Reactivation of latent human immunodeficiency virus 1 by human herpesvirus 6 infection.

Infection of the ACH-2 line of human leukemic T cells carrying latent Human immunodeficiency virus 1 (HIV-1) with Human herpesvirus 6 (HHV-6) resulted in an increase in reverse transcriptase (RT) activity, a marker of HIV-1 activation, in the culture supernatant. A similar effect was obtained with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The RT activity reached a peak at 24 hrs post infection (p.i.) and then declined, suggesting that the cells underwent lysis. The HIV-1 antigen was co-expressed with an early-late HHV-6 product, but not always with an immediate-early (IE) HHV-6 product, suggesting that one or more IE gene products were involved in the activation of latent HIV-1 in ACH-2 cells.

The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans.

In metazoans, most pre-messenger RNAs contain introns that are removed by splicing. The spliced mRNAs are then exported to the cytoplasm. Recent studies showed that splicing promotes efficient mRNA export, but the mechanism for coupling these two processes is not known. Here we show that Aly, the metazoan homologue of the yeast mRNA export factor Yralp (ref. 2), is recruited to messenger ribonucleoprotein (mRNP) complexes generated by splicing. In contrast, Aly does not associate with mRNPs assembled on identical mRNAs that already have no introns or with heterogenous nuclear RNP (hnRNP) complexes. Aly is recruited during spliceosome assembly, and then becomes tightly associated with the spliced mRNP. Aly shuttles between the nucleus and cytoplasm, and excess recombinant Aly increases both the rate and efficiency of mRNA export in vivo. Consistent with its splicing-dependent recruitment, Aly co-localizes with splicing factors in the nucleus. We conclude that splicing is required for efficient mRNA export as a result of coupling between the splicing and the mRNA export machineries.

Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin-3, a tight junction integral membrane protein.

Claudins (claudin-1 to -18) with four transmembrane domains and two extracellular loops constitute tight junction strands. The peptide toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to claudin-3 and -4, but not to claudin-1 or -2. We constructed claudin-1/claudin-3 chimeric molecules and found that the second extracellular loop of claudin-3 conferred CPE sensitivity on L fibroblasts. Furthermore, overlay analyses revealed that the second extracellular loop of claudin-3 specifically bound to CPE at the K(a) value of 1.0x10(8) M(-1). We concluded that the second extracellular loop is the site through which claudin-3 interacts with CPE on the cell surface.

Nup116p associates with the Nup82p-Nsp1p-Nup159p nucleoporin complex.

Nup116p is a GLFG nucleoporin involved in RNA export processes. We show here that Nup116p physically interacts with the Nup82p-Nsp1p-Nup159p nuclear pore subcomplex, which plays a central role in nuclear mRNA export. For this association, a sequence within the C-terminal domain of Nup116p that includes the conserved nucleoporin RNA-binding motif was sufficient and necessary. Consistent with this biochemical interaction, protein A-Nup116p and the protein A-tagged Nup116p C-terminal domain, like the members of the Nup82p complex, localized to the cytoplasmic side of the nuclear pore complex, as revealed by immunogold labeling. Finally, synthetic lethal interactions were found between mutant alleles of NUP116 and all members of the Nup82p complex. Thus, Nup116p consists of three independent functional domains: 1) the C-terminal part interacts with the Nup82p complex; 2) the Gle2p-binding sequence interacts with Gle2p/Rae1p; and 3) the GLFG domain interacts with shuttling transport receptors such as karyopherin-beta family members.

Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

Identification of functional domains of Bordetella dermonecrotizing toxin.

Bordetella dermonecrotizing toxin (DNT) stimulates the assembly of actin stress fibers and focal adhesions by deamidating Gln63 of the small GTPase Rho. To clarify the functional and structural organization of DNT, we cloned and sequenced the DNT gene and examined the functions of various DNT mutants. Our analyses of the nucleotide and amino acid sequences revealed that the start codon of the DNT gene is a GTG triplet located 39 bp upstream of the reported putative initiation ATG codon; consequently, DNT contains an additional 13 amino acids at its N-terminal end. All of the N-terminally truncated mutants were found to modify Rho. The shortest fragment of DNT possessing the Rho modification activity consists of amino acids from Ile1176 to the C-terminal end. This fragment overlaps the region homologous to Escherichia coli cytotoxic necrotizing factors (CNFs), which show activity similar to that of DNT. The introduction of a mutation at Cys1305 located in the highly conserved region between CNFs and DNT eliminated the activity, indicating that this domain is the catalytic center of DNT. The N-terminal fragment (1 to 531) of DNT failed to modify Rho but reduced the DNT-induced polynucleation in MC3T3-E1 cells when simultaneously added with the holotoxin, suggesting competitive inhibition in the receptor-binding or internalizing step. Our finding that DNT consists of an N-terminal receptor-binding and/or internalizing domain and a C-terminal catalytically active domain may facilitate analysis of the overall action of the toxin on the mammalian target cells.

