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.

Characterization of human immunodeficiency virus type 1 resistant to modified cyclodextrin sulphate (mCDS71) in vitro.

Drug resistance of human immunodeficiency virus type 1 (HIV) to modified cyclodextrin sulphate (mCDS71) has been analysed with respect to both the in vitro appearance of resistance to the compound and the mechanism of the acquisition of resistance. Resistant strains could be obtained in all three strains (NL432, KK-1 and A018) tested after serial passages in MT-4 cells with a gradual increase of the concentration of mCDS71. Cross-resistance both to mCDS71 and dextran sulphate 8000 was observed. As a result of sequencing analysis of the gp120 V3-C5 region of resistant strains, the mechanism of resistance can be explained in several ways: (i) substitution of sugar chain-binding amino acids, N and S; (ii) three to five amino acid deletion in V4 loop; and (iii) several mutations in V3 and V4 regions. The real cause of the resistance may be a combination of these three mechanisms. The results suggest that the target of mCDS71 is relatively widely distributed on the viral surface glycoprotein.

Anti-HIV-1 activity of myo-inositol hexaphosphoric acid (IP6) and myo-inositol hexasulfate(IS6).

It is known that polysulfates have some anti-HIV-1 activity. We investigated the anti-HIV-1 activity of myo-inositol hexaphosphoric acid (IP6) and myo-inositol hexasulfate(IS6), low molecular weight carbohydrates. IP6 and IS6 inhibited the replication of HIV-1 in a T cell line as well as that of a freshly isolated strain in peripheral blood mononuclear cells. Neither substance inhibited HIV-1-induced giant cell formation, but addition of IS6 when infecting cells with HIV-1 inhibited the replication of HIV-1. Neither substance inhibited HIV-1 reverse transcriptase activity in vitro and no influence on late stage replication was noted. Although the mechanisms of IP6 and IS6 action remain unclear, it can be speculated that they act on HIV-1 early replicative stage. Although it is not possible to develop IP6 and IS6 themselves as anti-AIDS drugs, studies of these anti-HIV agents might be expected to provide seed for eventual production of superior drugs for AIDS treatment.

Slight difference in primary amino acid sequence of p17 matrix protein of HIV-1 exerts profound influence on its antigenicity.

HIV-1 p17 antigen has been studied for its biological significance in vitro as well as its immunological roles in vivo. By immunological approach of antibody-binding to HIV-1 p17 antigens of several subtypes in combination with computerized analysis of those tertial structures, it became evident that, irrelevant of similarity of linear amino acid sequence of different HIV-1 subtypes, a few amino acid substitutions close to or distant from specified epitope(s) affected their tertial structure resulting in change in ability of its binding to selected antibody. ELISA employing two monoclonal antibodies, A144 and C415, could detect p17 of subtypes A and B, but not of subtypes C, D, and E. Since the epitope site corresponding to A144 has been reported to be important for biological activity of p17 of HIV-1, change in tertial structure around this epitope may explain some difference in biology of HIV-1, such as infectivity of subtypes B and E.

Structure of a truncated human surfactant protein D is less effective in agglutinating bacteria than the native structure and fails to inhibit haemagglutination by influenza A virus.

Surfactant protein D (SP-D) is a lung-specific protein that is synthesized and secreted by lung epithelial cells and is believed to play an important role in lung host defence. This protein belongs to the C-type lectin family, which is characterized by an N-terminal cysteine-rich domain, a collagen-like domain, a neck domain and a carbohydrate recognition domain (CRD). To elucidate the biological actions of this animal lectin against such pathogens as micro-organisms, the biological activities of a recombinant partial SP-D lacking a collagen-like domain were examined. A recombinant human SP-D, consisting of a short collagen region (two repeats of Gly-Xaa-Yaa amino acid sequences), the neck domain and the CRD, was expressed in Escherichia coli. The recombinant SP-D was purified on a nickel column and then on a maltose-agarose column. This protein can form a trimeric structure owing to the neck domain and exhibits sugar-binding activity and specificity similar to those of native human SP-D. The recombinant SP-D caused dose-dependent and calcium-dependent agglutination of E. coli Y1088. The agglutination titre (the concentration required to achieve a 50% decrease in light transmission by agglutination) of recombinant SP-D was approx. 6-fold that of native SP-D. As for conglutination, the recombinant trimeric conglutinin required 8-16-fold higher concentrations than the native counterpart. In haemagglutination inhibition (HI) of influenza A virus, although native and recombinant conglutinin showed similar levels of HI activity, the recombinant SP-D was unable to inhibit haemagglutination, even at a concentration approx. 120-fold that of the native SP-D. The lectin precipitation and lectin blot assays showed that the truncated SP-D could bind to influenza A virus as well as native SP-D did. These results indicate that the agglutination activity of trimeric collectins can be largely retained, and furthermore that the oligomeric structure with several hands at opposite sites can enhance agglutination activity. The difference in HI activity against influenza A virus between native and recombinant SP-D suggests that SP-D uses a different mechanism from that of conglutinin to inhibit viral haemagglutination.

