Shinji Harada

Epstein-Barr virus-induced diseases in boys with the X-linked lymphoproliferative syndrome (XLP): Update on studies of the registry

Analyses of 100 subjects with the X-linked lymphoproliferative syndrome (XLP) in 25 kindreds revealed four major interrelated phenotypes: infectious mononucleosis, malignant B-cell lymphoma, aplastic anemia, and hypogammaglobulinemia. Eighty-one of the patients died. Two male subjects were asymptomatic but showed immunodeficiency to Epstein-Barr virus (EBV). Seventy-five subjects had the infectious mononucleosis phenotype and concurrently, 17 subjects of this group had aplastic anemia. All subjects with aplastic anemia died within a week. Aplastic anemia did not accompany hypogammaglobulinemia or malignant lymphoma phenotypes. Hypogammaglobulinemia had been detected before infectious mononucleosis in three subjects, after infectious mononucleosis in five subjects, and was not associated with infectious mononucleosis in 11 boys with hypogammaglobulinemia. In nine subjects infectious mononucleosis appeared to have evolved into malignant lymphoma; however, the majority of patients with malignant lymphoma showed no obvious antecedent infectious mononucleosis. One subject had infectious mononucleosis following recurrent malignant lymphoma. Twenty-six of 35 lymphomas were in the terminal ileum. Results of immunologic and virologic studies of 15 survivors revealed combined variable immunodeficiency and deficient antibody responses to EBV-specific antigens. Mothers of boys with XLP exhibited abnormally elevated titers of antibodies of EBV. Subjects of both sexes with phenotypes of XLP should be investigated for immunodeficiency to EBV. Persons with inherited or acquired immunodeficiency may be vulnerable to life-threatening EBV-induced diseases.

The broad anti-viral agent glycyrrhizin directly modulates the fluidity of plasma membrane and HIV-1 envelope

Cell entry of enveloped viruses requires a wide-fusion-pore mechanism, involving clustering of fusion-activated proteins and fluidization of the plasma membrane and viral envelope. In the present study, GL (glycyrrhizin) is reported to lower membrane fluidity, thus suppressing infection by HIV, influenza A virus and vesicular stomatitis virus, but not by poliovirus. GL-treated HIV-1 particles showed reduced infectivity. GL also inhibited cell-to-cell fusion induced by HIV-1 and HTLV-I (human T-cell leukaemia virus type I). However, when cells treated with 1 mg/ml GL were placed in GL-free medium, they showed increased susceptibility to HIV-1 infection and HTLV-I fusion due to enhancement of membrane fluidity. The membrane dependence of GL and GL removal experiments suggest that GL does affect the cell entry of viruses. HIVs with more gp120 were less dependent on temperature and less sensitive to GL treatment than those with less gp120, indicating that the existence of more gp120 molecules resulted in a higher probability of forming a cluster of fusion-activated proteins.

Involvement of both the V2 and V3 regions of the CCR5-tropic human immunodeficiency virus type 1 envelope in reduced sensitivity to macrophage inflammatory protein 1α

ABSTRACT To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage during the course of an infection in vivo, macrophage inflammatory protein (MIP)-1α-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1α (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat–β-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1β (>4-fold) and RANTES (regulated upon activation, normal T-cell expressed and secreted) (6-fold). The env sequence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the same V3 substitution appeared in virus passaged without MIP-1α. A single-round replication assay using a luciferase reporter HIV-1 strain pseudotyped with mutant envelopes confirmed that mutations in both V2 and V3 were necessary to confer the reduced sensitivity to MIP-1α, MIP-1β, and RANTES. However, the double mutant did not switch its chemokine receptor usage from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this mutant. These results indicated that V2 combined with the V3 region of the CCR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemokines without altering the ability to use chemokine receptors.

