Ultrastructural Analysis of ICP34.5? Herpes Simplex Virus 1 Replication in Mouse Brain Cells In Vivo

Replication-competent forms of herpes simplex virus 1 (HSV-1) defective in the viral neurovirulence factor infected cell protein 34.5 (ICP34.5) are under investigation for use in the therapeutic treatment of cancer. In mouse models, intratumoral injection of ICP34.5-defective oncolytic HSVs (oHSVs) has resulted in the infection and lysis of tumor cells, an associated decrease in tumor size, and increased survival times. The ability of these oHSVs to infect and lyse cells is frequently characterized as exclusive to or selective for tumor cells. However, the extent to which ICP34.5-deficient HSV-1 replicates in and may be neurotoxic to normal brain cell types in vivo is poorly understood. Here we report that HSV-1 defective in ICP34.5 expression is capable of establishing a productive infection in at least one normal mouse brain cell type. We show that γ34.5 deletion viruses replicate productively in and induce cellular damage in infected ependymal cells. Further evaluation of the effects of oHSVs on normal brain cells in animal models is needed to enhance our understanding of the risks associated with the use of current and future oHSVs in the brains of clinical trial subjects and to provide information that can be used to create improved oHSVs for future use.Several types of replication-competent neuroattenuated herpes simplex viruses (HSVs) are currently being evaluated in clinical cancer trials for safety and therapeutic activity (32), as well as for vaccine development (20). A critical safety concern associated with the clinical use of these oncolytic HSVs (oHSVs) is their ability to enter, replicate in, and spread to a wide range of cell types in different regions of the nervous system. One potential complication resulting from invasion of the central nervous system by HSV is herpes simplex encephalitis (HSE), an infection that causes lifelong neurological damage or death. A limited number of genes have been demonstrated to contribute to the virus''s ability to trigger HSE. The viral gene γ34.5 encodes the neurovirulence protein infected cell protein 34.5 (ICP34.5) (29). Viruses lacking the γ34.5 gene (e.g., R3616 and 1716) were found to be 5 logs less neurovirulent than wild-type strains of HSV-1 (4, 19, 36), as quantified by the intracranial LD₅₀, i.e., the lethal dose in 50% of mice inoculated intracerebroventricularly with the virus. The basis for this neuroattenuation was initially reported to be the inability of the γ34.5 deletion viruses to infect or replicate in brain cells (4). Subsequent immunohistochemical studies on infected brain tissue of intracerebroventricularly inoculated mice suggested that γ34.5 deletion viruses retained the ability to infect a wide range of brain cell types and to replicate in and, by day 7, destroy ependymal cells (ECs) (16, 21).To create a more neuroattenuated and thus safer virus, the virus G207 was constructed from the γ34.5 deletion virus R3616 by insertional mutagenesis of the U_L39 gene (25). The U_L39 gene encodes the large subunit of the viral ribonucleotide reductase (vRR) (29). Cellular ribonucleotide reductase is a DNA synthetic enzyme which is of low abundance in quiescent cells but is critical for the synthesis of DNA precursors and is thus abundant in mitotically active cells such as cancer cells. Based on the phenotype of viruses mutated in the vRR alone (13), this double-deletion virus lacking both ICP34.5 and vRR expression is predicted to restrict viral replication to cancer cells expressing cellular RR at levels sufficient to support viral replication (25). In preclinical studies with mice, inoculation with G207 via the intracerebroventricular route failed to destroy the EC layer at 5 days postinoculation (34). These studies supported the concept that a double-deletion virus may be safer in clinical trials than a virus lacking only ICP34.5 expression.To test the hypothesis that productive replication of γ34.5 deletion viruses is restricted to cancer cells, we developed sensitive methods to examine the ability of γ34.5 deletion viruses, with either intact or mutated vRR, to replicate productively in vivo and to complete the multistep process of virion assembly and egress.Common to most models of HSV virion assembly and egress is the observation that capsid proteins translated in the cytoplasm are imported to the nucleus, where a capsid shell is assembled and viral DNA is subsequently packaged. Capsids containing viral DNA are distinguished by an electron-dense (dark) center, whereas capsids lacking viral DNA contain a core protein visible by electron microscopy (EM) often as an inner concentric circle. In subsequent steps, DNA-filled capsids acquire an envelope by budding through the inner nuclear membrane into the perinuclear space. Capsids observed between the inner and outer nuclear membranes have an envelope and tegument and resemble mature extracellular virions (10).Consensus is lacking on the specific sequence of subsequent stages of viral egress, and multiple pathways may exist (3, 18, 24, 30). In the subsequent step of the envelopment-deenvelopment-reenvelopment model (18, 30), enveloped capsids in the perinuclear space lose their envelope by fusion with the outer nuclear membrane as the capsids enter the cytoplasm. In this model, progeny viruses are thus present in the cytoplasm as naked capsids. Cytoplasmic naked capsids acquire their mature envelope as they bud into a cytoplasmic organelle (e.g., a Golgi body).According to an alternative model, enveloped capsids move within the perinuclear space into the endoplasmic reticulum (ER), which is continuous with the perinuclear space (33). From this space, enveloped capsids, individually or in groups, bud off within a vesicle membrane characteristic of the outer nuclear membrane/ER. Within these vesicles, enveloped virions are transported through the cytoplasm. In a final step common to both models, the cytoplasmic vesicle releases mature enveloped virions into the extracellular space by fusing with the cell membrane.ECs are an ideal cell type for these studies due to their distinct morphology and location (described below) and their reported function as neural stem cells (15). We reasoned that since mitotic activity is the reported basis for the productive replication and selectivity of γ34.5 deletion viruses in cancer cells (9, 34), and ECs may be mitotically active, if any normal brain cell type were to support productive replication of γ34.5 deletion viruses, ECs would be the most likely candidate.ECs line the cerebral ventricles, acting as a semipermeable barrier between the brain parenchyma and the cerebrospinal fluid (CSF) in the ventricles (7, 12). Their location thus makes them easily exposed to the virus via intraventricular injections. Their location, combined with their morphologically distinct cuboid shape with kinocilia and microvilli that protrude into the CSF, allows them to be easily excised and recognized under both light microscopy and EM.Here we report the results of a side-by-side comparative study evaluating whether a double-deletion virus similar to G207 and a virus lacking only ICP34.5 expression differ from each other and from a wild-type virus in the ability to infect and replicate productively in ECs of the mouse brain in vivo. The results of these studies are consistent with results of other studies in that they demonstrate that viruses similar to those used in clinical trials (e.g., G207, HSV1716) have a greatly attenuated ability to replicate compared to that of a wild-type virus. However, our data also show very clearly that γ34.5 deletion viruses do replicate productively in infected mouse brain ECs in vivo. These studies suggest that (i) ECs can serve as an exquisitely sensitive model for future evaluations of the ability of oHSVs to replicate productively in normal mouse brain cells and (ii) the potential exists for double-deletion oHSVs to damage normal brain cells. Thus, further comparative studies are warranted to determine whether this risk is sufficiently high to restrict the administration of ICP34.5 deletion viruses in or near the cerebral ventricles in clinical studies.     