The Mex67p-mediated nuclear mRNA export pathway is conserved from yeast to human.

Human TAP is an orthologue of the yeast mRNA export factor Mex67p. In mammalian cells, TAP has a preferential intranuclear localization, but can also be detected at the nuclear pores and shuttles between the nucleus and the cytoplasm. TAP directly associates with mRNA in vivo, as it can be UV-crosslinked to poly(A)+ RNA in HeLa cells. Both the FG-repeat domain of nucleoporin CAN/Nup214 and a novel human 15 kDa protein (p15) with homology to NTF2 (a nuclear transport factor which associates with RanGDP), directly bind to TAP. When green fluorescent protein (GFP)-tagged TAP and p15 are expressed in yeast, they localize to the nuclear pores. Strikingly, co-expression of human TAP and p15 restores growth of the otherwise lethal mex67::HIS3/mtr2::HIS3 double knockout strain. Thus, the human TAP-p15 complex can functionally replace the Mex67p-Mtr2p complex in yeast and thus performs a conserved role in nuclear mRNA export.

Beard restoration using a free puboinguinal hair-bearing flap.

A free puboinguinal hair-bearing flap was transferred with anastomosis of the external pudendal vessels, for reconstruction of a bearded chin in an adult male. Although the flap does not match the skin of the chin in texture or color, it can provide an excellent beard in terms of the color, density and quality of hair growth. Donorsite morbidity is minimal.

Bordetella bronchiseptica dermonecrotizing toxin induces reorganization of actin stress fibers through deamidation of Gln-63 of the GTP-binding protein Rho.

Bordetella dermonecrotizing toxin causes assembly of actin stress fibers and focal adhesions in some cultured cells and induces mobility shifts of the small GTP-binding protein Rho on electrophoresis. We attempted to clarify the molecular basis of the toxin action on Rho. Analysis of the amino acid sequence of toxin-treated RhoA revealed the deamidation of Gln-63 to Glu. The substitution of Glu for Gln-63 of RhoA by site-directed mutagenesis caused a mobility shift on electrophoresis, which was indistinguishable from that of the toxin-treated RhoA. Neither mutant RhoA-bearing Glu-63 nor toxin-treated RhoA significantly differed from untreated wild type RhoA in guanosine 5'-[gamma-thio]triphosphate binding activity but both showed a 10-fold reduction in GTP hydrolysis activity relative to untreated RhoA. C3H10T1/2 cells transfected with cDNA of the mutant RhoA bearing Glu-63 showed extensive formation of actin stress fibers similar to the toxin-treated cells. These results indicate that the toxin catalyzes deamidation of Gln-63 of Rho and renders it constitutively active, leading to formation of actin stress fibers.

Clostridium perfringens enterotoxin utilizes two structurally related membrane proteins as functional receptors in vivo.

Human and mouse cDNAs showing homology to the Clostridium perfringens enterotoxin (CPE) receptor gene (CPE-R) from Vero cells (DDBJ/EMBL/GenBankTM accession no. D88492) (Katahira, J., Inoue, N., Horiguchi, Y., Matsuda, M., and Sugimoto, N. (1997) J. Cell Biol. 136, 1239-1247) were cloned. They were classified into two groups, the Vero cell CPE receptor homologues and rat androgen withdrawal apoptosis protein (RVP1; accession no. M74067) homologues, based on the similarities of primary amino acid sequences. L929 cells that were originally insensitive to CPE became sensitive to CPE on their transfection with cDNAs encoding either the CPE receptor or RVP1 homologues, indicating that these gene products are not only structurally similar but also functionally active as receptors for CPE. By binding assay, the human RVP1 homologue showed differences in affinity and capacity of binding from those of the human CPE receptor. Northern blot analysis showed that mouse homologues of the CPE receptor and RVP1 are expressed abundantly in mouse small intestine. The expression of CPE-R mRNA in the small intestine was restricted to cryptic enterocytes, indicating that the CPE receptor is expressed in intestinal epithelial cells. These results are consistent with reports that CPE binds to the small intestinal cells via two different kinds of receptors. High levels of expression of CPE-R and/or RVP1 mRNA were also detected in other organs, including the lungs, liver, and kidneys, but only low levels were expressed in heart and skeletal muscles. These results indicate that CPE uses structurally related cellular proteins as functional receptors in vivo and that organs that have not so far been recognized as CPE-sensitive have the potential to be targets of CPE.

Molecular cloning and functional characterization of the receptor for Clostridium perfringens enterotoxin.

