keizo Tomonaga

GENETIC DIVERSITY OF ARGENTINE ISOLATES OF FELINE IMMUNODEFICIENCY VIRUS

We report the nucleotide sequence and genetic diversity of part of the envelope (env) gene of four strains of feline immunodeficiency virus (FIV) isolated from Argentine domestic cats. The DNA encoding the V3 to V5 regions of the env gene of the FIV isolates were amplified by PCR, cloned and sequenced. Phylogenetic analysis revealed that the Argentine isolates did not cluster into a single group; one isolate clustered with subtype B FIV isolated in the USA and Japan, whereas the others formed a new cluster of FIV which might represent a prototype sequence for subtype E.

Isolation of Borna Disease Virus from Human Brain Tissue

ABSTRACT Serological and molecular epidemiological studies indicate that Borna disease virus (BDV) can infect humans and is possibly associated with certain neuropsychiatric disorders. We examined brain tissue collected at autopsy from four schizophrenic patients and two healthy controls for the presence of BDV markers in 12 different brain regions. BDV RNA and antigen was detected in four brain regions of a BDV-seropositive schizophrenic patient (P2) with a very recent (2 years) onset of disease. BDV markers exhibited a regionally localized distribution. BDV RNA was found in newborn Mongolian gerbils intracranially inoculated with homogenates from BDV-positive brain regions of P2. Human oligodendroglia (OL) cells inoculated with brain homogenates from BDV-positive gerbils allowed propagation and isolation of BDVHuP2br, a human brain-derived BDV. Virus isolation was also possible by transfection of Vero cells with ribonucleoprotein complexes prepared from BDV-positive human and gerbil brain tissues. BDVHuP2br was genetically closely related to but distinct from previously reported human- and animal-derived BDV sequences.

The genome of feline immunodeficiency virus.

SummaryFeline immunodeficiency virus (FIV) is a member of the genusLentivirus of the familyRetroviridae. FIV can infect T lymphocytes and monocytes/macrophages in vitro and in vivo, and causes an acquired immunodeficiency syndrome-like disease in cats. Several isolates of FIV from geographically distant countries have been molecularly cloned. There is considerable heterogeneity especially in Env gene among the FIV isolates and they can be divided into two or more subgroups. Like other lentiviruses, FIV has a complex genome structure. Gag gene encodes matrix, capsid and nucleocapsid proteins, and Pol gene encodes protease, reverse transcriptase, dUTPase and integrase. The dUTPase is not present in the primate lentiviruses but present in the nonprimate lentiviruses. Env gene encodes surface and transmembrane envelope glycoproteins. In addition to the structural and enzymatic proteins, at least three more genes (Vif, ORF A, Rev) are present in FIV. Vif is related to the infectivity of the cell-free viruses. Rev functions in the stability and transport of incompletely spliced viral RNAs from the nucleus to cytoplasm and is indispensable for virus replication. Although the Tat protein of the primate lentiviruses is essential for virus replication, ORF A (putative Tat gene) of FIV is not essential for virus replication in established feline T lymphoblastoid cell lines. However, the ORF A gene product is related to the efficient replication of the virus in primary peripheral blood lymphocytes. In the long terminal repeat (LTR) of FIV, there are many putative binding sites for enhancer/promoter proteins. Among these binding sites, the putative AP-1 site is important for basal promoter activity of the LTR and responsible for the T cell activation signal through protein kinase C, however the site is not required for the virus replication in established feline T lymphoblastoid cell lines. Comparative study of the molecular biology of lentiviruses revealed that the genome structure, splicing pattern and functional enhancer protein-binding sites of FIV are more similar to those of the ruminant lentiviruses than those of the primate lentiviruses.

Characterization of an integrase mutant of feline immunodeficiency virus

SummaryThe role of the integrase region of feline immunodeficiency virus (FIV) in viral replication was examined using an integrase mutant clone of FIV which carries a frameshift mutation in the region. Upon transfection, although the integrase mutant was able to release virus-like particles into the supernatant from the transfected cells, the virions produced by the mutant contained unprocessed gag precursor protein and undetectable levels of reverse transcriptase activity. Furthermore, the mutant virions were unable to direct the synthesis of viral DNA after infection in target cells. To understand this phenotype of the integrase mutant in more detail, we constructed a gag-pol expression plasmid from an FIV molecular clone and assayed roles of the integrase region on virus particle formation following transfection. When an inframe deletion was introduced into the protease region of the expression plasmid, the mutant was able to efficiently release gag- and gag-pol precursor proteins into the supernatant from the transfected cells. An expression plasmid with mutations in both the protease and integrase regions, however, failed to release the gag-pol precursor protein from the cells. These results suggested an essential role for the integrase region for efficient incorporation of the gag-pol precursor into the virions.

Feline immunodeficiency virus can infect a human cell line (MOLT-4) but establishes a state of latency in the cells.

Infectivity of feline immunodeficiency virus (FIV) in feline and human lymphoblastoid cell lines was examined using homogeneous populations of FIV derived from infectious molecular clones of strains TMZ and Petaluma, and two recombinant chimeric clones carrying gag, pol, vif and ORF-A from the heterologous virus. FIV from the clones with the env region of the Petaluma strain was shown to infect and establish provirus in a human lymphoid cell line (MOLT-4), although the FIV-infected cells did not produce any infectious viruses. By treatment of the infected MOLT-4 cells with a phorbol ester, infectious virus was rescued. To examine which stage of the life-cycle of FIV is blocked in these cells, we analysed transcription of FIV-14 in the cells by RT-PCR. FIV-specific RNA expression could not be detected. These results strongly suggest that latency of the virus in MOLT-4 cells is due to a failure in transcription.