Human Tau Isoforms Assemble into Ribbon-like Fibrils That Display Polymorphic Structure and Stability

Fibrous aggregates of Tau protein are characteristic features of Alzheimer disease. We applied high resolution atomic force and EM microscopy to study fibrils assembled from different human Tau isoforms and domains. All fibrils reveal structural polymorphism; the “thin twisted” and “thin smooth” fibrils resemble flat ribbons (cross-section ∼10 × 15 nm) with diverse twist periodicities. “Thick fibrils” show periodicities of ∼65–70 nm and thicknesses of ∼9–18 nm such as routinely reported for “paired helical filaments” but structurally resemble heavily twisted ribbons. Therefore, thin and thick fibrils assembled from different human Tau isoforms challenge current structural models of paired helical filaments. Furthermore, all Tau fibrils reveal axial subperiodicities of ∼17–19 nm and, upon exposure to mechanical stress or hydrophobic surfaces, disassemble into uniform fragments that remain connected by thin thread-like structures (∼2 nm). This hydrophobically induced disassembly is inhibited at enhanced electrolyte concentrations, indicating that the fragments resemble structural building blocks and the fibril integrity depends largely on hydrophobic and electrostatic interactions. Because full-length Tau and repeat domain constructs assemble into fibrils of similar thickness, the “fuzzy coat” of Tau protein termini surrounding the fibril axis is nearly invisible for atomic force microscopy and EM, presumably because of its high flexibility.     

pH-Induced Conformational Change of the β-Barrel-Forming Protein OmpG Reconstituted into Native E. coli Lipids

A gating mechanism of the β-barrel-forming outer membrane protein G (OmpG) from Escherichia coli was recently presented. The mechanism was based on X-ray structures revealed from crystals grown from solubilized OmpG at both neutral pH and acidic pH. To investigate whether these conformations represent the naturally occurring gating mechanism, we reconstituted OmpG in native E. coli lipids and applied high-resolution atomic force microscopy. The reconstituted OmpG molecules assembled into both monomers and dimers. Single monomeric and dimeric OmpG molecules showed open channel entrances at pH 7.5 and at room temperature. The extracellular loops connecting the β-strands that form the transmembrane β-barrel pore exhibited elevated structural flexibility. Upon lowering the pH to 5.0, the conformation of OmpG molecules changed to close the extracellular entrance of their channel. It appears that one or more of the extracellular loops collapsed onto the channel entrance. This conformational change was fully reversible. Our data confirm that the previously reported gating mechanism of OmpG occurs at physiological conditions in E. coli lipid membranes.     