Interleukin-8 is constitutively and commonly produced by various human carcinoma cell lines

SummaryWe examined the production of interleukin-8 and interleukin-6 by 30 human carcinoma cell lines. Serum levels of interleukin-8 were measured in 14 patients with hepatocellular carcinoma by enzyme-linked immunosorbent assay and Northern blotting. Furthermore, serum interleukin-8 was also investigated in a nude mouse bearing a tumor of the HuH7 hepatoma cell line producing interleukin-8. Of the 30 cell lines, 29 (96.7%) constitutively produced interleukin-8, and 19 of the 29 (65.5%) were high producers (>1 ng/ml culture supernatant). Among the high producers, 4 cell lines released both interleukin-8 and interleukin-6. Interleukin-6 was constitutively produced by 17 of the 30 (56.7%) cell lines, 4 of which (23.5%) were high producers (>1 ng/ml). By Northern blot analysis, mRNAs of interleukin-8 and interleukin-6 were detected in producing cell lines. Of 14 patients with hepatocellular carcinoma, 4 (28.5%) showed increased levels of serum interleukin-8. Furthermore, inoculation of the HuH7 hepatoma cell line which produced the highest amount of interleukin-8 into a nude mouse resulted in tumor production accompanied by an elevated level of human interleukin-8 (646 pg/ml) in the peripheral blood. Thus, interleukin-8 is constitutively and commonly produced by various carcinoma cell lines. The production of interleukin-8 by carcinoma cells may be related to the elevation of serum interleukin-8 in patients with hepatocellular carcinoma. Finally, these cell lines may be valuable for studying the relationship between interleukin-8 and cancer.

Isolation of TAK-779-resistant HIV-1 from an R5 HIV-1 GP120 V3 Loop Library

The human immunodeficiency virus (HIV-1) envelope glycoprotein (GP) 120 interacts with CD4 and the CCR5 coreceptor for viral entry. The V3 loop in GP120 is a crucial region for determining coreceptor usage during viral entry, and a variety of amino acid substitutions has been observed in clinical isolates. To construct an HIV-1 V3 loop library, we chose 10 amino acid positions in the V3 loop and incorporated random combinations (27,648 possibilities) of the amino acid substitutions derived from 31 R5 viruses into the V3 loop of HIV-1JR-FL proviral DNA. The constructed HIV-1 library contained 6.6 × 106 independent clones containing a set of 0–10 amino acid substitutions in the V3 loop. To address whether restricted steric alteration in the V3 loop could confer resistance to an entry inhibitor, TAK-779, we selected entry inhibitor-resistant HIV-1 by increasing the concentration of TAK-779 from 0.10 to 0.30 μm in PM1-CCR5 cells with high expression of CCR5. The selected viruses at passage 8 contained five amino acid substitutions in the V3 loop without any other mutations in GP120 and showed 15-fold resistance compared with the parental virus. These results indicated that a certain structure of the V3 loop containing amino acid substitutions derived from 31 R5 viruses can contribute to the acquisition of resistance to entry inhibitors binding to CCR5. Taken together, this type of HIV-1 V3 loop library is useful for isolating and analyzing the specific biological features of HIV-1 with respect to alterations of the V3 loop structure.

The antiretroviral potency of APOBEC1 deaminase from small animal species

Although the role of the APOBEC3-dependent retroelement restriction system as an intrinsic immune defense against human immunodeficiency virus type1 (HIV-1) infection is becoming clear, only the rat ortholog of mammalian APOBEC1s (A1) thus far has been shown to possess antiviral activity. Here, we cloned A1 cDNAs from small animal species, and showed that similar to rat A1, both wild-type and Δvif HIV-1 infection was inhibited by mouse and hamster A1 (4- to 10-fold), whereas human A1 had negligible effects. Moreover, rabbit A1 significantly reduced the infectivity of both HIV-1 virions (>300-fold), as well as that of SIVmac, SIVagm, FIV and murine leukemia virus. Immunoblot analysis showed that A1s were efficiently incorporated into the HIV-1 virion, and their packaging is mediated through an interaction with the nucleocapsid Gag domain. Interestingly, there was a clear accumulation of particular C-T changes in the genomic RNAs of HIV-1 produced in their presence, with few G-A changes in the proviral DNA. Together, these data reveal that A1 may function as a defense mechanism, regulating retroelements in a wide range of mammalian species.

