bluuebunny/crossref_metadata_2025 huggingface dataset:
Software agents and the route to the information economy
Authors: Kephart Jeffrey O.
Published: December 2002

Humans are on the verge of losing their status as the sole economic species on the planet. In private laboratories and in the Internet laboratory, researchers and developers are creating a variety of autonomous economically motivated software agents endowed with algorithms for maximizing profit or utility. Many economic software agents will function as miniature businesses, purchasing information inputs from other agents, combining and refining them into information goods and services, and selling them to humans or other agents. Their mutual interactions will form the
information economy
: a complex economic web of information goods and services that will adapt to the ever-changing needs of people and agents. The information economy will be the largest multiagent system ever conceived and an integral part of the world's economy. I discuss a possible route toward this vision, beginning with present-day Internet trends suggesting that agents will charge one another for information goods and services. Then, to establish that agents can be competent price setters, I describe some laboratory experiments pitting software bidding agents against human bidders. The agents' superior performance suggests they will be used on a broad scale, which in turn suggests that interactions among agents will become frequent and significant. How will this affect macroscopic economic behavior? I describe some interesting phenomena that my colleagues and I have observed in simulations of large populations of automated buyers and sellers, such as price war cycles. I conclude by discussing fundamental scientific challenges that remain to be addressed as we journey toward the information economy.
Endogenizing geopolitical boundaries with agent-based modeling
Authors: Cederman Lars-Erik
Published: December 2002

Agent-based modeling promises to overcome the reification of actors. Whereas this common, but limiting, assumption makes a lot of sense during periods characterized by stable actor boundaries, other historical junctures, such as the end of the Cold War, exhibit far-reaching and swift transformations of actors' spatial and organizational existence. Moreover, because actors cannot be assumed to remain constant in the long run, analysis of macrohistorical processes virtually always requires “sociational” endogenization. This paper presents a series of computational models, implemented with the software package REPAST, which trace complex macrohistorical transformations of actors be they hierarchically organized as relational networks or as collections of symbolic categories. With respect to the former, dynamic networks featuring emergent compound actors with agent compartments represented in a spatial grid capture organizational domination of the territorial state. In addition, models of “tagged” social processes allows the analyst to show how democratic states predicate their behavior on categorical traits. Finally, categorical schemata that select out politically relevant cultural traits in ethnic landscapes formalize a constructivist notion of national identity in conformance with the qualitative literature on nationalism. This “finite-agent method”, representing both states and nations as higher-level structures superimposed on a lower-level grid of primitive agents or cultural traits, avoids reification of agency. Furthermore, it opens the door to explicit analysis of entity processes, such as the integration and disintegration of actors as well as boundary transformations.
Foundations of “new” social science: Institutional legitimacy from philosophy, complexity science, postmodernism, and agent-based modeling
Authors: Henrickson Leslie , McKelvey Bill
Published: December 2002

Since the death of positivism in the 1970s, philosophers have turned their attention to scientific realism, evolutionary epistemology, and the Semantic Conception of Theories. Building on these trends, Campbellian Realism allows social scientists to accept real-world phenomena as criterion variables against which theories may be tested without denying the reality of individual interpretation and social construction. The Semantic Conception reduces the importance of axioms, but reaffirms the role of models and experiments. Philosophers now see models as “autonomous agents” that exert independent influence on the development of a science, in addition to theory and data. The inappropriate molding effects of math models on social behavior modeling are noted. Complexity science offers a “new” normal science epistemology focusing on order creation by self-organizing heterogeneous agents and agent-based models. The more responsible core of postmodernism builds on the idea that agents operate in a constantly changing web of interconnections among other agents. The connectionist agent-based models of complexity science draw on the same conception of social ontology as do postmodernists. These recent developments combine to provide foundations for a “new” social science centered on formal modeling not requiring the mathematical assumptions of agent homogeneity and equilibrium conditions. They give this “new” social science legitimacy in scientific circles that current social science approaches lack.
Population growth and collapse in a multiagent model of the Kayenta Anasazi in Long House Valley
Authors: Axtell Robert L. , Epstein Joshua M. , Dean Jeffrey S. , Gumerman George J. , Swedlund Alan C. , Harburger Jason , Chakravarty Shubha , Hammond Ross , Parker Jon , Parker Miles
Published: December 2002