The Helicobacter pylori GroES Cochaperonin HspA Functions as a Specialized Nickel Chaperone and Sequestration Protein through Its Unique C-Terminal Extension

The transition metal nickel plays a central role in the human gastric pathogen Helicobacter pylori because it is required for two enzymes indispensable for colonization, the nickel metalloenzyme urease and [NiFe] hydrogenase. To sustain nickel availability for these metalloenzymes while providing protection from the metal''s harmful effects, H. pylori is equipped with several specific nickel-binding proteins. Among these, H. pylori possesses a particular chaperone, HspA, that is a homolog of the highly conserved and essential bacterial heat shock protein GroES. HspA contains a unique His-rich C-terminal extension and was demonstrated to bind nickel in vitro. To investigate the function of this extension in H. pylori, we constructed mutants carrying either a complete deletion or point mutations in critical residues of this domain. All mutants presented a decreased intracellular nickel content measured by inductively coupled plasma mass spectrometry (ICP-MS) and reduced nickel tolerance. While urease activity was unaffected in the mutants, [NiFe] hydrogenase activity was significantly diminished when the C-terminal extension of HspA was mutated. We conclude that H. pylori HspA is involved in intracellular nickel sequestration and detoxification and plays a novel role as a specialized nickel chaperone involved in nickel-dependent maturation of hydrogenase.Helicobacter pylori is a Gram-negative, microaerophilic bacterium that is the only persistent inhabitant of the human stomach. Its presence in humans is associated with a variety of pathologies, ranging from gastric and duodenal peptic ulcers to the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma (1, 39). Indeed, H. pylori is the only formally recognized bacterial carcinogen for humans (17), infecting half of the world''s population (19).In H. pylori, metal ions play a central role, since the transition metal nickel is the cofactor of the urease enzyme and is also required for [NiFe] hydrogenase. Urease catalyzes the hydrolysis of urea into the buffering compounds bicarbonate and ammonia, enabling H. pylori to persist in the acidic environment of the stomach. This enzyme accounts for up to 6% of the soluble cellular proteins and requires 24 nickel ions per active enzymatic complex (16). The uptake-type hydrogenase of H. pylori is a nickel-dependent enzyme containing a binuclear [NiFe] active site. This [NiFe] hydrogenase catalyzes the oxidation of molecular hydrogen and permits the utilization of hydrogen as an energy source during respiration-based energy production in the mucosa (21). Both enzymes are important for host colonization, as shown with several animal models (9, 10, 28, 42, 43). To sustain nickel availability for urease and hydrogenase while providing protection from the metal''s harmful effects, H. pylori possesses an elaborate and strictly controlled nickel metabolism.The incorporation of nickel ions into apohydrogenase requires the participation of the HypAB (HP0869 and HP0900) accessory proteins; for apourease, both the UreEFGH (HP0070-0067) accessory proteins and HypAB are necessary (4, 29). Besides these widely distributed accessory proteins, H. pylori possesses several specific proteins that are present in all H. pylori strains, namely, the histidine-rich proteins Hpn (HP1427) and Hpn-like (HP1432). These cytoplasmic and abundant proteins (Hpn represents 2% of the total protein content) bind nickel ions (five Ni²⁺ ions per monomer; dissociation constant [K_d] for nickel of 7.1 μM) and protect H. pylori against metal overload (15). Furthermore, it has recently been proposed that Hpn and Hpn-like can compete for nickel ions with the urease enzyme and thus regulate its enzymatic activity. In vivo and in vitro experiments indicate that Hpn and Hpn-like sequester nickel ions at neutral pH but donate them for urease activation under acidic pH conditions (14, 35, 44). Hydrogenase activity was unchanged in the Δhpn and Δhpn-like mutants (35).In addition to these proteins, H. pylori possesses a particular chaperone, HspA (HP0011), that is a homolog of the highly conserved and essential bacterial heat shock protein GroES (40). No other gene encoding a GroES homolog is found in the genome of H. pylori. GroES is the cochaperonin of the heptameric GroEL-GroES barrel complex, which mediates the correct folding of a variety of cellular proteins and which is conserved and essential in prokaryotes and eukaryotes (30). In addition to the conserved GroES chaperonin domain (domain A, amino acids 1 to 90) (Fig. ​(Fig.1A),1A), HspA contains a C-terminal extension of 28 amino acids (domain B, amino acids 91 to 118) (Fig. 1A and B) that contains 8 His and 4 Cys residues. Based on this high number of His and Cys residues known to bind transition metal ions, the purified recombinant HspA protein specifically binds two nickel ions per molecule (K_d of 1.1 to 1.8 μM) (7, 18). This domain also contains an HX₄DH motif (boxed in Fig. ​Fig.1B)1B) that is considered to be a nickel-binding signature sequence in the nickel-cobalt (NiCoT) transporter family (11). In addition, Loguercio et al. (20) observed that in vitro, the HspA C-terminal domain is folding into two vicinal disulfide bounds engaging two cysteine pairs that form a unique closed-loop structure. However, since HspA is a cytoplasmic protein, the in vivo relevance of this structure is uncertain.Open in a separate windowFIG. 1.(A) Representation of the HspA protein of H. pylori with the GroES-like domain A and the nickel-binding domain B. (B) Amino acid sequence of domain B of wild-type HspA and of three mutants: HspA-ΔC, with a complete deletion of this domain, and HspA-NB and -CC, each carrying two substitutions that are underlined. Cysteine and histidine residues are in blue and red, respectively. The HX₄DH motif, which in the nickel-cobalt (NiCoT) transporter family is considered to be a nickel-binding signature sequence, is boxed. (C) Immunoblot experiment with whole-cell lysates from the H. pylori wild-type strain and from the three hspA mutants after denaturing SDS-PAGE and using the monoclonal antibody P1-1, which specifically recognizes a conserved epitope of HspA domain A. The predicted molecular mass of the wild-type HspA monomer is 13 kDa, and that of HspA-ΔC is 9.8 kDa. The monomeric (M) and dimeric (D) forms of the HspA wild type (WT) are indicated on the left side of the blot. A cross-reacting unspecific protein band is marked with a star (*) and served as a loading control. Molecular mass standards are indicated at right.The domain B sequence is conserved in and restricted to H. pylori and the closely related Helicobacter acinonychis species but is absent from all other available sequenced Helicobacter species (see Fig. S1 in the supplemental material). When expressed in Escherichia coli, HspA protected bacteria from nickel overload (7) and increased urease activity 4-fold from the coexpressed H. pylori urease gene cluster (18). Therefore, HspA was hypothesized to function in nickel sequestration and as a specialized nickel donor protein for urease (18). However, no functional characterization of the C terminus was carried out for H. pylori due to the essential nature of HspA (40).In this study, we investigated the role of the nickel-binding C terminus of HspA in H. pylori. We found that the unique C terminus of HspA is involved in nickel sequestration and protection against nickel overload. Contrary to previous data from heterologous studies of E. coli, HspA seemed not to provide nickel ions for urease activation. In contrast, we have found an unexpected and specific function of the HspA C-terminal region in the nickel-dependent maturation of the important colonization factor hydrogenase.     

