Shigeharu Ueda

Selective amplification of cDNA sequence from total RNA by cassette-ligation mediated polymerase chain reaction (PCR): Application to sequencing 6·5 kb genome segment of hantavirus strain B-1

A method, referred to as cassette-ligation mediated polymerase chain reaction (PCR), has been developed to permit selective and specific amplification of cDNA sequence from total cellular RNA. This technique comprises (i) digestion of cDNA with multiple restriction enzymes, (ii) ligation of cleavage products to double-stranded DNA cassettes possessing a corresponding restriction site and (iii) amplification of cassette-ligated restriction fragments containing a short, known sequence (but not all the other ligation products) by PCR using the specific and cassette primers; the specific primer is designed to prime synthesis from the known sequence of the cDNA whereas the cassette primer anneals to one strand of the cassette. Sequencing from the cassette primer provides information to design a new primer for the next walking step. The amplified cDNA fragments are often larger than the maximum DNA fragments (500-600 bp) that can be sequenced without the need of synthesizing internal sequencing primer. Each of such large cDNA fragments is dissected into smaller DNA fragments by repeating cassette-ligation mediated PCR exploiting different restriction sites and different sets of cassette primers. This dissection process reduces the number of specific primers to a minimum, thereby increasing the speed of sequencing and minimizing the overall cost. We have successfully applied this cDNA walking and sequencing by the cassette-ligation mediated PCR to the sequencing of an entire 6.5 kb genome segment of hantavirus strain B-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation and characterization of the human immunodeficiency virus type 1 mutants

SummaryMutations were introduced by recombinant DNA techniques into 9 genes of an infectious molecular clone of human immunodeficiency virus type 1. The 24 mutants generated were characterized biochemically and biologically by transfection and infection experiments. None of the mutants which have mutations ingag (p17, p24, and p15 regions),pol (protease, reverse transcriptase, and endonuclease domains),env (gp120 region),tat, orrev were infectious, whereasvif, vpr, vpu, some ofenv (gp41) andnef mutants could grow in human CD4+ cells to various degrees. Of the non-infectious mutants, only endonuclease (pol) and gp41 mutants exhibited normal phenotypes with respect to the production of functional reverse transcriptase, the expression ofgag, pol, andenv proteins, and the generation of progeny virions, when examined in transient assays. All infectious mutants killed the CD4+ cells with the exception of a mutant carrying a defect in thevif gene.

Human membrane cofactor protein (MCP, CD46): multiple isoforms and functions.

Human membrane cofactor protein (MCP, CD46) is a 45-70 kDa protein with genetic and tissue-specific heterogeneity, and is expressed on all nucleated cells. MCP consists from N-terminus of 4 short consensus repeats (SCRs), 1-3 serine/threonine-rich (ST) domains, a transmembrane domain (TM) and a cytoplasmic tail (CYT). More than 8 isoforms are generated secondary to alternative splicing due to combinations of various exons encoding the ST, TM and CYT domains. It serves as a cofactor of serine protease factor I for inactivation of complement C3b and C4b. Its primary role is to protect host cells from homologous complement attack by inactivating C3b/C4b deposited on the membrane. It also acts as receptors for measles virus (MV), some kinds of bacteria and for a putative ligand on oocytes. MV infection causes temporal host immune suppression, which may appear secondary to signaling events through MCP on macrophages and dendritic cells. These functional properties of human MCP may facilitate xenotransplantation and may be useful in the generation of animal models of measles by creating human MCP-expressing animals.

Functional Modulation of Human Macrophages Through CD46 (Measles Virus Receptor): Production of IL-12 p40 and Nitric Oxide in Association with Recruitment of Protein-Tyrosine Phosphatase SHP-1 to CD46

