Biologic importance of neuraminidase stalk length in influenza A virus.

To investigate the biologic importance of the neuraminidase (NA) stalk of influenza A virus, we generated mutant viruses of A/WSN/33 (H1N1) with stalks of various lengths (0 to 52 amino acids), by using the recently developed reverse genetics system. These mutant viruses, including one that lacked the entire stalk, replicated in tissue culture to the level of the parent virus, whose NA stalk contains 24 amino acid residues. In eggs, however, the length of the stalk was correlated with the efficiency of virus replication: the longer the stalk, the better the replication. This finding indicates that the length of the NA stalk affects the host range of influenza A viruses. The NA stalkless mutant was highly attenuated in mice; none of the animals died even after intranasal inoculation of 10(6) PFU of the virus (the dose of the parent virus required to kill 50% of mice was 10(2.5) PFU). Moreover, the stalkless mutant replicated only in the respiratory organs, whereas the parent virus caused systemic infection in mice. Thus, attenuation of the virus with the deletion of the entire NA stalk raises the possibility of its use as live vaccines.     

Immunohistochemical localization of S100A1 and S100A6 in postnatally developing salivary glands of rats

 S100 proteins are calcium-binding proteins of the EF-hand superfamily and are involved in the regulation of a number of cellular processes. The present study deals with the immunohistochemical expression of S100A1 and S100A6 in the rat submandibular and sublingual glands during postnatal development from day 0 to 12 weeks. Expression of S100A1 was particularly concentrated in pillar and transition cells in the granular convoluted tubule (GCT) and in striated duct cells of the submandibular gland age 4 weeks to maturity. None or only weak staining for S100A1 was observed in the duct segment at 0–5 days. On the contrary, immunostaining of S100A6 was present in proacinar cells in undifferentiated submandibular gland at age 3 days to 2 weeks. S100A6 immunoreactivity in rat submandibular gland coexisted with chromogranin reactivity. It is possible that S100A6 regulates secretion of chromogranin in proacinar cells. Secretion of growth factors and biologically active peptides in the GCT are regulated by calcium signals, and S100A1 may be involved in the secretory mechanism of granular cells. S100A1 and S100A6 are potentially of great importance in secretory functions of granular cells and proacinar cells, as well as in rat salivary glands. Accepted: 14 July 1998     

cDNA cloning and predicted primary structure of scallop sarcoplasmic reticulum Ca-ATPase

Sarcoplasmic reticulum (SR) Ca²⁺-ATPase of the scallop cross-striated adductor muscle was purified with deoxycholate and digested with lysyl endopeptidase for sequencing of the digested fragments. Overlapping cDNA clones of the ATPase were isolated by screening the cDNA library with an RT-PCR product as a hybridization probe, which encodes the partial amino acid sequence of the ATPase. The predicted amino acid sequence of the ATPase contained all the partial sequences determined with the proteolytic fragments and consisted of the 993 residues with 70% overall sequence similarity to those of the SR ATPases from rabbit fast-twitch and slow-twitch muscles. An outline of the structure of the scallop ATPase molecule is predicted to mainly consist of ten transmembrane and five ‘stalk’ domains with two large cytoplasmic regions as observed with the rabbit ATPase molecules. The sequence relationship between scallop and other sarco/endoplasmic reticulum-type Ca²⁺-ATPases is discussed.     

EVOLUTIONARY DIVERGENCE IN THE RED ALGAE CALOGLOSSA LEPRIEURII AND C. APOMEIOTICA

Morphological comparisons, hybridization experiments, and molecular phylogenetic analyses using the RUBISCO spacer region were undertaken on 12 populations of Caloglossa leprieurii (Montagne) J. Agardh in order to clarify their relationships. In addition, data from one population of the morphologically similar but asexual species, C. apomeiotica (West et Zuccarello), were included in the assessment. Three morphological types were recognized on the basis of the number of cell rows at nodes of the main axis opposite to the lateral branch and blade width: single/ broad (with three mating groups), multiple/broad (three mating groups) and multiple/slender (one mating group). In the molecular analyses, C. leprieurii was resolved as two clusters that correspond phenetically to the single and multiple cell row types. Both the morphological and molecular data indicate that the asexual species was derived from sexual plants of the multiple cell row type. The reproductive compatibility correlates with genetic distance rather than geographical distance. Sympatric mating groups are completely incompatible and have 10–21 nucleotide changes in the examined region, whereas mating groups that produce abnormal progeny or pseudocystocarps are allopatrically distributed with 5–7 nucleotide changes. The present data suggest that the two populations, one with single and the other with multiple cell rows, which are sympatrically distributed in southeastern Japan, have probably evolved by allopatric speciation. The single/broad type that is restricted to the western Pacific, may have diverged genetically between eastern and western Australia, with subsequent dispersal from the western population as far as Japan.     

71-Kilodalton Heat Shock Cognate Protein Acts as a Cellular Receptor for Syncytium Formation Induced by Human T-Cell Lymphotropic Virus Type 1

We previously reported that the region corresponding to amino acids 197 to 216 of the gp46 surface glycoprotein (gp46-197) served as a binding domain for the interaction between gp46 and trypsin-sensitive membrane components of the target cell, leading to syncytium formation induced by human T-cell lymphotropic virus type 1 (HTLV-1)-bearing cells. Our new evidence shows that the 71-kDa heat shock cognate protein (HSC70) acts as a cellular receptor for syncytium formation. Using affinity chromatography with the peptide gp46-197, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we isolated three components (bands A, B, and C) from MOLT-4 cell lysate which exhibited specific interactions with gp46 and inhibitory activities for syncytium formation induced by HTLV-1-bearing cells. Band A and B components were identified as HSC70 and β-actin, respectively, through amino acid sequencing by tandem mass spectrometry and immunostaining with specific monoclonal antibodies. Band C is likely to be a nonprotein component, because full activity for syncytium formation was seen after extensive trypsin digestion. Anti-HSC70 monoclonal antibody clearly blocked syncytium formation in a coculture of HTLV-1-bearing cells and indicator cells, whereas no inhibition was seen with anti-β-actin monoclonal antibody. Furthermore, flow cytometric analysis indicated that anti-HSC70 antibody reacted with MOLT-4 cells. Thus, we propose that HSC70 expressed on the target cell surface acts as a cellular acceptor to gp46 exposed on the HTLV-1-infected cell for syncytium formation, thereby leading to cell-to-cell transmission of HTLV-1.     

