Asato Kojima

Two species of human CRK cDNA encode proteins with distinct biological activities.

Two distinct human CRK cDNAs, designated CRK-I and CRK-II, were isolated from human embryonic lung cells by polymerase chain reaction and by screening of a human placenta cDNA library, respectively. CRK-I differed from CRK-II in that it lacked a 170-nucleotide sequence, suggesting that CRK-I and CRK-II were the products of alternative splicing. The amino acid sequences deduced from these two cDNAs differed in the carboxyl termini and contained one SH2 and either one or two SH3 domains. RNAse protection analysis demonstrated both CRK-I and CRK-II mRNAs in various human cells. Three CRK proteins, of 42, 40, and 28 kDa, were identified in human embryonic lung cells by means of antibodies against the SH2 region and the SH3 region of the bacterially expressed CRK-I protein. Transient expression of CRK-I and CRK-II cDNAs in COS7 cells showed that the former encoded the 28-kDa protein and the latter encoded the 40- and 42-kDa proteins. All human cell lines so far examined expressed the 40-kDa protein; however, expression of the 28- and the 42-kDa proteins was variable. In a comparison of the biological activity of the two human CRK proteins, both proteins were stably expressed in rat 3Y1 cells. All cell lines expressing CRK-I protein showed altered morphology, proliferated in soft agar, and grew as massive tumors in nude mice. Although CRK-II-expressing cells showed a slight morphologic change, they did not make colonies in soft agar or grow in nude mice. These results demonstrate that the two species of human CRK cDNA encode proteins which differ in their biological activities.

All APOBEC3 family proteins differentially inhibit LINE-1 retrotransposition

Approximately 17% of the human genome is comprised of long interspersed nuclear element 1 (LINE-1, L1) non-LTR retrotransposons. L1 retrotransposition is known to be the cause of several genetic diseases, such as hemophilia A, Duchene muscular dystrophy, and so on. The L1 retroelements are also able to cause colon cancer, suggesting that L1 transposition could occur not only in germ cells, but also in somatic cells if innate immunity would not function appropriately. The mechanisms of L1 transposition restriction in the normal cells, however, are not fully defined. We here show that antiretroviral innate proteins, human APOBEC3 (hA3) family members, from hA3A to hA3H, differentially reduce the level of L1 retrotransposition that does not correlate either with antiviral activity against Vif-deficient HIV-1 and murine leukemia virus, or with patterns of subcellular localization. Importantly, hA3G protein inhibits L1 retrotransposition, in striking contrast to the recent reports. Inhibitory effect of hA3 family members on L1 transposition might not be due to deaminase activity, but due to novel mechanism(s). Thus, we conclude that all hA3 proteins act to differentially suppress uncontrolled transposition of L1 elements.

Impaired Processing and Presentation of Cytotoxic-T-Lymphocyte (CTL) Epitopes Are Major Escape Mechanisms from CTL Immune Pressure in Human Immunodeficiency Virus Type 1 Infection

ABSTRACT Investigating escape mechanisms of human immunodeficiency virus type 1 (HIV-1) from cytotoxic T lymphocytes (CTLs) is essential for understanding the pathogenesis of HIV-1 infection and developing effective vaccines. To study the processing and presentation of known CTL epitopes, we prepared Epstein-Barr virus-transformed B cells that endogenously express the gag gene of six field isolates by adopting an env/nef-deletion HIV-1 vector pseudotyped with vesicular stomatitis virus G protein and then tested them for the recognition by Gag epitope-specific CTL lines or clones. We observed that two field variants, SLFNTVAVL and SVYNTVATL, of an A*0201-restricted Gag CTL epitope SLYNTVATL, and three field variants, KYRLKHLVW, QYRLKHIVW, and RYRLKHLVW, of an A24-restricted Gag CTL epitope KYKLKHIVW escaped from being killed by the CTL lines, despite the fact that they were recognized when the synthetic peptides corresponding to these variant sequences were exogenously loaded onto the target cells. Thus, their escape is likely due to the changes that occur during the processing and presentation of epitopes in the infected cells. Mutations responsible for this mode of escape were located within the epitope regions rather than the flanking regions, and such mutations did not influence the virus replication. The results suggest that the impaired antigen processing and presentation often occur in HIV-1 field isolates and thus are one of the major mechanisms that enable HIV-1 to escape from CTL recognition. We emphasize the importance of testing HIV-1 variants in an endogenous expression system.

An infectious DNA clone of HIV type 1 subtype C.

