Hisatoshi Shida

Sequence requirements for nucleolar localization of human T cell leukemia virus type I pX protein, which regulates viral RNA processing

The posttranscriptional regulator (p27x-III) of human T cell leukemia virus type I (HTLV-I) is located predominantly in the cell nucleolus. A highly basic amino-terminal sequence (NH2-Met-Pro-Lys-Thr-Arg-Arg-Arg-Pro-Arg-Arg-Ser-Gln-Arg-Lys-Arg-Pro-Pro -Thr- Pro) in this protein, when fused to the amino termini of beta-galactosidase and p40x of HTLV-I, acts as an autonomous signal capable of directing the hybrid proteins to the cell nucleolus.

Nucleotide sequence of the vaccinia virus hemagglutinin gene

Vaccinia virus hemagglutinin (HA) is expressed at late time of infection cycle, and it is nonessential for virus growth. Location of the HA structural gene was determined by hybrid-arrested and hybrid-selected translation methods at the right terminus of the HindIII A fragment. The position of the HA gene was confirmed by the production of the complete HA protein in the cells transfected with the plasmid containing that region. Examination of this nucleotide sequence revealed the positions of cleavage sites for a number of restriction endonucleases. The deduced amino acid sequence revealed that the HA protein is a member of typical surface membrane glycoproteins. Comparison of the nucleotide sequence upstream of the HA coding region with corresponding region of other late genes suggested the existence of the consensus decanucleotides TTCATTTa/tGT between 34 to 18 bp upstream to the initiation codon followed by a cluster of A or T, a unique feature of the late genes of vaccinia virus. These results in conjunction with the ease of isolating HA- mutants provide a basis for a new site suitable for inserting foreign genes.

Hemadsorption and fusion inhibition activities of hemagglutinin analyzed by vaccinia virus mutants

Vaccinia virus IHD-J strain induces hemagglutinin (HA) on the surface membrane of infected cells and does not elicit cell-cell fusion (F-). We isolated 21 independent hemadsorption-negative (HAD-) mutant viruses from IHD-J and five HAD+ revertants from one of these mutants. Of the 21 mutants, 19 that synthesized either no or little HA at the cell surface caused cell-cell fusion (F+), whereas none of the five revertants that synthesized HA at the cell surface induced cell-cell fusion. Furthermore, anti-HA monoclonal antibody B2D10 induced extensive polykaryocytosis of IHD-J-infected cells and suppressed the ability of the IHD-J-infected cell extract to inhibit the polykaryocytosis induced by IHD-W. The other 2 of the 21 HAD- mutants, B1 and A2, which induced HAs at the cell surface, showed F- and F+ phenotype, respectively. The HA molecule of mutant B1 had a single amino acid substitution of Lys for Glu-121 in its extracellular domain, whereas that of mutant A2 had a single substitution mutation of Tyr for Cys-103. We conclude that the vaccinia HA is a fusion inhibition protein, that the active sites for the two activities reside separately in its extracellular domain, and that cysteine-103 is important in forming the proper tertiary structure of the protein to exert both activities.

Mechanism of virus occlusion into A-type inclusion during poxvirus infection

Abstract The A-type inclusion (ATI), a product as a late function of some poxviruses, has different strain-specific properties, some containing mature viruses (V + ATI) and some without (V − ATI). The properties of virus-occluding factor (VO factor) responsible for the V + trait were examined either employing a cell fusion technique by inactivated Sendai virus or under the cell-free condition. The VO factor was synthesized late in the (V − virus-infection corresponding to the time of ATI formation. A proportion of the VO factor synthesized may rapidly attach to the surface of mature V+ viruses (vaccinia) so as to acquire the ability of occlusion within ATI, and a residual proportion of the VO factor released throughout the cytoplasm may attach similarly to that of (V − viruses carried in by cell fusion or added in vitro . Neither protein synthesis nor energy was required to occlude mature viruses into ATIs, and the V + trait occurred in the presence of rifampicin. Comparison of the electrophoresis distribution of proteins constituting V + and (V − virions revealed that the VO factor was identical with the VP 4c polypeptide located at the surface of vaccinia virion. The polypeptide showing the same mobility on polyacrylamide gel electrophoresis was also found in the postviral supernatant fraction prepared from V + virus-infected cells.

In vitro Induction of Cytotoxic T Lymphocytes against HTLV-I-infected T-Cells from Adult T-Cell Leukemia Patients, Asymptomatic HTLV-I Carriers and Seronegative Healthy Donors

We investigated an in vitro method to produce cytotoxic T lymphocytes (CTLs) against HTLV‐I‐infected T‐cells using peripheral blood mononuclear cells (PBMC) of adult T‐cell leukemia (ATL) patients, asymptomatic HTLV‐I carriers (AC) and seronegative healthy donors. The PBMC were restimulated repeatedly for 4 weeks with HLA‐matched HTLV‐I‐infected T‐cells which had been pretreated at 56deg;C for 30 min to inactivate infectious HTLV‐I. The culture medium included 10–100 units/ml of recombinant lymphokines (rIL‐1, rIL‐2, rIL‐4, rIL‐6 and rIL‐7) and 10% fetal calf serum in RPMI‐1640 medium. The cytotoxic activity was measured against HLA‐matched HTLV‐I‐infected T‐cell lines after CD4+ or CD8+ cells were positively panned from the cultured PBMC. The PBMC of ATL, AC and healthy donors were able to produce either CD4+ or CD8+ CTLs against HTLV‐I‐related antigens (env, gag, p21x, p27rex and p40tax) as well as the antigen(s) of as‐yet unknown specificity expressed on HTLV‐I‐infected T‐cells. All the CTLs recognized the specific antigens in the context of either class I or class II HLA types. These results indicated that ATL patients, AC and healthy donors were immunocompetent to generate CTLs against HTLV‐I‐infected T‐cells and probably against HTLV‐I‐transformed T‐cells.

