Hideaki Tsuchie

Analysis of a human immunodeficiency virus type 1 isolate carrying a truncated transmembrane glycoprotein

We have recently reported the isolation of a human immunodeficiency virus type 1 (HIV-1), KB-1gp32 carrying a shorter size (32 kDa) of transmembrane glycoprotein (TMP) from TALL-1 cells persistently infected with KB-1gp41 virus strain (Shimizu et al., 1990a). Endoglycosidase treatments showed that the different size of the TMP between the two strains was due to a truncation of 9 kDa of polypeptide in the KB-1gp32 TMP coding region. Sequence analysis revealed the substitution of a CAG codon to a TAG stop codon just downstream of the putative membrane-spanning domain of the TMP of KB-1gp32. This resulted in a truncation of some 133 amino acids of the cytoplasmic domain of TMP. The data indicate that a premature stop codon in KB-1gp32 has been introduced during adaption of the parental virus to TALL-1 cells. We have constructed two chimeric clones between the env region of a clone pKB-1, derived from KB-1gp32, and an infectious molecular clone pNL-432. We have also constructed a site-directed mutant of pNL-432 carrying a premature stop codon at the same position as the env stop codon of pKB-1. Among the three clones carrying a premature stop codon in env, only one chimeric clone was infectious to TALL-1 but not MT-2 cells. This clone contained the entire tat, rev, vpu, and env genes of pKB-1. The pNL-432 mutant was not infectious. The results suggest that some sequences of pKB-1 might compensate for the truncation of the TMP during replication in TALL-1 cells.

Genetic subtypes of HIV-1 in the Philippines

Objectives:To determine the genetic variability of HIV-1 amongst infected Filipinos and to analyze phylogenetic relationships, temporal introductions and transmission dynamics of identified variants. Methods:Polymerase chain reaction amplification and direct sequencing of a 204 base-pair fragment of the env C2–V3 region from uncultured peripheral blood mononuclear cells obtained from 51 HIV-1-positive Filipinos infected from 1987 to mid-1996. Evolutionary distance and phylogenetic relationships among the DNA sequences were estimated. Results:The 51 Philippine strains were classified into five env V3 subtypes, namely subtype B (n = 37), subtype E (n = 8), subtype A (n = 3), subtype C (n = 2) and subtype D (n = 1). The overall env nucleotide divergence ranged from 11.7 to 32.2%. The nucleotide variation appeared to be random and no temporal ordering was observed. The variation of the sequences at the tip of the V3 loop was very broad. Subtypes B and C isolates did not show close genetic relationship to other Asian variants. Only three of the subtype E strains had close affinity to known Asian sequences. The majority (94%) of the subjects acquired the infection by sexual transmission. About two-thirds were presumably infected outside the Philippines, whereas the remaining were infected indigenously. Information was limited to allow segregation of the identified subtypes by mode of transmission or risk groups. Conclusion:Our findings demonstrate the presence of multiple genetic subtypes of HIV-1 in the Philippines. The apparent geographic range of previously reported genotypes in South and South-east Asia was extended and has obvious implications for env-based antiviral interventions.

Inhibitory effect of novel 1-deoxynojirimycin derivatives on Hiv-1 replication

The effect of 11 derivatives of 1-deoxynojirimycin (DNM) on the replication of HIV-1 was studied. Compared with DNM, seven of them showed remarkable inhibition of HIV-1-induced syncytium formation at significantly low concentrations which were not cytotoxic. Two derivatives were found to markedly reduce the infectious virus yields from cell lines chronically infected with HIV. Analysis of HIV-1 envelope glycoproteins showed that the derivatives induced modification of the processing of not only gp120/160 but also the transmembrane glycoprotein gp41. The modification of the processing of the transmembrane glycoprotein gp41 might play an important role in the inhibition of virus replication at a step after the binding of gp120 to CD4. The enhanced anti-HIV activity of DNM derivatives reported here could increase the possibility of non-toxic therapeutic intervention in HIV infections.

HIV-1 variants in South and South-East Asia

HIV spread in South and South-East Asia is most alarming, and genetic variability of HIV-1 is an important consideration in vaccine development. In this study, we examined the third variable (V3) region of env gene of HIV-1 variants prevalent in Thailand, Malaysia, India, and the Philippines. By phylogenetic tree analyses, an HIV-1 variant from an injecting drug user (IDU) in Thailand belonged to subtype B, and HIV-1 variants from 2 IDUs in Malaysia were classified into 2 subtypes, B and E. One HIV-1 variant from a male homosexual in the Philippines belonged to subtype B. Out of 8 HIV-1 variants from sexually transmitted disease patients in India, 7 belonged to subtype C, and one to subtype A. Although the total number of individuals examined in this study was limited, 4 HIV-1 subtypes were found in South and South-East Asia and large international movements of HIV-1-infected individuals in this region could induce global dissemination of these HIV-1 variants.

Shorter size of transmembrane glycoprotein of an HIV-1 isolate.