A cDNA encoding the Clostridium perfringens enterotoxin receptor gene (CPE-R) was cloned from an expression library of enterotoxin-sensitive Vero cells. The nucleotide sequence of CPE-R showed that the enterotoxin receptor consists of 209 amino acids with a calculated molecular mass of 22,029 D. This receptor is highly hydrophobic, contains four putative transmembrane segments, and has significant similarity to the rat androgen withdrawal apoptosis protein RVP1 and the mouse oligodendrocyte specific protein, the functions of which are unknown. The expression of CPE-R was detected in the enterotoxin-sensitive Vero, Hep3B, and Intestine 407 cell lines, but not in the enterotoxin-insensitive K562 and JY cell lines. The CPE-R gene product expressed in enterotoxin-resistant L929 cells bound to enterotoxin specifically and directly and with high affinity and rendered the cells sensitive to the toxin, indicating that the cloned receptor is functional. Results showed that enterotoxin could not assemble into a complex with a defined structure unless it interacted with the receptor. From these results, it is proposed that the enterotoxin receptor is required for both target cell recognition and pore formation in the cell membrane.

Bordetella bronchiseptica dermonecrotizing toxin stimulates assembly of actin stress fibers and focal adhesions by modifying the small GTP-binding protein rho.

We studied the biochemical mechanism of morphological changes in cells treated with Bordetella dermonecrotizing toxin (DNT). DNT caused the morphological changes of serum-starved MC3T3-E1 cells from flat shapes to reflactile ones. These changes were accompanied by the assembly of actin stress fibers and focal adhesions, which is known to be regulated by the small GTP-binding protein rho. Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and inactivates rho protein, 'rounded' the cells within 2 hours after addition to the extracellular fluid and their rounded shapes were maintained for at least 10 hours. However, when the cells were co-treated with C3 exoenzyme and DNT, they were rounded at 2 hours but recovered an apparently intact morphology after 3-8 hours of incubation. rho proteins in lysates from DNT-treated cells and untreated cells were radiolabeled by [32P]ADP-ribosylation with C3 exoenzyme and analyzed by SDS-polyacrylamide gel electrophoresis. Whereas the lysate from untreated cells showed a single band of [32P]ADP-ribosylated rho protein, the lysate from DNT-treated cells showed an additional two bands as well as the band identical to that of the lysate from untreated cells. Recombinant rhoA protein treated with DNT in vitro also showed a mobility shift in SDS-polyacrylamide gel electrophoresis. These results indicate that DNT causes the assembly of actin stress fibers and focal adhesions by directly modifying rho protein.

Effects of Bordetella bronchiseptica dermonecrotizing toxin on bone formation in calvaria of neonatal rats.

The effects of Bordetella bronchiseptica dermonecrotizing toxin on bone formation were investigated using a purified toxin preparation. Single injection of 4.3 ng of the toxin into the subcutaneous tissue overlying the calvariae of neonatal rats necrotized periosteum of parietal bone and degenerated osteoblasts within two days. Nine days after the injection, the lesion of the bone tissue became severe; the bone matrix became thin and fragmented. These observations indicate that dermonecrotizing toxin without other factors produced by the organisms impairs bone formation.

Effects of translation initiation factor eIF-5A on the functioning of human T-cell leukemia virus type I Rex and human immunodeficiency virus Rev inhibited trans dominantly by a Rex mutant deficient in RNA binding.

The viral transactivator proteins Rex and Rev are necessary for the expression of structural proteins of human T-cell leukemia virus type I and human immunodeficiency virus type 1, respectively. Although the interaction of Rex/Rev with a cellular cofactor(s) has been thought to be required for Rex/Rev action, there is no suitable system to search for the cofactor(s) in mammalian cells. We found that a Rex mutant, TAgRex, which contains a simian virus 40 nuclear localization signal in place of the N-terminal 19 amino acids of Rex, could dominantly inhibit wild-type Rex/Rev functions. The inhibition did not require either Rev response element/Rex response element binding or the oligomerization ability of the mutant, but it did require a region around amino acid 90 of the Rex protein, suggesting that TAgRex sequestered the cellular cofactor. Complementation with the eukaryotic translation initiation factor 5A (eIF-5A) in this system could restore the impaired Rex function. These results indicate that eIF-5A is the cofactor indispensable for Rex function. Additionally, by using a two-hybrid system, the homo-oligomer formation of Rex was found to be mediated by the region around amino acid 90 in addition to Tyr-64 and Trp-65 of Rex protein. Thus, eIF-5A may play a part in the formation of the Rex homo-oligomer.

A cellular protein which is coprecipitated with HTLV-I rex protein in the presence of the target mRNA.