Localization of the viral antigen of feline immunodeficiency virus in the lymph nodes of cats at the early stage of infection

SummaryImmunohistochemical examinations of localization of feline immunodeficiency virus (FIV) Gag protein were performed on lymph nodes of cats experimentally inoculated with three different strains of FIV (infectious molecular clone of TM 1, Petaluma, and KYO-1 strains), using rabbit anti-FIV Gag serum. The FIV Gag antigens were observed in many follicular dendritic cells (FDCs) and sparsely in small lymphocytes of paracortical area in the lymph nodes of cats inoculated with Petaluma and KYO-1 strains. However, the antigens were present only in small lymphocytes, and not in FDCs of a cat inoculated with infectious molecular clone of the TM 1 strain. The cell type differences in expression of the viral antigen in vivo might reflect on the cell tropisms of the FIV strains in vitro. By double immunohistochemical staining with rabbit anti-FIV Gag serum and monoclonal antibodies which recognize feline CD 4, feline CD 8 or feline pan-T molecules, the FIV Gag-positive lymphocytes were characterized as feline CD 4-positive T cells. Since the distributions of FIV Gag antigens were mainly in the FDCs, the FDCs may play an important role as a major reservoir and may be a primary target of FIV at early stages of infection.

Feline CD 4 molecules expressed on feline non-lymphoid cell lines are not enough for productive infection of highly lymphotropic feline immunodeficiency virus isolates

SummaryTo investigate whether the feline CD 4 (fCD 4) molecules are involved in infections of highly lymphotropic feline immunodeficiency virus (FIV) isolates, we expressed fCD 4 stably on Crandell feline kidney cells andFelis catus whole foetus 4 cells by transfection of a cDNA encoding the fCD 4 glycoprotein, and then infected them with TM 1 and TM 2 strains of FIV, which are unable to infect these cells productively. In spite of fCD 4 being expressed on these cells, no virus production was observed. This result indicates that fCD 4 expression alone cannot induce a productive infection of the FIV TM 1 and TM 2 strains.

The AP-1 binding site in the feline immunodeficiency virus long terminal repeat is not required for virus replication in feline T lymphocytes

Sequences of 31 bp containing putative AP-1 and AP-4 binding sequences in the U3 region of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) were deleted and the basal promoter activity of the LTR was measured by the chloramphenicol acetyltransferase (CAT) assay. The activity of the FIV LTR was reduced in Felis catus whole foetus 4 (fcwf-4) cells and Crandell feline kidney cells by this deletion. Cotransfection of murine c-Fos or c-Jun expression plasmids with the FIV LTR-CAT reporter plasmid into fcwf-4 cells revealed that FIV LTR could be activated by c-Fos but not c-Jun in the cells. The mutated LTR was introduced into an infectious molecular clone of FIV and the replication rate and the cytopathogenic activity of the mutant were compared with those of the wild-type in two feline CD4-positive T lymphoblastoid cell lines. It was found that the rate and activity of the mutant were almost the same as those of the wild-type. From these data, we conclude that the 31 bp fragment is important for achieving maximal expression of the FIV genome, but not required for the replication of FIV in feline T lymphocytes.

Feline immunodeficiency virus gene expression : analysis of the RNA splicing pattern and the monocistronic rev mRNA

The transcription pattern of the feline immunodeficiency virus (FIV) genome in a feline CD4+ cell line was examined. In addition to the genomic RNA (9.2 kb), at least five FIV-specific transcripts [5.2, 4.4 (doublet), 1.7 and 1.4 kb] were detected by using subgenomic restriction enzyme fragments of an FIV molecular clone or FIV-specific oligonucleotides as probes. Among these transcripts, the 9.2, 5.2 and 4.4 (doublet) kb mRNAs were not expressed in the cytoplasm of cells transfected with a rev- mutant. To determine the location of splice junctions in the FIV genome, we used PCR to amplify and clone cDNAs corresponding to the viral mRNAs from infected cells. The region between pol and env was found to contain at least two splice donor and three splice acceptor sites. Two splice acceptor sites were detected in the 3 region of env. By hybridization analysis and sequencing of cDNA clones, it was revealed that the medium sized mRNAs are derived from a single splice event, with different splice acceptor sites, and that the two smaller transcripts are doubly or triply spliced mRNAs. Our results demonstrate a complex pattern of alternative splicing of FIV mRNAs. Furthermore, we identified monocistronic rev mRNA species that employ a unique splice acceptor site.

Molecular Chaperone BiP Interacts with Borna Disease Virus Glycoprotein at the Cell Surface

ABSTRACT Borna disease virus (BDV) is characterized by highly neurotropic infection. BDV enters its target cells using virus surface glycoprotein (G), but the cellular molecules mediating this process remain to be elucidated. We demonstrate here that the N-terminal product of G, GP1, interacts with the 78-kDa chaperone protein BiP. BiP was found at the surface of BDV-permissive cells, and anti-BiP antibody reduced BDV infection as well as GP1 binding to the cell surface. We also reveal that BiP localizes at the synapse of neurons. These results indicate that BiP may participate in the cell surface association of BDV.