Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties

A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA14-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA14-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10(6) PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein.     

Homogeneous, structurally defined heparin-oligosaccharides with low anticoagulant activity inhibit the generation of the amplification pathway C3 convertase in vitro

This paper demonstrates that heparin-oligosaccharides with low anticoagulant activity have a high capacity to inhibit activation of the amplification pathway of complement in vitro. We prepared heparin-oligosaccharides by partial depolymerization of heparin using purified flavobacterial heparinase. The resulting oligosaccharide mixture was then fractionated using strong anion exchange-high pressure liquid chromatography to produce individual oligosaccharide components of this mixture, with degree of polymerization ranging from 2 to 16. These heparin-oligosaccharides were examined for both their anticoagulant activity and capacity to inhibit activation of the amplification pathway of complement. Although there was little difference among commercial heparins, a correlation between molecular weight and activity to inhibit convertase generation was clearly established for heparin-oligosaccharides between degree of polymerization 2 through 16. Heparin-oligosaccharides of degree of polymerization 10-16 (Mr 3888-5320) demonstrated up to 54% of heparin's activity on a molar basis (and up to 163% of heparin's activity on a weight basis) in inhibiting the amplification pathway of complement in vitro while showing almost no anticoagulant activity. These studies, for the first time, completely separate heparin's ability to inhibit complement activation from its anticoagulant activity.     

Phorbol ester-induced differentiation permits productive human cytomegalovirus infection in a monocytic cell line

The susceptibility of four different human cell lines (HUT 102, THP-1, MOLT-4, and HL-60) to infection by human CMV (HCMV) was studied. Only HUT 102 was susceptible and only immediate-early gene products were produced. However, THP-1, a monocytic cell line, could be infected by HCMV with a full cycle of replication after treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which produced differentiation of the cell line into cells with characteristics of mature macrophages. Late (structural) Ag were demonstrated, as were infectious virions as detected by electron microscopy and infectious center assay. HL-60, a promyelocytic cell line, was not susceptible to HCMV infection after treatment with TPA despite differentiation into adherent cells with properties of macrophages, suggesting that cellular lineage was important. Treatment with TPA after infection resulted in a greatly reduced frequency of infected cells, suggesting that pretreatment was essential. Furthermore, continued presence of TPA was unnecessary after differentiation was induced. This study establishes the precedent of productive HCMV infection in human monocytic cells. The potential mechanism and relevance of enhanced replication induced by TPA are discussed.     

Heterogeneity of cellular glutathione among cells derived from a murine fibrosarcoma or a human renal cell carcinoma detected by flow cytometric analysis

Monochlorobimane (syn-(ClCH2, CH3)-1,5-diazabicyclo-[3.3.0]-octa-3,6-dione-2,8-dione; mBCl) forms a fluorescent adduct with glutathione (GSH), which has been used as a basis for flow cytometric analysis. While mBCl will react nonspecifically with many different thiols, preferential derivatization of GSH can be achieved by using a low concentration of mBCl, since the reaction with GSH is catalyzed by GSH S-transferase, and the nonenzymatic reaction is very slow (k = 3.3 x 10(-1) M-1 s-1 at 37 degrees C, pH 7.5). The rate of derivatization of cellular GSH can be 1000 times greater than predicted from the nonenzymatic reaction rate, although this factor can vary among cell lines. GSH values obtained by flow cytometry (FCM) agree well with those obtained by an enzymatic assay, over a wide range of GSH values, for EMT6/SF cells treated with L-buthionine sulfoximine to vary GSH content. FCM analysis of the GSH content of cells obtained by disaggregation of EMT6/SF tumors, grown in BALB/c mice, revealed a wide variation in single-cell GSH content. The data suggest that there are distinct subpopulations within these tumors, which can be partially characterized by GSH content, but may also have other distinguishing characteristics, such as enhanced sensitivity or resistance to cytotoxic agents. Heterogeneity in single-cell GSH content was also observed by FCM analysis of cells obtained by disaggregation of a biopsy of a human renal cell carcinoma. This result points to the potential value of FCM analysis of GSH in the identification and characterization of human tumor subpopulations which may be of clinical significance in the treatment of cancer by radiation or chemotherapeutic agents.     