Delayed Onset of Infectious Mononucleosis Associated with Acquired Agammaglobulinemia and Red Cell Aplasia

In 1974, an 11-year-old white boy with the X-linked lymphoproliferative syndrome developed hyper-IgM after becoming infected with Epstein-Barr virus. However, he failed to develop normal immune responses against the virus. In December 1981, when red cell aplasia occurred, he was given packed erythrocytes and gammaglobulin. Nine weeks later, acute infectious mononucleosis developed. Concurrently, his T4/T8 helper/suppressor ratio decreased from 2.7 to 0.2, and IgM antibodies to Epstein-Barr virus appeared. Subsequently, circulating B cells became undetectable in his blood, and agammaglobulinemia appeared. Red cell aplasia abated transiently. This patients course was complicated by Haemophilus influenzae and Mycobacterium tuberculosis pneumonias, and red cell aplasia and agammaglobulinemia have persisted. Epstein-Barr virus acting as a slow virus probably induced the red cell aplasia and agammaglobulinemia because of the aberrant immune responses to Epstein-Barr virus. Immunodeficient responses to Epstein-Barr virus should be sought in other patients with the diseases documented in our patient.

Entry of human immunodeficiency virus (HIV) into MT-2, human T cell leukemia virus carrier cell line

SummaryThe ultrastructural features of early events in human immunodeficiency virus (HIV) infection of HTLV-I-carrying MT-2 lymphocytes were investigated by electron microscopy. Within 10 min after virus inoculation at 37°C, the virus entered the cell in two ways; (1) the virus attached to the lymphocyte membrane and the viral core entered the cell after fusion of the viral envelope with the cell membrane, and (2) part of the cell membrane to which the virus was attached became invaginated, the virus became trapped in a phagosome and the viral core entered after the fusion of viral membrane with the vacuolar membrane. Thereafter, some cells were observed to form syncytia with multiple nuclei. When the proportion of anti-HIV antibody-reactive cells present exceeded 90%, virus production was strongly activated, and budding on the cell membrane was frequently observed.

A combination of polymorphic mutations in V3 loop of HIV-1 gp120 can confer noncompetitive resistance to maraviroc

Maraviroc binds to the pocket of extracellular loops of the cell surface CCR5 and prevents R5 HIV-1 from using CCR5 as a coreceptor for entry into CD4-positive cells. To evaluate the contribution of the V3 loop structure in gp120 to maraviroc resistance, we isolated maraviroc-resistant variants from the V3 loop library virus (HIV-1(V3Lib)) containing a set of random combinations of 0-10 polymorphic mutations in vitro. HIV-1(V3Lib) at passage 17 could not be suppressed even at 10 μM (>1400-fold resistance), while HIV-1(JR-FL) at passage 17 revealed an 8-fold resistance to maraviroc. HIV-1(V3Lib-P17) contained T199K and T275M plus 5 mutations in the V3 loop, I304V/F312W/T314A/E317D/I318V. The profile of pseudotyped virus containing I304V/F312W/T314A/E317D/I318V in V3 loop alone revealed a typical noncompetitive resistance, although T199K and/or T275M could not confer noncompetitive resistance. This type of library virus is useful for isolation of escape viruses from effective entry inhibitors.

Acquisition of multi-PI (protease inhibitor) resistance in HIV-1 in vivo and in vitro.

Protease inhibitors are effective antiviral agents which can lead to a severe decrease in HIV RNA copies in plasma of naive patients, however even successful suppression of the virus with antiretroviral agents including protease inhibitor(s) (PI(s)) generates PI-resistant HIV-1 after long term treatment. Occasionally HIV-1 acquires cross-resistance to other PIs with which the patients have not been treated. Cross-resistance to multiple PIs (multi-PI resistance) leads to a restricted salvage strategy; therefore multi-PI resistance is one of the serious obstacles to efficient antiretroviral chemotherapy. The most common PI-resistance mechanism in HIV-1 is the emergence and accumulation of multiple amino acid substitutions within the viral protease. As well, additional substitutions in protease cleavage sites or substitutions in the Gag protein at non-cleavage sites are involved in recovery of the reduced replication fitness of HIV-1 caused by these mutations in the viral protease. To address or predict the resistance mechanisms of PIs, resistant HIV-1 variants have been intensively studied in vitro. However, the profiles of the amino acid substitutions obtained in PI resistant variants are more diverse and complex than that found in vitro. More elaborate in vitro systems for further analysis of acquisition of PI resistance mechanisms are needed.