Human CD46, formerly membrane cofactor protein, binds and inactivates complement C3b and serves as a receptor for measles virus (MV), thereby protecting cells from homologous complement and sustaining systemic measles infection. Suppression of cell-mediated immunity, including down-regulation of IL-12 production, has been reported on macrophages (Mφ) by cross-linking their CD46. The intracellular events responsible for these immune responses, however, remain unknown. In this study, we found that 6- to 8-day GM-CSF-treated peripheral blood monocytes acquired the capacity to recruit protein-tyrosine phosphatase SHP-1 to their CD46 and concomitantly were able to produce IL-12 p40 and NO. These responses were induced by stimulation with mAbs F(ab′)2 against CD46 that block MV binding or by a wild-type MV strain Kohno MV strain (KO; UV treated or untreated) that was reported to induce early phase CD46 down-regulation. Direct ligation of CD46 by these reagents, but not intracellular MV replication, was required for these cellular responses. Interestingly, the KO strain failed to replicate in the 6- to 8-day GM-CSF-cultured Mφ, while other MV strains replicated to form syncytia under the same conditions. When stimulated with the KO strain, rapid and transient dissociation of SHP-1 from CD46 was observed. These and previous results provide strong evidence that CD46 serves as a signal modulatory molecule and that the properties of ligands determine suppression or activation of an innate immune system at a specific maturation stage of human Mφ.

Possible role of dimerization in human immunodeficiency virus type 1 genome RNA packaging

ABSTRACT The dimer initiation site/dimer linkage sequence (DIS/DLS) region in the human immunodeficiency virus type 1 (HIV-1) RNA genome is suggested to play important roles in various steps of the virus life cycle. However, due to the presence of a putative DIS/DLS region located within the encapsidation signal region (E/psi), it is difficult to perform a mutational analysis of DIS/DLS without affecting the packaging of RNA into virions. Recently, we demonstrated that duplication of the DIS/DLS region in viral RNA caused the production of partially monomeric RNAs in virions, indicating that the region indeed mediated RNA-RNA interaction. We utilized this system to assess the precise location of DIS/DLS in the 5′ region of the HIV-1 genome with minimum effect on RNA packaging. We found that the entire lower stem of the U5/L stem-loop was required for packaging, whereas the region important for dimer formation was only 10 bases long within the lower stem of the U5/L stem-loop. The R/U5 stem-loop was required for RNA packaging but was completely dispensable for dimer formation. The SL1 lower stem was important for both dimerization and packaging, but surprisingly, deletion of the palindromic sequence at the top of the loop only partially affected dimerization. These results clearly indicated that the E/psi of HIV-1 is much larger than the DIS/DLS and that the primary DIS/DLS is completely included in the E/psi. Therefore, it is suggested that RNA dimerization is a part of RNA packaging, which requires multiple steps.

A VP4 sequence highly conserved in human rotavirus strain AU-1 and feline rotavirus strain FRV-1

The primary amino acid sequence of the VP4 proteins of human rotavirus strain AU-1 and feline rotavirus strain FRV-1 was deduced from nucleotide sequence analysis of full-length genome segment 4 cDNAs produced by a combined reverse transcription-polymerase chain reaction. The VP4 genes were 2359 nucleotides in length and contained one long open reading frame capable of encoding a protein of 775 amino acids. Strain AU-1 and FRV-1 VP4s were 98.8% similar at both the nucleotide sequence and amino acid level. Given that most of the genome segments of strains AU-1 and FRV-1 formed hybrids under stringent hybridization conditions, the relationship between their VP4 gene sequences is best explained by feline rotavirus being transmitted to human hosts as whole virions relatively recently. Of added interest is that AU-1 and FRV-1 VP4 both exhibit high degrees of similarity (96.0% nucleotide identity and 97.2 to 97.5% amino acid identity) with serotype G1 human rotavirus strain K8 VP4, which is distinct from any other sequenced VP4 allele. This suggests that strain K8 VP4 was derived by natural gene reassortment from a feline rotavirus or a strain AU-1-like human rotavirus.

Isolation and characterization of conglutinin as an influenza A virus inhibitor.