The plant metabolite, 6-methoxybenzoxazolinone, stimulates an increase in secretion of follicle-stimulating hormone and size of reproductive organs in Microtus pinetorum

The plant metabolite, 6-methoxybenzoxazolinone (6-MBOA), occurring in leaf tissue of rapidly growing monocots, cues reproduction in some mammals. In the pine vole, Microtus pinetorum, peripubertal females respond to this nonestrogenic compound with a 40% increase in serum levels of follicle-stimulating hormone (FSH). In addition, 6-MBOA significantly increases the weight of the ovary and uterus in both peripubertal and mature voles. This study is the first to offer evidence that 6-MBOA interacts with the pituitary to stimulate reproduction in voles.     

Determinants of the Hepatitis C Virus Nonstructural Protein 2 Protease Domain Required for Production of Infectious Virus

The hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a dimeric multifunctional hydrophobic protein with an essential but poorly understood role in infectious virus production. We investigated the determinants of NS2 function in the HCV life cycle. On the basis of the crystal structure of the postcleavage form of the NS2 protease domain, we mutated conserved features and analyzed the effects of these changes on polyprotein processing, replication, and infectious virus production. We found that mutations around the protease active site inhibit viral RNA replication, likely by preventing NS2-3 cleavage. In contrast, alterations at the dimer interface or in the C-terminal region did not affect replication, NS2 stability, or NS2 protease activity but decreased infectious virus production. A comprehensive deletion and mutagenesis analysis of the C-terminal end of NS2 revealed the importance of its C-terminal leucine residue in infectious particle production. The crystal structure of the NS2 protease domain shows that this C-terminal leucine is locked in the active site, and mutation or deletion of this residue could therefore alter the conformation of NS2 and disrupt potential protein-protein interactions important for infectious particle production. These studies begin to dissect the residues of NS2 involved in its multiple essential roles in the HCV life cycle and suggest NS2 as a viable target for HCV-specific inhibitors.An estimated 130 million people are infected with hepatitis C virus (HCV), the etiologic agent of non-A, non-B viral hepatitis. Transmission of the virus occurs primarily through blood or blood products. Acute infections are frequently asymptomatic, and 70 to 80% of the infected individuals are unable to eliminate the virus. Of the patients with HCV-induced chronic hepatitis, 15 to 30% progress to cirrhosis within years to decades after infection, and 3 to 4% of patients develop hepatocellular carcinoma (17). HCV infection is a leading cause of cirrhosis, end-stage liver disease, and liver transplantation in Europe and the United States (7), and reinfection after liver transplantation occurs almost universally. There is no vaccine available, and current HCV therapy of pegylated alpha interferon in combination with ribavirin leads to a sustained response in only about 50% of genotype 1-infected patients.The positive-stranded RNA genome of HCV is about 9.6 kb in length and encodes a single open reading frame flanked by 5′ and 3′ nontranslated regions (5′ and 3′ NTRs). The translation product of the viral genome is a large polyprotein containing the structural proteins (core, envelope proteins E1 and E2) in the N-terminal region and the nonstructural proteins (p7, nonstructural protein 2 [NS2], NS3, NS4A, NS4B, NS5A, and NS5B) in the C-terminal region. The individual proteins are processed from the polyprotein by various proteases. The host cellular signal peptidase cleaves between core/E1, E1/E2, E2/p7, and p7/NS2, and signal peptide peptidase releases core from the E1 signal peptide. Two viral proteases, the NS2-3 protease and the NS3-4A protease, cleave the remainder of the viral polyprotein in the nonstructural region (22, 27). The structural proteins package the genome into infectious particles and mediate virus entry into a naïve host cell; the nonstructural proteins NS3 through NS5B form the RNA replication complex. p7 and NS2 are not thought to be incorporated into the virion but are essential for the assembly of infectious particles (14, 36); however, their mechanisms of action are not understood.NS2 (molecular mass of 23 kDa) is a hydrophobic protein containing several transmembrane segments in the N-terminal region (5, 9, 32, 39). The C-terminal half of NS2 and the N-terminal third of NS3 form the NS2-3 protease (10, 11, 26, 37). NS2 is not required for the replication of subgenomic replicons, which span NS3 to NS5B (20). However, cleavage at the NS2/3 junction is necessary for replication in chimpanzees (16), the full-length replicon (38), and in the infectious tissue culture system (HCVcc) (14). Although cleavage can occur in vitro in the absence of microsomal membranes, synthesis of the polyprotein precursor in the presence of membranes greatly increases processing at the NS2/3 site (32). In vitro studies indicate that purified NS2-3 protease is active in the absence of cellular cofactors (11, 37). In addition to its role as a protease, NS2 has been shown to be required for assembly of infectious intracellular virus (14). The N-terminal helix of NS2 was first implicated in infectivity by the observation that an intergenotypic breakpoint following this transmembrane segment resulted in higher titers of infectious virus (28). Structural and functional characterization of the NS2 transmembrane region has shown that this domain is essential for infectious virus production (13). In particular, a central glycine residue in the first NS2 helix plays a critical role in HCV infectious virus assembly (13). The NS2 protease domain, but not its catalytic activity, is also essential for infectious virus assembly, whereas the unprocessed NS2-3 precursor is not required (13, 14).The crystal structure of the postcleavage NS2 protease domain (NS2^pro, residues 94 to 217), revealed a dimeric cysteine protease containing two composite active sites (Fig. 2C; [21]). Two antiparallel α-helices make up the N-terminal subdomain, followed by an extended crossover region, which positions the β-sheet-rich C-terminal subdomain near the N-terminal region of the partner monomer. Two of the conserved residues of the catalytic triad (His 143, Glu 163) are located in the loop region after the second N-terminal helix of one monomer, while the third catalytic residue, Cys 184, is located in the C-terminal subdomain of the other monomer. Creation of this unusual pair of composite active sites through NS2 dimerization has been shown to be essential for autoproteolytic cleavage (21). The structure of NS2^pro further demonstrated that the C-terminal residue of NS2 remains bound in the active site after cleavage, suggesting a possible mechanism for restriction of this enzyme to a single proteolytic event (21). Here we have used the crystal structure of NS2^pro, along with sequence alignments, to target conserved residues in each of the NS2^pro structural regions. Our mutational analysis revealed that the residues in the dimer crossover region and the C-terminal subdomain are important for infectious virus production. In contrast, the majority of amino acids in the active site pocket were not required for infectivity. Interestingly, we observed that the extreme C-terminal leucine of NS2 is absolutely essential for generation of infectious virus, as mutations, deletions, and extensions into NS3 are very poorly tolerated. This analysis begins to dissect the determinants of the multiple functions of this important protease in the HCV life cycle.     