Analysis of the hemagglutinin glycoprotein from mutants of vaccinia virus that accumulates on the nuclear envelope

We isolated mutants whose vaccinia hemagglutinin (HA) accumulates on nuclear envelopes and the rough endoplasmic reticulum. Mutant HA must be blocked at a pre-Golgi step because it has high-mannose-type carbohydrates but no fucose. Neither N- nor O-glycosidically linked carbohydrates are involved in the transport defect of the mutant HA, because tunicamycin, an inhibitor of N-type glycosylation, has no effect, and O-type glycosylation takes place in the Golgi organelle. The unglycosylated form of the mutant HA synthesized in the presence of tunicamycin is 3000 daltons larger than the wild type. This higher molecular weight is related to the transport defect. HAs translated in vitro also show this difference, evidence that it reflects mutation in the HA structural gene. Portions of HAs that project into the cytoplasm seem to account for this weight difference. Thus the cytoplasmic tail of glycoprotein has an important function in transport out of the rough endoplasmic reticulum.

The function of the vaccinia hemagglutinin in the proteolytic activation of infectivity

The vaccinia virus hemagglutinin (HA) has specific affinity for the structural protein, VP37K. The nature of this affinity and its relationship to the function of the HA were analyzed using HA mutants. The VP37K reactive site of the HA molecule is located in its transmembrane region, and the vaccinia virus HA associates with the viral particle via the VP37K-HA affinity. The viruses possessing an HA with fusion inhibitor activity were largely of the low infectivity form, whereas the viruses that associated mutant HAs defective in the activity were of the high infectivity form. D1 mutant virus does not produce HA. When it was incubated with the HA of the IHD-J strain, the HA associated with the virus particle. The HA-loaded D1 mutant virus acquired a high affinity not only for chick erythrocytes but also for KB and Vero cells. At the same time, the infectivity for Vero cells was decreased. The original high infectivity was recovered by treatment with trypsin. The virion-associated vaccinia HA has two functions; the HA protects the infectivity of the virus by the fusion inhibitor activity and exhibits affinity against host cells. Vaccinia virus first adsorbs to the cell via HA, and then proteolysis of the HA activates the second adsorption site which seems to be the fusogenic site of the virus. Proteolytic activation represents removal of the fusion inhibitor activity of the HA.

Vaccinia Virus Hemagglutinin

Several viruses agglutinate erythrocytes of various animal species. The components responsible for this phenomenon have been termed hemagglutinins (HA), which in many cases have been identified as glycoproteins present in virus envelopes. The HA titers have often been used as convenient indicators of the amounts of viruses. Recent studies on virus HA dealt not only with their structures and functions in virus replication but with the molecular mechanisms of their biosynthesis as well, applicable in general to membrane glycoproteins.

Immunogenicity of a recombinant vaccinia virus expressing envelope a glycoprotein of bovine leukaemia virus.

We constructed a recombinant vaccinia virus (RVV) expressing envelope (env) glycoprotein (gp51) of bovine leukaemia virus (BLV): the expression of gp51 was detected by Western blot of the lysates of rabbit kidney cells infected with the RVV. The rabbits inoculated intradermally with the RVV alone failed to induce detectable anti-gp51 antibodies even 10 weeks after immunization. However, when these animals were boosted with inactivated BLV virion in saline, significant levels of anti-gp51 antibodies were induced as shown both in Western blot and immunodiffusion analyses. In these animals, antibodies against gag product (p24) were not detected. On the other hand, the rabbits inoculated with wild-type vaccinia virus and boosted similarly three times with the BLV virion in saline did not induce detectable anti-gp51 antibodies at all. The present experiment revealed that the RVV possessed the capability to endow immunological memory without inducing apparent anti-gp51 antibody responses, meaning that the RVV activated helper T cells far more strongly than B cells. The applicability of the RVV to vaccine development is discussed.

Two Distinct Pathways for Intronless mRNA Expression: One Related, the Other Unrelated to Human Immunodeficiency Virus Rev and Human T Cell Leukemia Virus Type I Rex Functions

Intronless mRNAs were classified into two classes based on the sensitivities of their expression to the inhibitory effect of TAgRex, a dominant-negative mutant of the Rex protein of human T cell leukemia virus type I, and their abilities to express the genes encoded in the intron of the human immunodeficiency virus (HIV) genome. Interferon-alpha mRNA could not induce the expression of the env gene of HIV, and its expression was resistant to TAgRex. In contrast, the posttranscriptional regulatory element (PRE), necessary for the nucleo-cytoplasmic export of mRNAs of hepatitis B virus, induced expression of the chloramphenicol acetyl transferase gene located within the intron of the HIV genome. PRE-mediated expression was inhibited by TAgRex. Thus, these results suggest that there are at least two distinct pathways for intronless mRNA expression, one related to and the other unrelated to Rev and Rex functions.