Long House Valley in the Black Mesa area of northeastern Arizona (U.S.) was inhabited by the Kayenta Anasazi from about 1800 before Christ to about
anno Domini
1300. These people were prehistoric ancestors of the modern Pueblo cultures of the Colorado Plateau. Paleoenvironmental research based on alluvial geomorphology, palynology, and dendroclimatology permits accurate quantitative reconstruction of annual fluctuations in potential agricultural production (kg of maize per hectare). The archaeological record of Anasazi farming groups from
anno Domini
200-1300 provides information on a millennium of sociocultural stasis, variability, change, and adaptation. We report on a multiagent computational model of this society that closely reproduces the main features of its actual history, including population ebb and flow, changing spatial settlement patterns, and eventual rapid decline. The agents in the model are monoagriculturalists, who decide both where to situate their fields as well as the location of their settlements. Nutritional needs constrain fertility. Agent heterogeneity, difficult to model mathematically, is demonstrated to be crucial to the high fidelity of the model.
Energetics of nanocrystalline TiO
2
Authors: Ranade M. R. , Navrotsky A. , Zhang H. Z. , Banfield J. F. , Elder S. H. , Zaban A. , Borse P. H. , Kulkarni S. K. , Doran G. S. , Whitfield H. J.
Published: December 2002

The energetics of the TiO
2
polymorphs (rutile, anatase, and brookite) were studied by high temperature oxide melt drop solution calorimetry. Relative to bulk rutile, bulk brookite is 0.71 ± 0.38 kJ/mol (6) and bulk anatase is 2.61 ± 0.41 kJ/mol higher in enthalpy. The surface enthalpies of rutile, brookite, and anatase are 2.2 ± 0.2 J/m
2
, 1.0 ± 0.2 J/m
2
, and 0.4 ± 0.1 J/m
2
, respectively. The closely balanced energetics directly confirm the crossover in stability of nanophase polymorphs inferred by Zhang and Banfield (7). An amorphous sample with surface area of 34,600 m
2
/mol is 24.25 ± 0.88 kJ/mol higher in enthalpy than bulk rutile.
The housekeeping gene xanthine oxidoreductase is necessary for milk fat droplet enveloping and secretion: gene sharing in the lactating mammary gland
Authors: Vorbach Claudia , Scriven Alistair , Capecchi Mario R.
Published: December 2002

Xanthine oxidoreductase (XOR) is the rate-limiting enzyme in purine catabolism occurring in most cell types. However, this housekeeping gene is expressed at very high levels in a number of mammalian tissues including the lactating mammary epithelium, suggesting additional roles for XOR in these tissues. Mice with targeted disruption ofXOR were generated to assess these potential additional roles. XOR−/− mice are runted and do not live beyond 6 wk of age. Strikingly, however, XOR+/− females, although of healthy appearance and normal fertility, are unable to maintain lactation and their pups die of starvation 2 wk postpartum. Histological and whole-mount analyses showed that inXOR+/− females the mammary epithelium collapses, resulting in premature involution of the mammary gland. Electron microscopy showed that XOR is specifically required for enveloping milk fat droplets with the apical plasma membrane prior to secretion from the lactating mammary gland. We present evidence that XOR may have primarily a structural role, as a membrane-associated protein, in milk fat droplet secretion and thus XOR provides another example of “gene sharing”. About 5% of women experience primary lactation insufficiency. The above observations suggest that human females suffering from xanthinuria, a deficiency in XOR, are potential candidates for lactation problems.
Lipoatrophic diabetes in Irs1−/−/Irs3−/− double knockout mice
Authors: Laustsen Palle G. , Michael M. Dodson , Crute Barbara E. , Cohen Shmuel E. , Ueki Kohjiro , Kulkarni Rohit N. , Keller Susanna R. , Lienhard Gustav E. , Kahn C. Ronald
Published: December 2002