Dynamics of social and energetic stress in wild female chimpanzees

Stress hormone measurements can reinforce and refine hypotheses about the costs of particular contexts or behaviors in wild animals. For social species, this is complicated because potential stressors may come from the physical environment, social environment, or some combination of both, while the stress response itself is generalized. Here, we present a multivariate examination of urinary cortisol dynamics over 6 years in the lives of wild female chimpanzees in the Kanyawara community of Kibale National Park, Uganda. We hypothesized that chimpanzee socioecology provides strong indications of both energetic and social stress to females, but that the salience of these stressors might vary over a female's life history in accordance with their changing reproductive costs and social interactions. Using linear mixed models, we found that urinary cortisol levels increased significantly with age but were also elevated in young immigrants to the community. Across reproductive states, cycling, non-estrous females had relatively low cortisol compared to lactating, estrous, or pregnant females. Aggression from males led to higher cortisol levels among estrous females, frequent targets of aggressive sexual coercion. In contrast, energetic stress was most salient to lactating females, who experienced higher cortisol during months of low fruit consumption. Low dominance rank was associated with increased cortisol, particularly during the energetically demanding period of lactation. The effects of female conflict were felt widely, even among those who were the primary aggressors, providing further evidence that long-term resource competition, while apparently muted, exerts a far-reaching impact on the lives of chimpanzee females.     

On the determination of empirical absolute chiral structure: chiroptical spectrum correlations for D3 lanthanide (III) complexes

Circularly polarized luminescence (CPL) from selected transitions of Eu(III) in resolved single crystals of Na3[Eu(ODA)3].2NaClO4.6H2O are compared to CPL results obtained from solutions containing perturbed racemic mixtures of Eu(2,6-pyridine-dicarboxylate)3 (3-) and enantiomerically pure d-f helicate LambdaLambda-(-)EuCr(L8)3] in order to determine an empirical relationship between helicity and CPL spectra. Comparison of the CPL results, even for the magnetic dipole allowed transitions of Eu(III) where one measures large chiral discrimination, shows that the signs and magnitudes do not correlate with the overall helicity of the Eu(III) site. It is concluded that the symmetry of the Eu(III) site in LambdaLambda-(-)EuCr(L8)3 is not close enough to D3 to allow for the confirmation of the presumed spectra:structure correlation.