Mosquito-borne alphaviruses, which replicate alternately and obligately in mosquitoes and vertebrates, appear to experience lower rates of evolution than do many RNA viruses that replicate solely in vertebrates. This genetic stability is hypothesized to result from the alternating host cycle, which constrains evolution by imposing compromise fitness solutions in each host. To test this hypothesis, Sindbis virus was passaged serially, either in one cell type to eliminate host alteration or alternately between vertebrate (BHK) and mosquito (C6/36) cells. Following 20 to 50 serial passages, mutations were identified and changes in fitness were assessed using competition assays against genetically marked, surrogate parent viruses. Specialized viruses passaged in a single cell exhibited more mutations and amino acid changes per passage than those passaged alternately. Single host-adapted viruses exhibited fitness gains in the cells in which they specialized but fitness losses in the bypassed cell type. Most but not all viruses passaged alternately experienced lesser fitness gains than specialized viruses, with fewer mutations per passage. Clonal populations derived from alternately passaged viruses also exhibited adaptation to both cell lines, indicating that polymorphic populations are not required for simultaneous fitness gains in vertebrate and mosquito cells. Nearly all passaged viruses acquired Arg or Lys substitutions in the E2 envelope glycoprotein, but enhanced binding was only detected for BHK cells. These results support the hypothesis that arbovirus evolution may be constrained by alternating host transmission cycles, but they indicate a surprising ability for simultaneous adaptation to highly divergent cell types by combinations of mutations in single genomes.Sindbis virus (SINV) is the type species of the genus Alphavirus, a group of RNA viruses with nonsegmented singlestranded genomes of approximately 11.7 kb (23, 25). The alphavirus genome is capped at the 5Ј end and contains a 3Ј poly(A) tail. The 5Ј two-thirds of the genome encode the nonstructural proteins 1 to 4, which are necessary for viral replication. The structural proteins, capsid and E1 and E2 envelope glycoproteins, are translated from a subgenomic mRNA (26S) which is identical in sequence to the 3Ј one-third of the genome (Fig. 1). The Sindbis virion contains an icosahedral nucleocapsid that consists of 240 copies of the capsid protein surrounded by a lipid envelope derived from the plasma membrane of infected cells, into which the glycoprotein E1/E2 heterodimers are embedded (4).Nearly all alphaviruses rely on horizontal mosquito-borne transmission among vertebrate hosts, requiring alternating replication in highly divergent hosts and cell types (7,34). Alphaviruses and other arthropod-borne viruses also appear to undergo lower rates of evolution than many other animal RNA viruses that replicate solely in vertebrates (6,33,35). Rates of SINV nucleotide substitution, deduced from oligonucleotide fingerprinting, are approximately 4 ϫ 10 Ϫ4 ...

Electrophiles generated endogenously, or via the metabolic bioactivation of drugs and other environmental chemicals, are capable of binding to a variety of nucleophilic sites within proteins. Factors that determine site selective susceptibility to electrophile-mediated post-translational modifications, and the consequences of such alterations, remain largely unknown. To identify and characterize chemical-mediated protein adducts, electrophiles with known toxicity were utilized. Hydroquinone, and its mercapturic acid pathway metabolites, cause renal proximal tubular cell necrosis and nephrocarcinogenicity in rats. The adverse effects of HQ and its thioether metabolites are in part a consequence of their oxidation to the corresponding electrophilic 1,4-benzoquinones (BQ). We now report that BQ and 2-(N-acetylcystein-S-yl)benzoquinone (NAC-BQ) preferentially bind to solvent-exposed lysine-rich regions within cytochrome c. Furthermore, we have identified specific glutamic acid residues within cytochrome c as novel sites of NAC-BQ adduction. The microenvironment at the site of adduction governs both the initial specificity and the structure of the final adduct. The solvent accessibility and local pKa of the adducted and neighboring amino acids contribute to the selectivity of adduction. Postadduction chemistry subsequently alters the nature of the final adduct. Using molecular modeling, the impact of BQ and NAC-BQ adduction on cytochrome c was visualized, revealing the spatial rearrangement of critical residues necessary for protein-protein interactions. Consequently, BQ-adducted cytochrome c fails to initiate caspase-3 activation in native lysates and also inhibits Apaf-1 oligomerization into an apoptosome complex in a purely reconstituted system. In summary, a combination of mass spectroscopic, molecular modeling, and biochemical approaches confirms that electrophile-protein adducts produce structural alterations that influence biological function.