An HIV-1 strain carrying a shorter form of the transmembrane glycoprotein (TM) with a mobility of 32 kD, named KB-1, was isolated from a Japanese male hemophiliac by coculture of his peripheral blood mononuclear cells (PBMCs) with MT-2 cells and adaption to TALL-1 cells. Another HIV-1 strain, named KB-2, was isolated from his seropositive spouse by coculture of her PBMCs with MT-2 cells. The KB-2 strain carried a TM of ordinary size, with a mobility of 41 kD. The KB-1 strain carrying a truncated form of the TM could replicate in MT-2, MT-4, TALL-1 and MOLT-4 cells. The KB-1 strain is a useful HIV-1 isolate for investigating the function of the cytoplasmic domain of the TM and the significance of the presence of an in-frame stop codon in HIV env gene.

Conservation of the central proline-rich (PxxP) motifs of human immunodeficiency virus type 1 Nef protein during the disease progression in two hemophiliac patients

The nef gene is considered to play a crucial role in the development of acquired immunodeficiency syndrome (AIDS). In this study, we analyzed the sequence of nef quasispecies obtained from replication‐competent HIV‐1 isolates from two Japanese hemophiliac patients infected with HIV‐1. At least 10 nef clones were isolated at each time point and a total of 75 individual nef quasispecies were sequenced. We observed a gradual increase in genetic diversity of the nef gene over time. Among the various functional regions of Nef protein, myristoylation site and the central PXXP (SH3 ligand) motifs were well conserved. The scattered regions responsible for downregulation of CD4 and class I MHC were also conserved. These data suggest that these functions of Nef may be involved throughout the disease process.

Oligonucleotide Fingerprint Analysis of Coxsackievirus A10 Isolated in Japan

Eight coxsackievirus A10 strains isolated in 1978 and in 1981 and 1982 from patients with hand, foot-and-mouth disease and with herpangina at a dispensary in Matsue city were compared by RNA fingerprinting techniques. The oligonucleotide maps of the four 1978 isolates were related to each other by 85 to 93% with respect to their large T1 oligonucleotides. In contrast, the oligonucleotide maps of the four 1981 and 1982 isolates were very different from each other. Co-electrophoresis experiments revealed that the 1981 and 1982 strains shared only 17 to 34% of their large oligonucleotides. In addition, some large oligonucleotides were found in most of the fingerprint maps of isolates from 1978 to 1982, suggesting that there are regions in the genome of coxsackievirus A10 which are not subject to mutational changes.

Removal of HIV antigens and HIV-infected cells in vitro using immunomagnetic beads

Human anti-HIV antibody-coated magnetic beads and magnetic particle concentrators were used to eliminate HIV antigens and the infected cells in vitro. Fifty micrograms/ml of the antibody coated on 30 mg/ml of tosyl-activated beads, and 10 min of reaction time between the antigens and the immunobeads were sufficient to eliminate the virus antigens from phosphate buffered saline, serum and peripheral blood. HIV-infected cells were eliminated in vitro, but not completely. This method will be useful in eliminating viral antigens and infected cells from clinical material such as blood and blood products.

Characterization of Cells of the Myeloid-Monocytic Lineage (ML-1, HL-60, THP-1, U-937) Chronically Infected with the Human Immunodeficiency Virus-1

The myeloid-monocytic cells ML-1, HL-60, THP-1 and U-937 were chronically infected (for more than 2 years) with the lymphotropic HIV-1 strain HTLV-IIIB. Reinfection experiments revealed that viruses obtained from chronically infected ML-1/HIV-1 and HL-60/HIV-1 cells show a low infectivity if tested with uninfected ML-1 and HL-60 cells in contrast to virus preparations from chronically infected THP-1/HIV-1 and U-937/HIV-1 with their corresponding uninfected cell lines. Analyses of selected cell surface markers showed a differential expression of CD4, CD8, CD11c, CD14, CD15, CD20, HLA-DR and HLA-DQ in non- or chronically infected cells. During chronical infection, the myeloid-monocytic cells lost their reactivity with peroxidase and esterase. In chronically infected cells, the steady-state levels for TNF-alpha mRNA remained unchanged while those for IL-6 decreased. The half-lives of transcripts of both TNF-alpha (t1/2: 70 min) and IL-6 (t1/2: 100 min) were nearly the same in uninfected and chronically infected HL-60 cells.

Human Immunodeficiency Virus Infection in Cells of Myeloid‐Monocytic Lineage

We established persistent infection with a strain of human immunodeficiency virus type I, HTLV‐IIIB, in a promyelomonocytic cell line, ML‐1 (CD4 antigen nearly negative and CD4 mRNA negative), and a promonocytic cell line, THP‐1 (CD4 antigen positive). Different reaction of giant cell formation was found after co‐cultivation of infected and uninfected cells of ML‐1, HL‐60, THP‐1 and U‐937 cell lines with uninfected and infected MOLT4 (a T‐lymphoma cell line).