Yasuo Inoshima

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.

Serosurvey for Selected Virus Infections of Wild Carnivores in Taiwan and Vietnam

Serum samples from two leopard cats (Felis bengalensis) and four Formosan gem-faced civets (Paguma larvata taivana) in Taiwan, September 1995, and nine leopard cats in Vietnam, August and December 1997, were examined for the prevalence of antibodies against feline parvovirus, feline herpesvirus type 1, feline calicivirus and feline immuno-deficiency virus. All civets and nine of 11 leopard cats were shown to have antibodies against feline parvovirus (FPV), and FPVs were isolated from mononuclear cells in the peripheral blood of the six leopard cats.

Phylogenetic analysis of feline immunodeficiency virus isolated from cats in Taiwan

SummaryFeline immunodeficiency virus was isolated from four cats from Taiwan. The isolates were designated TI-1, TI-2, TI-3 and TI-4. Each was isolated from PBMCs following co-cultivation of PBMCs with a feline T-lymphoblastoid cell line (MYA-1 cells). However, the Taiwanese isolates did not grow in a feline kidney cell line (CRFK cells). The nucleotide sequences of the V3-V5 region of the envelope gene of the Taiwanese isolates were determined and compared with those of previously described isolates. Phylogenetic analysis of this region indicates that Taiwanese isolates belong to subtype C.

TEMPORAL PATTERNS OF FELINE IMMUNODEFICIENCY VIRUS TRANSCRIPTS IN PERIPHERAL BLOOD CELLS DURING THE LATENT STAGE OF INFECTION

We have investigated the in vivo state of feline immunodeficiency virus (FIV) transcription in peripheral blood mononuclear cells (PBMC) of chronically FIV-infected, asymptomatic cats. FIV was detected in a high percentage of PBMC but not in the plasma of these cats. By quantitative reverse transcription-PCR (RT-PCR) analysis. FIV transcriptional status in the PBMC was characterized by extremely low or undetectable levels of unspliced or singly spliced mRNAs and predominantly multiply spliced mRNAs. Upon stimulation in vitro, however, the larger mRNA species and infectious virus production were rapidly induced in the PBMC. Furthermore, we demonstrated that viral production was induced in association with differential increases in the levels of each multiply spliced mRNA coding for FIV regulatory proteins. From these results, we suggest that replication of FIV is blocked at an early stage of gene expression in vivo, as described in asymptomatic human immunodeficiency virus (HIV) -infected patients, and that FIV infection in cats may be a useful model for clinical latency of HIV infection in man. Moreover, we propose that the replication of FIV in vivo may be controlled by the differential expression of each multiply spliced mRNA.

In vivo functions of the auxiliary genes and regulatory elements of feline immunodeficiency virus

Feline immunodeficiency virus (FIV) is a widespread lentivirus of domestic cats that causes an acquired immunodeficiency syndrome (AIDS)-like disease similar to human AIDS caused by human immunodeficiency virus. FIV has a complex genome structure including structural, enzymatic and auxiliary genes and regulatory elements. In this article, we review the in vivo roles of some of these FIV auxiliary genes and regulatory elements, especially focusing on the dUTPase, vif, and ORF-A genes and AP-1 binding site in the enhancer region of the long terminal repeat, by comparison with those of other non-primate lentiviruses. These genes and elements are considered to be important for viral replication, immunological response and pathogenesis in cats.

Replication of Feline Syncytial Virus in Feline T-Lymphoblastoid Cells and Induction of Apoptosis in the Cells

Feline syncytial virus (FSV) was isolated from feline peripheral blood mononuclear cells of FSV‐seropositive cats. When the susceptibility of feline T‐lymphocytes to FSV was examined using three strains of FSV, FSV antigens were detected in the FSV‐infected T‐lymphoblastoid cells. Further, a diversity of biological properties, including replication kinetics and syncytia formation, was noted among the strains, and condensation of chromatin and the fragmentation of cellular DNA were observed in the infected cells. From these data, we conclude that FSV is lymphotropic and can induce apoptosis in the lymphocytes.

The roles of vif and ORF-A genes and AP-1 binding site in in vivo replication of feline immunodeficiency virus

SummaryTo examine the in vivo roles of auxiliary genes and regulatory elements of feline immunodeficiency virus (FIV), the provirus load in various tissues of cats infected with each of the mutant viruses (Δvif, ΔORF-A and ΔAP-1) was studied. Although all mutant viruses could infect various tissues, provirus loads in various tissues especially those in cats infected with Δvif virus were lower than those with the wild-type virus. Our results indicate the significance of vif and ORF-A genes and AP-1 binding site of FIV for efficient viral replication and full pathogenicity in cats.

Regulatory properties of the integrated long terminal repeat of the feline immunodeficiency virus.

To examine the regulatory properties of feline immunodeficiency virus (FIV) long terminal repeat (LTR) integrated into host chromatin, Crandell feline kidney cells were stably transfected with the FIV LTR that directs the bacterial chloramphenicol acetyltransferase (CAT) gene. Using these cells, we examined the effects of treatment with several chemical agents, infection with feline viruses, or transfection with effector plasmids expressing FIV gene products on FIV LTR-directed gene expression. Among them, treatment with the phorbol ester (a strong activator of protein kinase C), forskolin (an inducer of cyclic-AMP), 5-azacytidine (a DNA methylation antagonist), or infection with feline herpesvirus type 1 (FHV-1), resulted in induction of CAT activity in the cells. These results suggest that the integrated FIV LTR is stimulated by cellular transcriptional factors induced by phorbol ester, forskolin and FHV-1, and is also inactivated by DNA methylation. Furthermore, this permanent cell line can be used as a screening system of activators of the FIV LTR.

Construction of a recombinant feline herpesvirus type 1 expressing Gag precursor protein of feline immunodeficiency virus

SummaryWe constructed a deletion mutant of feline herpesvirus type 1 (FHV-1) and a recombinant FHV-1. The deletion mutant is the virus with a region (367 bp) deleted from the start codon of thymidine kinase (TK) gene to the SmaI site within the TK gene, and the other is a recombinant FHV-1 expressing Gag protein of feline immunodeficiency virus (FIV), in which a cDNA encoding the Gag protein of FIV was inserted at the TK deletion site of the former deletion mutant. These viruses were designated as C7301ddlTK and C7301ddlTK-gag, respectively. Growth kinetics of these viruses in Crandell feline kidney cells was similar to that of the parent C7301 strain. By immunoblot analysis, C7301ddlTK- gag was confirmed to express the FIV Gag precursor protein in the cells.