Adaptive responses to mild heat shock are among the most widely conserved and studied in nature. More intense heat shock, however, induces apoptosis through mechanisms that remain largely unknown. Herein, we present evidence that heat shock activates an apical protease that stimulates mitochondrial outer membrane permeabilization and processing of the effector caspase-3 in a benzyloxycarbonyl-VAD-fluoromethyl ketone (polycaspase inhibitor)-and Bcl-2-inhibitable manner. Surprisingly, however, neither FADD⅐caspase-8 nor RAIDD⅐caspase-2 PIDDosome (p53-induced protein with a death domain) complexes were detected in dying cells, and neither of these initiator caspases nor the endoplasmic reticulum stress-activated caspases-4/12 were required for mitochondrial outer membrane permeabilization. Similarly, although cytochrome c was released from mitochondria following heat shock, functional Apaf-1⅐ caspase-9 apoptosome complexes were not formed, and caspase-9 was not essential for the activation of caspase-3 or the induction of apoptosis. Thus, heat shock does not require any of the known initiator caspases or their activating complexes to promote apoptotic cell death but instead relies upon the activation of an apparently novel apical protease with caspase-like activity.Two pathways, referred to as the death receptor (extrinsic) and mitochondrial (intrinsic) pathways, are widely regarded as being responsible for most, if not all, caspase-dependent apoptosis (1, 2). In both cases, caspase cascades are initiated through formation of a protein complex that contains a specific adapter protein and its associated "initiator" caspase (i.e. caspase-2, -8, -9, -10, or -12) (3). Once activated, most initiator caspases proteolytically activate the downstream "effector" caspases-3, -6, and/or -7, which in turn cleave specific cellular substrates resulting in chromatin compaction, membrane blebbing, and cell shrinkage.Stimulation of death receptors (such as CD95 and tumor necrosis factor (TNF) 3 receptor 1) with their cognate ligands or agonistic antibodies induces receptor aggregation and formation of a "death-inducing signaling complex" (4). Although important differences exist among the various death receptor complexes, they all utilize the adapter protein Fas-associated death domain (FADD) to recruit and induce a conformational change in caspase-8 (and -10), which results in its activation and autoprocessing (induced proximity model) (3). In so-called type I cells, it is thought that death receptor stimulation activates sufficient amounts of caspase-8 within the death-inducing signaling complex so as to efficiently activate caspase-3 and execute the apoptotic program. In contrast, in type II cells, such as Jurkat T cells, caspase-8 indirectly mediates caspase-3 activation by first cleaving and activating the proapoptotic Bcl-2 family member Bid (5). Similar to Bid, other BH3-only proteins, such as Bim, Puma, etc., serve as "stress sensors" that initiate the mitochondrial pathway to apoptosis by either inhibiting anti-apoptotic B...

Hippocrates' assertion that 'what the lance does not heal, fire will' underscores the fact that for thousands of years heat has been used to treat a variety of diseases, including cancer. Indeed, spontaneous tumor remission has been observed in patients following feverish infection [1], and expression of activated oncogenes, such as Ras, can render tumor cells sensitive to heat compared with normal cells [2, 3]. In the past, a primary drawback to the use of heat as a clinical therapy was the inability to selectively focus heat to tumors in situ. Of late, however, several approaches have been devised to deliver heat more precisely, including the use of heated nanoparticles, making hyperthermia a more clinically tractable treatment option [4, 5]. Despite these practical advances, the mechanisms responsible for heat shock-induced cell death remain controversial and ill-defined. In this Visions and Reflections we discuss recent findings surrounding the initiation of heat shock-induced apoptosis, and propose future areas of research.

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