Based on the phenotypes of knockout mice and cell lines, as well as pathway-specific analysis, the insulin receptor substrates IRS-1, IRS-2, IRS-3, and IRS-4 have been shown to play unique roles in insulin signal transduction. To investigate possible functional complementarity within the IRS family, we generated mice with double knockout of the genes for IRS-1/IRS-3 and IRS-1/IRS-4. Mice with a combined deficiency of IRS-1 and IRS-4 showed no differences fromIrs1−/− mice with respect to growth and glucose homeostasis. In contrast, mice with a combined deficiency of IRS-1 and IRS-3 developed early-onset severe lipoatrophy associated with marked hyperglycemia, hyperinsulinemia, and insulin resistance. However, in contrast to other models of lipoatrophic diabetes, there was no accumulation of fat in liver or muscle. Furthermore, plasma leptin levels were markedly decreased, and adenovirus-mediated expression of leptin in liver reversed the hyperglycemia and hyperinsulinemia. The results indicate that IRS-1 and IRS-3 play important complementary roles in adipogenesis and establish theIrs1−/−/Irs3−/− double knockout mouse as a novel model of lipoatrophic diabetes.
A cytoskeleton-like role for the bacterial cell wall during engulfment of the Bacillus subtilis forespore
Authors: Abanes-De Mello Angelica , Sun Ya-Lin , Aung Stefan , Pogliano Kit
Published: December 2002

A hallmark of bacterial endospore formation is engulfment, during which the membrane of one cell (the mother cell) migrates around the future spore, enclosing it in the mother cell cytoplasm. Bacteria lack proteins required for eukaryotic phagocytosis, and previously proteins required for membrane migration remained unidentified. Here we provide cell biological and genetic evidence that three membrane proteins synthesized in the mother cell are required for membrane migration as well as for earlier steps in engulfment. Biochemical studies demonstrate that one of these proteins, SpoIID, is a cell wall hydrolase, suggesting that membrane migration in bacteria can be driven by membrane-anchored cell wall hydrolases. We propose that the bacterial cell wall plays a role analogous to that of the actin and tubulin network of eukaryotic cells, providing a scaffold along which proteins can move.
ORC and the intra-S-phase checkpoint: a threshold regulates Rad53p activation in S phase
Authors: Shimada Kenji , Pasero Philippe , Gasser Susan M.
Published: December 2002

The intra-S-phase checkpoint in yeast responds to stalled replication forks by activating the ATM-like kinase Mec1 and the CHK2-related kinase Rad53, which in turn inhibit spindle elongation and late origin firing and lead to a stabilization of DNA polymerases at arrested forks. A mutation that destabilizes the second subunit of the Origin Recognition Complex, orc2-1, reduces the number of functional replication forks by 30% and severely compromises the activation of Rad53 by replication stress or DNA damage in S phase. We show that the restoration of the checkpoint response correlates in a dose-dependent manner with the restoration of pre-replication complex formation in G1. Other forms of DNA damage can compensate for the reduced level of fork-dependent signal in the orc2-1 mutant, yet even in wild-type cells, the amount of damage required for Rad53 activation is higher in S phase than in G2. Our data suggest the existence of an S-phase-specific threshold that may be necessary to allow cells to tolerate damage-like DNA structures present at normal replication forks.
Prevention of Disease Induced by a Partially Heterologous AIDS Virus in Rhesus Monkeys by Using an Adjuvanted Multicomponent Protein Vaccine
Authors: Voss Gerald , Manson Kelledy , Montefiori David , Watkins David I. , Heeney Jonathan , Wyand Michael , Cohen Joe , Bruck Claudine
Published: December 2002