Normal horse and guinea pig sera contain alpha 2-macroglobulin which inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H2 and H3 subtypes. On the other hand, normal bovine serum contains a component termed beta inhibitor that inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H1 and H3 subtypes. To investigate the nature of the beta inhibitor of influenza A virus, we purified the conglutinin and examined its characteristics. First, we found a high correlation between the hemagglutination inhibition(HI) titer and conglutinin titer in several bovine sera (r = 0.906, p less than 0.005). The HI of bovine serum was mainly dependent on conglutinin because the HI activity was abrogated by N-acetylglucosamine but not by D-mannose. The conglutinin, purified from bovine serum, had neutralizing-activity as well as HI activity on influenza A viruses of the H1 and H3 subtypes. The HI activity of conglutinin was heat stable (56 degrees C, 30 min), Ca(++)-dependent, and resistant to both neuraminidase and periodate treatments. The HI activity of purified conglutinin was blocked by N-acetylglucosamine but not by D-mannose. The conglutinin was bound to hemagglutinin which had high mannose and complex sugar chains and its binding was inhibited by N-acetylglucosamine and dependent on divalent cations. These data indicate that the beta-like inhibitor activity of bovine serum is mainly dependent on conglutinin which inhibits hemagglutination and neutralizes the virus infectivity by its binding to a carbohydrate site at the HA.

Analysis of Marek's disease virus serotype 1-specific phosphorylated polypeptides in virus-infected cells and Marek's disease lymphoblastoid cells

By use of monoclonal antibodies, a virus-specific cytoplasmic antigen related to phosphorylated polypeptides specific to serotype 1 of Mareks disease virus (MDV)-related viruses (MDV1) has been identified in all MD tumour cell lines examined, as well as in infected cells and in tumour lesions of chickens with MD. At least two phosphorylated polypeptides with mol. wt. 39,000 (39K) to 36K and 24K (pp39/36 and pp24, respectively) were identified in the MD tumour cell line H10 cultured at 33 degrees C by immunoprecipitation with monoclonal antibody M21 which reacts with virus-specific phosphorylated polypeptides. These polypeptides were not detected in cells infected with MDV-related viruses of serotype 2 or 3. Immunoblot analysis indicated that these two polypeptides contained a serotype 1-specific epitope recognized with M21. An additional 41K polypeptide appeared in different virus strains of serotype 1. These polypeptides were found to contain phosphorylated serine but no detectable phosphorylated tyrosine or phosphorylated threonine. Cell fractionation indicated that the two phosphorylated polypeptides were mainly associated with smooth and rough endoplasmic reticulum fractions of cells infected with MDV1. Furthermore, the mRNA coding for pp39/36 could be separated from that coding for pp24 on a sucrose density gradient. These results suggest that pp24 and pp39/36 are translated from distinct mRNAs and encoded from overlapping genes or separate regions with partial DNA homology in the MDV1 genome.

Most Virus-specific Polypeptides in Cells Productively Infected with Marek's Disease Virus or Herpesvirus of Turkeys Possess Cross-reactive Determinants

Most virus-specific polypeptides in cells productively infected with Mareks disease virus (MDV) or herpesvirus of turkeys (HVT) possess cross-reactive antigenic determinants, although the two viruses have very little DNA homology. The cross-reactivity appeared to be more evident when [35S]methionine-labelled polypeptides were immunoprecipitated than when the [3H]glucosamine-labelled polypeptides were immunoprecipitated, suggesting that the glycoproteins of MDV and HVT may be less related in structure than other proteins. The major cross-reactive glycoproteins excreted into the culture mediums of MDV- and HVT-infected cells had molecular weights of 64 000 (gp64) and 56 000 (gp56) respectively.

Association of serine in position 1124 of Hantaan virus glycoprotein with virulence in mice

Hantaan virus (HV) of the genus Hantavirus causes a fatal disease in suckling mice following intraperitoneal or intracerebral infection. HV cl-1, which was obtained from the 76-118 strain of HV by growth in Vero E6 cells, exhibited high mortality rates in mice whereas mice infected with HV cl-2 survived without any clinical signs. To determine the molecular basis for the marked difference in virulence, we compared the nucleotide sequences of the large (L), medium (M) and small (S) segments of HV cl-1 genome with those of HV cl-2 and found that there was only one predicted amino acid substitution. This amino acid substitution was in position 1124 of the glycoprotein encoded by the M genome segment, in which serine in HV cl-1 was replaced by glycine in HV cl-2. Although there were several nucleotide and amino acid differences between the parental 76-118 strain and HV cl-1, the serine in position 1124 of the glycoprotein was common to the pathogenic parent and the pathogenic mutant. These results suggest that this substitution may be responsible for the virulence of this hantavirus.