Contribution to the regulation of virus replication in cells latently infected with human immunodeficiency virus 1.

Monocytes/macrophages have been known to play an important role in the initiation and propagation of human immunodeficiency virus 1 (HIV-1) infection. To analyze the function of these cells during the clinical asymptomatic period of infection, we examined the effect of murine peritoneal macrophages and human peripheral blood macrophages on two cell lines latently infected with HIV-1, a promonocytic cell line, U1, and a T-cell line, ACH-2. Monokines of the murine peritoneal macrophages induced significant viral expression in U1, but not in ACH-2 cells. Experiments employing transient transfection of U937 and CEM cells with HIV long terminal repeat (LTR)-chloramphenicol acetyl transferase (CAT) plasmids indicated that the effect of these monokines was due to specific activation of the HIV LTR. In contrast, supernatants of human macrophages induced viral expression in both ACH-2 and U1 cells. These results suggest that several monokines are active in regulating the transition from the clinical asymptomatic period of HIV infection to progression to acquired immunodeficiency syndrome (AIDS).

Neutralization of human immunodeficiency virus type 1 (HIV-1) with antibody from carriers' plasma against HIV-1 protein p17.

It was investigated whether human antibody against HIV-1 protein p17 (anti-p17) in HIV carriers' plasma has the ability to neutralize the infectivity of HIV. By the pretreatment of HIV-1 with anti-p17 from HIV carriers, progeny HIV-1 production from cells infected with virus pretreated with anti-p17 was suppressed and/or delayed. The neutralizing activity of anti-p17 was decreased in the presence of recombinant p17. The latter obviously masked the neutralizing activity of anti-p17. The relevant epitope(s) on p17 is located apparently on the surface of HIV virions and the binding of anti-p17 to p17 impairs the infectivity of HIV. This implies that anti-p17, if stably present in HIV carriers' plasma, may also play an important role in reducing the infectivity of HIV-1 in vivo.

Recombinant bovine conglutinin, lacking the N-terminal and collagenous domains, has less conglutination activity but is able to inhibit haemagglutination by influenza A virus.

Conglutinin is a bovine serum protein which was first described as a vertebrate lectin. This protein belongs to the family of C-type lectins. These lectins are composed of four characteristic domains: (1) an N-terminal cysteine-rich domain, (2) a collagen-like domain, (3) a neck domain and (4) a carbohydrate recognition domain (CRD). Recently lectins have been shown to function as immunoglobulin-independent defence molecules due to a complement-mediated mechanism or opsonization. Our previous study showed that bovine conglutinin can inhibit haemagglutination by influenza A viruses and act by directly neutralizing them due to its lectin properties. In order to elucidate the biological role of the collagen-like domain, a recombinant partial conglutinin lacking this collagen-like domain was produced in an Escherichia coli system and its biological activities were examined. A 497 bp sequence, consisting of a short collagen region (two repeats of G-X-Y amino acid sequences), the neck domain, and the CRD of conglutinin cDNA, was amplified by the reverse-transcriptase PCR technique. The cDNA was transferred to a bacterial expression vector system (pRSET-A) and stable transfectants with a high level of conglutinin production were obtained. SDS/PAGE and Western blotting analyses showed a recombinant fusion protein of 27 kDa. Results of a cross-linking study and gel-filtration assay indicated that the recombinant conglutinin can form a trimeric structure and that it has sugar binding activity and specificity similar to that of native conglutinin. The recombinant conglutinin was also found to inhibit haemagglutination caused by influenza A virus as well as to possess less conglutination activity. These results suggest that in order for conglutinin to inhibit haemagglutination caused by the influenza virus, as well as to have sugar binding activity or to form trimers, it does not require the N-terminal and collagenous domains; however, they are essential for full conglutination activity.

Prevalence of blood-borne viruses among intravenous drug users and alcoholics in Hiroshima, Japan.

We investigated the prevalence of human immunodeficiency viruses-1 and 2 (HIV-1 and HIV-2), human T-lymphotropic virus type I and II, hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus among intravenous drug users (IVDU) in Hiroshima, Japan, where little is known about their present levels. From June to December 1993, serum samples were collected from 47 IVDU and 98 alcoholics in Hiroshima, Japan, and examined for markers of virus infection. The prevalence of antibody to HCV (anti-HCV) and/or HCV-RNA was significantly higher in IVDU than alcoholics (74.5% vs 20.4%, 44.7% vs 10.2% respectively, P < 0.001). In contrast, the prevalence of antibody to hepatitis B surface antigen and/or core antigen (anti-HBs and/or anti-HBc) showed no significant difference between the 2 groups (57.4% vs 66.3%). HIV-1 infection was found in one (2.1%) IVDU and genome analysis indicated that it was subtype B according to Myers' classification. Thus, an extremely low level of HIV infection and a high level of HCV infection was found in IVDU. Careful follow-up of this group is thought to be needed to minimize an outbreak of HIV-1 infection in Japan.