Among the 10 subtypes of the M group of human immunodeficiency virus type 1, subtype C is the most prevalent in India and may dominate worldwide in the near future; however, there has been no report on the infectious DNA clone of this subtype. We have isolated an infectious DNA clone of the 93IN101 strain of HIV-1 subtype C, which was isolated in India in 1993. MAGIC5 cells, which are derived from HeLa-CD4-LTR-beta-gal (MAGI) cells and express CCR5, were inoculated with the 93IN101 strain of HIV-1 subtype C. The genomic DNA of the infected cells was used as a template for amplification of the HIV-1 genome. The genome DNA obtained was subcloned into pBR322, and the resulting plasmid was designated as pIndie-C1. The insert of pIndie-C1 was 9680 bp in length and had an intact genomic organization with open reading frames of all structural, regulatory, and accessory proteins. Phylogenetic analysis confirmed that the nucleotide sequence of pIndie-C1 is closely related to those of HIV-1 subtype C isolated in India. Transfection of pIndie-C1 into 293T cells yielded as much virus as did pNL432, one of the most widely used HIV DNA clones. The recovered Indie-C1 virus infected MAGIC5 but not the parent MAGI cells, indicating that Indie-C1 is CCR5 tropic. Expressed Env protein was reacted efficiently with the sera of HIV-1-infected patients of India, but not of Japan. Expression of Nef and Vpr was also confirmed by immunoblotting.

Rapid and Simple Phenotypic Assay for Drug Susceptibility of Human Immunodeficiency Virus Type 1 Using CCR5-Expressing HeLa/CD4+ Cell Clone 1-10 (MAGIC-5)

ABSTRACT We describe a rapid and simple novel phenotypic assay for drug susceptibility of human immunodeficiency virus type-1 (HIV-1) using a CCR5-expressing HeLa/CD4+ cell clone 1-10 (MAGIC-5). MAGIC-5 cells produced large amounts of HIV-1 in culture supernatants, which enabled us to perform the phenotypic resistance assay. Determination of HIV-1 susceptibility to various protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors was completed within 15 days in T-cell-tropic (X4) and macrophage-tropic (R5) viruses using fresh plasma samples containing at least 104copies/ml. The nucleotide sequence of the envelope V3 region of HIV-1 in plasma was almost identical to that of the virus isolated by MAGIC-5 cells, suggesting a lack of selection bias in our assay. The assay variability was confined to within five-fold in all drugs examined. Accordingly, we used a 10-fold increase in the 50% inhibitory concentration as the cutoff value for viral resistance in the present assay. HIV-1 resistant to lamivudine, which was not detected by conventional genotypic assays, was isolated. In HIV-1 with PI-associated primary amino acid substitutions, our assay showed that drug resistance profiles correlated well with previously reported genotypic-assay data. Furthermore, our assay provided comprehensive results regarding PI resistance in the presence of multiple mutations. The novel assay successfully quantified the level of resistance of clinical HIV-1 isolates to a battery of anti-HIV drugs, indicating its clinical usefulness, particularly in patients who failed to respond to antiretroviral chemotherapy.

An Attenuated LC16m8 Smallpox Vaccine: Analysis of Full-Genome Sequence and Induction of Immune Protection

ABSTRACT The potential threat of smallpox bioterrorism has made urgent the development of lower-virulence vaccinia virus vaccines. An attenuated LC16m8 (m8) vaccine was developed in 1975 from the Lister strain used in the World Health Organization smallpox eradication program but was not used against endemic smallpox. Today, no vaccines can be tested with variola virus for efficacy in humans, and the mechanisms of immune protection against the major intracellular mature virion (IMV) and minor extracellular enveloped virion (EEV) populations of poxviruses are poorly understood. Here, we determined the full-genome sequences of the m8, parental LC16mO (mO), and grandparental Lister (LO) strains and analyzed their evolutionary relationships. Sequence data and PCR analysis indicated that m8 was a progeny of LO and that m8 preserved almost all of the open reading frames of vaccinia virus except for the disrupted EEV envelope gene B5R. In accordance with this genomic background, m8 induced 100% protection against a highly pathogenic vaccinia WR virus in mice by a single vaccination, despite the lack of anti-B5R and anti-EEV antibodies. The immunogenicity and priming efficacy with the m8 vaccine consisting mainly of IMV were as high as those with the intact-EEV parental mO and grandparental LO vaccines. Thus, mice vaccinated with 107 PFU of m8 produced low levels of anti-B5R antibodies after WR challenge, probably because of quick clearance of B5R-expressing WR EEV by strong immunity induced by the vaccination. These results suggest that priming with m8 IMV provides efficient protection despite undetectable levels of immunity against EEV.