The relative importance of the deep and superficial vascular systems for delay of the transverse rectus abdominis musculocutaneous flap as demonstrated in a rat model

The use of some form of delay maneuver for "high-risk" patients before transfer of the superior pedicled lower transverse rectus abdominis musculocutaneous (TRAM) flap for breast reconstruction has augmented the rate of success in both the experimental and clinical arenas. A common method of vascular delay has been the bilateral division of both the superficial inferior epigastric and deep inferior epigastric vessels. Whether all of these must be divided to adequately effect the delay is unknown. For that matter, the relative importance of the superficial versus the deep vascular systems is unclear. To investigate this uncertainty, a delay was attempted in 61 Sprague-Dawley rats by division of either the superficial inferior epigastric or deep cranial epigastric vessels (the latter is the homologue to the human deep inferior epigastric) in unilateral or bilateral fashion. Division of the contralateral superficial inferior epigastric vessel resulted in significantly greater TRAM flap survival than either ipsilateral or bilateral superficial inferior epigastric vessel division (p = 0.0034 or p = 0.0093, respectively). Division of the ipsilateral or bilateral deep cranial epigastric vessel resulted in significantly greater flap survival than just contralateral deep cranial epigastric vessel division (p = 0.0034 or p = 0.006, respectively). No significant difference was observed between the group having contralateral superficial inferior epigastric or groups with ipsilateral deep cranial epigastric division, implying that either alone would be efficacious to achieve the desired delay effect. This would allow the other vascular system to be retained intact for later potential salvage maneuvers as needed.     

Characterization of benzodiazepine receptors with fluorescent ligands

Fluorescein conjugates of the high-affinity benzodiazepine receptor ligands Ro 15-1788 and Ro 7-1986 were synthesized. The binding of these fluorescent ligands (BD 621 and BD 607) to benzodiazepine receptors was characterized by direct fluorescence measurement. Both the equilibrium dissociation constants (KD) of BD 621 and BD 607 and the maximum number of binding sites (Bmax) estimated by fluorescence monitoring were consistent with values obtained by using radioligand binding techniques. The binding of BD 621 and BD 607 assessed by fluorescence measurement was reversible, abolished by photoaffinity labeling with Ro 15-4513, and unaffected by a variety of substances that do not bind to benzodiazepine receptors. The potencies of chemically diverse benzodiazepine receptor compounds to inhibit fluorescent ligand binding were highly correlated (r = 0.94, P less than 0.001), with potencies obtained from radioligand binding techniques. These findings demonstrate the feasibility of using direct fluorescence measurement techniques to quantitate ligand-receptor interactions.