We examined the cellular protein(s) which can associate with Rex protein of human T cell leukemia virus type I (HTLV-I), using Rex-maltose binding protein (MBP) fusion protein. Immunoprecipitation of RexMBP with anti-MBP antibody revealed that a 24 kD protein (p24) associated with RexMBP only in the presence of Rex-responsive mRNA. The fact that p24 was present in both the nucleus and the cytoplasm is consistent with a role of Rex in the nucleo-cytoplasmic transport of viral mRNAs. P24 did not interact with nonfunctional Rex mutant proteins even if they had RNA binding activity in vitro. These results suggest the possible involvement of p24 in the Rex function through a complex formation with Rex on Rex-responsive mRNA.

Postantibiotic effects and postantibiotic sub-MIC effects of benzylpenicillin on viridans streptococci isolated from patients with infective endocarditis.

We investigated the postantibiotic effects (PAEs) and the postantibiotic sub-MIC effects of benzylpenicillin on three strains of viridans streptococci isolated from infective endocarditis patients. The PAEs of benzylpenicillin on penicillin tolerant Streptococcus sanguis TW-70 (0.4-3.9 h), penicillin tolerant S. sanguis TW-80 (0.3-6.3 h) and nontolerant Streptococcus oralis TW-186 (0.5-3.1 h) were dependent on exposure time. The PAEs were not concentration dependent for S. sanguis TW-70 and S. sanguis TW-80 above the MIC, and for S. oralis TW-186 above 16 x MIC. The antimicrobial effects of benzylpenicillin at sub-MIC concentrations were examined in bacteria pretreated with benzylpenicillin (8 x MIC) for 2 h and compared with untreated bacteria. At the sub-MICs tested, the regrowth of pretreated S. oralis TW-186 cells was more prolonged than that of untreated cells and bactericidal action was seen only in pretreated cells. These effects (so-called 'postantibiotic' sub-MIC effects') were not observed in penicillin tolerant S. sanguis TW-70. The presence of the postantibiotic sub-MIC effect may be an important factor in determining the dosing regimen for infective endocarditis.

Isolation and characterization of simian immunodeficiency virus from African white-crowned mangabey monkeys (Cercocebus torquatus lunulatus).

Forty-eight of 236 sera from seven species of African non-human primates in Kenya, including those of white-crowned mangabey monkeys (Cercocebus torquatus lunulatus) had antibodies to simian immunodeficiency viruses (SIVs). Isolates of simian lentivirus were obtained from seropositive white-crowned mangabey monkeys which are indigenous in West Africa. This virus, designated as SIVWCM, appeared morphologically similar to HIV by electron microscopy, showed Mg(2+)-dependent reverse transcriptase activity, and induced cytopathic effects in human CD4-positive cells. Western blotting analysis revealed that env products of SIVWCM cross-reacted with those of SIVAGM more strongly than with those of HIV-1 and SIVMAC, and clear hybridization bands were detected with an SIVAGM probe. For comparison of the virus sequence with those of other primate lentiviruses, part of the pol gene and the long terminal repeats (LTRs) were amplified and cloned. Sequencing showed that SIVWCM isolates were closely related to SIVAGM isolates. This study suggested that SIVAGM from the Cercopithecus genus and SIVWCM from the Cercocebus genus may be members of an SIV group that is genetically distinct from the SIV from a sooty mangabey monkey (SIVSMM) of the genus Cercocebus, to which the white-crowned mangabey monkey also belongs.

Isolation and characterization of a highly divergent HIV-2[GH-2]: generation of an infectious molecular clone and functional analysis of its rev-responsive element in response to primate retrovirus transactivators (Rev and Rex).

A highly divergent HIV-2 designated as HIV-2[GH-2] was obtained from an AIDS-related complex (ARC) patient in Ghana. A full-length molecular clone of this isolate was obtained and a biologically active clone was constructed. Its restriction pattern differed from that of prototype HIV-2[GH-1] in 25 of 35 restriction sites, but was strikingly similar to a previously characterized HIV-2 isolate from a Ghanaian (HIV-2ALT). The conserved integrase region (288-bp fragment) previously displayed 95% identity with that of ALT but 17-20% divergence from the HIV-2 prototype member, and a new distinct subgroup (HIV-2b) of HIV-2 consisting of GH-2 and ALT was postulated (Miura et al. 1991.) These isolates, however, were biologically distinguishable from each other by its replication capacity in a monocyte line, U937, in which GH-2 could not grow but ALT grew well. In addition, the nucleotide sequence of the LTR of this new isolate displays 21% divergence from that of prototype HIV-2[GH-1], but the core enhancer, Sp1 binding sites and TATA box were conserved. Although the 3' half of the env gene sequence which is deleted in HIV-2ALT clone showed 27% diversity from the prototype, functional differences in the rev-responsive element were not observed.