Amino Acid 36 in the Human Immunodeficiency Virus Type 1 gp41 Ectodomain Controls Fusogenic Activity: Implications for the Molecular Mechanism of Viral Escape from a Fusion Inhibitor

ABSTRACT We have previously described a human immunodeficiency virus type 1 (HIV-1) proviral clone, pL2, derived from defective viral particles with higher fusogenicity than the prototypic NL4-3 virus. In this study, we attempted to determine the region that confers the enhanced fusion activity by creating envelope recombinants between pL2 and pNL4-3, as well as point mutants based on pNL4-3. The results indicate that amino acid 36 of gp41 is key for the fusogenic activity and infectivity enhancement and that glycine 36 (36G) of gp41 in pL2 is conserved in nearly all HIV-1 isolates except for pNL4-3. The mutation 36G→D in a primary-isolate-derived Env decreased syncytium-forming activity and infectivity. The assays for cell-cell fusion and viral binding suggested that the enhanced fusion mediated by the 36D→G mutation is not due to increased binding efficiency but is directly due to actual enhancement of viral fusion activity. Interestingly, this amino acid position is exactly equivalent to that at which the mutation of HIV-1 isolates that have escaped from a fusion inhibitor, enfuvirtide (T-20), has been frequently observed. The correlation between these previous findings and our findings was suggested by structural analysis. Our finding, therefore, has implications for a molecular basis of the viral escape from this drug.

Stable High-Producer Cell Clone Expressing Virus-Like Particles of the Japanese Encephalitis Virus E Protein for a Second-Generation Subunit Vaccine

ABSTRACT We produced and characterized a cell clone (J12#26 cells) that stably expresses Japanese encephalitis virus (JEV) cDNA, J12, which encodes the viral signal peptide, premembrane (prM), and envelope (E) proteins (amino acid positions 105 to 794). Rabbit kidney-derived RK13 cells were transfected with a J12 expression plasmid, selected by resistance to marker antibiotics, and cloned by two cycles of a limiting-dilution method in the presence of antibiotics, a procedure that prevents the successful generation of E-producing cell clones. J12#26 cells secreted virus-like particles containing the authentic E antigen (E-VLP) into the culture medium in a huge enzyme-linked immunosorbent assay-equivalent amount (2.5 μg per 104 cells) to the internationally licensed JE vaccine JE-VAX. E-VLP production was stable after multiple cell passages and persisted over 1 year with 100% expressing cells without detectable cell fusion, apoptosis, or cell death, but was suspended when the cells grew to 100% confluency and contact inhibition occurred. Mice immunized with the purified J12#26 E-antigen without adjuvant developed high titers of neutralizing antibodies for at least 7 months and 100% protection against intraperitoneal challenge with 5 × 106 PFU of JEV when examined according to the JE vaccine standardization protocol. These results suggest that the recombinant E-VLP antigen produced by the J12#26 cell clone is an effective, safe, and low-cost second-generation subunit JE vaccine.

Immunogenicity and efficacy of two types of West Nile virus-like particles different in size and maturation as a second-generation vaccine candidate.

Virus-like particles (VLPs) of flaviviruses generated from the prM and E genes are a promising vaccine candidate. We have established cell clones continuously releasing VLPs of West Nile virus (WNV) in serum-free conditions. Two types of VLPs were distinguished by sedimenting analyses in sucrose density gradients. Fast sedimenting VLPs (F-VLPs) were large (40-50 nm) and composed of the E and processed mature M proteins, whereas slowly sedimenting VLPs (S-VLPs) were small (20-30 nm) particles consisting of the E and immature prM proteins. F-VLPs induced higher neutralizing antibody and anti-WNV IgG titers than S-VLPs. Furthermore, IgG2a was dominant over IgG1 by immunization with F-VLPs as with whole virion-derived antigens. Mice vaccinated with a low dose (3 ng) of F-VLPs showed higher protective efficacy (83% survivals) against WNV infection than S-VLP-immune mice (17% survivals). These results indicate that F-VLPs more closely resemble the virions and take a better immunogenic form than S-VLPs as WNV vaccine candidates.

Experimental infection of cynomolgus and African green monkeys with human herpesvirus 6

Cynomolgus and African green monkeys were inoculated with human herpesvirus 6 (HHV-6). An antibody response was first observed 10 days and 5 days after inoculation of cynomolgus monkeys and African green monkeys, respectively, and was detectable for the duration of the experiment (33 days). HHV-6 DNA was first detected by the polymerase chain reaction in mononuclear cells of one cynomolgus monkey and one African green monkey 10 days after virus inoculation, and in a total of three of four cynomolgus monkeys (75%) and four of five African green monkeys (80%) later after inoculation. Furthermore, HHV-6 DNA was detected in the lymph nodes and spleen of monkeys killed 33 days after virus inoculation. A rash was observed on the trunk of one African green monkey 13 days after virus inoculation, otherwise the infection was asymptomatic. When mononuclear cells from both groups of monkeys were cultured in medium containing concanavalin A and interleukin 2, and infected with HHV-6 in vitro, virus replication was observed. The data suggest that HHV-6 infects these species of monkey and that this system could be useful as an animal model of HHV-6 infection.