Nuevo método para la determinación del anticuerpo anti GP 41 / Annual reports 1993 / New method for the detection of anti GP 41 antibodies

Actualmente se emplean los métodos ELISA y Aglutinación de particículas de gelatina para la detección de anticuerpo anti HIV. Sin embargo, se hace necesario encontrar un método rápido, fácil y accesible al operador. El método de SimpliRed que fue desarrolladopor científicos australianos (Agen Biomedical Limited) para la determinación del anticuerpo anti HIV se basa en la aglutinación de glóbulos rojos, lo que se puede determinar por visualización directa utilizando 10 ul de sangre total. El método utiliza un anticuerpo monoclonal de doble especifidad en donde uno de los fragmentos Fab está dirigido exclusivamente contra la glicoforina (glicoproteina de membrana de los glóbulos rojos), lo cuál permite la unión al eritrocito sin producir aglutinación. El otro fragmento Fab. es conjugado a péptidos sintéticos correspondientes a epítopes de HIV cubiertos con proteínas gp41. El fragmento Fab. es reconocido por el anticuerpo anti gp41 si estuviese presente en la sangre o suero, ocurriendo en este caso aglutinación en dos minutos. Se han analizado 30 sueros y plasmas de portadores HIV provenientes del Japón por método de Aglutinación de partículas de gelatina (Serodia HIV Fujirebio), Western Blot (Lab Blot), (Diagnostic Pasteur) y el método SimpliRed cuyos resultados demostraron 78 por ciento de sensibilidad y una especifidad del 100 por ciento utilizando el método de SimpliRed. Este método ofrece muchas expectativas para futuros ensayos, puede ser empleado en los bancos de sangre juntamente con la tipificación sanguínea y en los centros de asistencias de urgencias

Three antigenic regions in p17 of human immunodeficiency virus type 1 (HIV-1) revealed by mouse monoclonal antibodies and human antibodies in HIV-1 carrier sera.

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (KA = 1.9 x 10(5)-1.4 x 10(8) M-1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (KA = 2.0 x 10(5)-1.8 x 10(7) M-1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.

Hepatitis C virus genotypes, reactivity to recombinant immunoblot assay 2 antigens and liver disease.

To clarify the relationship between hepatitis C virus (HCV) genotypes and liver disease, we typed HCV genomes in the sera of 151 blood donors, 180 patients with type C chronic liver disease (CLD), and 30 haemophiliacs residing in Hiroshima, Japan. All of the subjects were positive for anti-HCV and HCV-RNA, and were examined for seroreactivity to HCV-specific antigens. The HCV genotypes were determined by polymerase chain reaction (PCR) with type-specific primers deduced from the putative core region of the HCV genome. Significantly more (P < 0.001) type III HCV was found in the samples from the CLD patients (80%) than in those from the blood donors (55%). Significantly more (P < 0.001) type III HCV was found in the samples from the blood donors (29.1%) than in those from the CLD patients (11.7%). There was no significant difference in the distribution of the HCV types among the patients with chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma. A four-antigen recombinant immunoblot assay (RIBA-2) assay was used to compare the serum samples for their reactivity to a range of structural and nonstructural peptides specific for HCV (5-1-1, C100-3, C33c, and C22-3). The frequency of seropositivity to 5-1-1 and C100-3 was significantly higher (P < 0.001) in type II HCV-infected blood donors than in type III HCV-infected donors (68.2% and 65.9% vs. 4.5% and 22.7%, respectively). Among the type III HCV-infected individuals, the CLD patients had a significantly higher (P < 0.01) frequency of seropositivity to 5-1-1 than the blood donors (33.3% vs. 4.5%).(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and genomic analysis of human T lymphotropic virus type II from Ghana.

An HTLV-II was isolated from a Ghanaian female patient with ARC and anti-HTLV-II antibody by cocultivation of PBMC with Molt-4 cells. A part of the 5' LTR of this virus was sequenced. Sequence homology between this virus and the corresponding sequence of Mo, the prototype of HTLV-II, is 97.4%. Furthermore, this virus is a member of the HTLV-IIa group, which was proposed by Hall et al. This is the first report of an HTLV-II sequence from Africa confirmed not only by serological evidence, but also by genomic analysis. This finding will provide useful information for considering the spread of HTLV-II in the world and the evolution of the HTLV group.