Archetype JC Virus Efficiently Replicates in COS-7 Cells, Simian Cells Constitutively Expressing Simian Virus 40 T Antigen

JC polyomavirus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), is ubiquitous in humans, infecting children asymptomatically and then persisting in the kidney. Renal JCV is not latent but replicates to excrete progeny in the urine. The renal-urinary JCV DNAs carry the archetype regulatory region that generates various rearranged regulatory regions occurring in JCVs derived from the brains of PML patients. Tissue cultures that support the efficient growth of archetype JCV have not been reported. We studied whether archetype JCV could replicate in COS-7 cells, simian cells transformed with an origin-defective mutant of simian virus 40 (SV40). Efficient JCV replication, as detected by a hemagglutination assay, was observed in cultures transfected with five of the six archetype DNAs. The progeny JCVs could be passaged to fresh COS-7 cells. However, when the parental cells of COS-7 not expressing T antigen were transfected with archetype JCV DNAs, no viral replication was detected, indicating that SV40 T antigen is essential for the growth of JCV in COS-7 cells. The archetype regulatory region was conserved during viral growth in COS-7 cells, although a small proportion of JCV DNAs underwent rearrangements outside the regulatory region. We then attempted to recover archetype JCV from urine by viral culture in COS-7 cells. Efficient JCV production was observed in COS-7 cells infected with five of the six JCV-positive urine samples examined. Thus, COS-7 cells should be of use not only for the production of archetype JCV on a large scale but also for the isolation of archetype JCV from urine.     

Immunization with a Single Major Histocompatibility Complex Class I-Restricted Cytotoxic T-Lymphocyte Recognition Epitope of Herpes Simplex Virus Type 2 Confers Protective Immunity

We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8⁺ cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2K^b-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8⁺ T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8⁺ CTL in HSV vaccine design.

Both humoral and cell-mediated components of the immune response are involved in controlling herpes simplex virus (HSV) infection (51, 61). Studies of humans and of mice have implicated a role for both CD8⁺ (6, 25, 32, 33, 47, 65–67) and CD4⁺ (27, 37–39, 52, 53) T-lymphocyte subsets in mediating protection against HSV infection. For example, CD8⁺ T cells have been shown to be important in limiting replication of HSV in the footpad (6) and colonization of the spinal dorsal root ganglia (6, 66). In contrast, other studies using a zosteriform model of infection have primarily indicated a role for CD4⁺ T cells in the clearance of HSV (37–39). Both CD4⁺ and CD8⁺ (56, 72, 74–76) HSV-specific T lymphocytes have been detected in humans seropositive for HSV. However, the contribution of each subset in the control of HSV infection has not been clearly defined. This illustrates the controversy regarding the relative roles of each subset in the resolution of HSV infection.To address the role of the CD8⁺ T-cell subset in providing acquired immunity to HSV infection, we examined the protection afforded by HSV-specific, CD8⁺ cytotoxic T lymphocytes (CTL) directed to a single CTL recognition epitope. In previous studies by others, immunization with single CTL epitopes has been effective in controlling viral pathogens including lymphocytic choriomeningitis virus (14, 54, 62, 73), murine cytomegalovirus (15), influenza virus (55), and Sendai virus (28). Although HSV-encoded CTL recognition epitopes have been identified by their ability to serve as targets for HSV-specific CTL (3, 8, 24, 64), the ability of CTL directed to these individual epitopes to confer protection against HSV infection has not been determined. We have designed two separate vaccination strategies which permit the exclusive induction of a single HSV epitope-specific, CD8⁺ T-lymphocyte response and have evaluated the ability of this response to confer protective immunity to HSV infection.Hanke et al. (24) broadly identified an immunodominant, H-2K^b-restricted epitope within HSV glycoprotein B (gB). The minimal amino acid sequence of this epitope, gB498-505 (SSIEFARL), was demonstrated by Bonneau et al. (8), using synthetic peptides and an epitope-specific CTL clone. The amino acid sequence, SSIEFARL, is identical in both HSV type 1 (HSV-1) (gB498-505) and HSV-2 (gB496-503) (11). CTL specific for gB498-505 are readily induced by immunization with synthetic peptide (8), a cell line expressing gB498-505 in the context of simian virus 40 (SV40) T antigen (5), and a recombinant viral vector expressing this epitope in the context of a cellular protein (19). In the present study, two recombinant vaccinia viruses (rVV-ES-gB498-505 and rVV-gB498-505) and a recombinant influenza virus (WSN/NA/gB) were generated to express a single HSV-encoded epitope, HSV-1 gB498-505, and were characterized for the ability to induce a potent, HSV-specific CTL response upon mucosal immunization. To determine the protection afforded by immunization with each of the individual recombinant viruses, we used a lethal model of HSV-2 encephalitis. Our findings suggest that the induction of a CTL response directed against a single HSV-specific CTL recognition epitope is sufficient to confer significant protective immunity to HSV infection.