Recombinant protein subunit AIDS vaccines have been based predominantly on the virus envelope protein. Such vaccines elicit neutralizing antibody responses that can provide type-specific sterilizing immunity, but in most cases do not confer protection against divergent viruses. In this report we demonstrate that a multiantigen subunit protein vaccine was able to prevent the development of disease induced in rhesus monkeys by a partially heterologous AIDS virus. The vaccine was composed of recombinant human immunodeficiency virus type 1 (HIV-1) gp120, NefTat fusion protein, and simian immunodeficiency virus (SIV) Nef formulated in the clinically tested adjuvant AS02A. Upon challenge of genetically unselected rhesus monkeys with the highly pathogenic and partially heterologous SIV/HIV strain SHIV89.6pthe vaccine was able to reduce virus load and protect the animals from a decline in CD4-positive cells. Furthermore, vaccination prevented the development of AIDS for more than 2.5 years. The combination of the regulatory proteins Nef and Tat together with the structural protein gp120 was required for vaccine efficacy.
Long-Range Effects of Retroviral Insertion on c
-myb
: Overexpression May Be Obscured by Silencing during Tumor Growth In Vitro
Authors: Hanlon L. , Barr N. I. , Blyth K. , Stewart M. , Haviernik P. , Wolff L. , Weston K. , Cameron E. R. , Neil J. C.
Published: December 2002

The c
-myb
oncogene is a frequent target for retroviral activation in hemopoietic tumors of avian and mammalian species. While insertions can target the gene directly, numerous clusters of retroviral insertion sites have been identified which map close to c
-myb
and outside the transcription unit in T-lymphomas (
Ahi-1
,
fit-1
, and
Mis-2
) and monocytic and myeloid leukemias (
Mml1
,
Mml2
,
Mml3
, and
Epi-1
). Previous analyses showed no consistent effect of these insertions on c
-myb
expression, raising the possibility that other nearby genes were the true targets. In contrast, our analysis of four cell lines established from lymphomas bearing insertions at
fit-1
(
fti-1
) (feline leukemia virus) and
Ahi-1
(Moloney murine leukemia virus) shows that these display higher expression levels of c
-myb
RNA and protein compared to a panel of phenotypically similar cell lines lacking such insertions. An interesting feature of the cell lines with long-range c-
myb
insertions was that each also carried an activated
Myc
allele. The potential for oncogenic synergy between
Myb
and
Myc
in T-cell lymphoma was confirmed in transgenic mice overexpressing alleles of both genes in the T-cell compartment, lending further credence to the case for c-
myb
as the major target for long-range activation. In contrast, mapping and analysis of c-
myb
neighboring genes (
HBS1
and
FLJ20069
) showed that the expression of these genes did not correlate well with the presence of proviral insertions. A possible explanation for the paradoxical behavior of c
-myb
was provided by one of the murine T-lymphoma lines bearing an insertion at
Ahi-1
(p/m16i) that reproducibly down-regulated c
-myb
RNA and protein to very low levels or undetectable levels on prolonged culture. Our observations implicate c
-myb
as a key target of upstream and downstream retroviral insertions. However, overexpression may become dispensable during outgrowth in vitro, and perhaps during tumor progression in vivo, providing a potential rationale for the previously observed discordance between retroviral insertion and c
-myb
expression levels.
Initiation of Poliovirus Negative-Strand RNA Synthesis Requires Precursor Forms of P2 Proteins
Authors: Jurgens Christy , Flanegan James B.
Published: December 2002

The replication proteins encoded in the P2 region of the poliovirus genome induce extensive rearrangement of cellular membranes into vesicles and are a required component of viral RNA replication complexes. To identify distinct viral protein(s) from the P2 region of the genome that were required to form functional RNA replication complexes, the P2 proteins were expressed in addition to P3 in HeLa S10 translation-RNA replication reactions. Membrane-associated preinitiation replication complexes were isolated from these reactions and used to measure negative-strand synthesis. The formation of replication complexes capable of initiating negative-strand synthesis was observed when either P23 or when P2 and P3 were expressed in the HeLa S10 translation-replication reactions. The amount of negative-strand RNA synthesized with P2 and P3 was approximately 50% of that observed with P23. Negative-strand synthesis was not observed when the processed forms of the P2 proteins (e.g., 2A, 2B, 2C, 2AB, and 2BC) were used in various combinations in place of P2. In contrast, the expression of 2A and 2BCP3 supported negative-strand synthesis at the same level observed with P23. Therefore, functional replication complexes were formed in reaction mixtures that contained either 2A and 2BCP3 or P2 and P3. Genetic complementation analysis of P23 RNA that contained a lethal mutation in 2C confirmed these results. The expression of 2BCP3 in
trans
restored the replication of P23-2C(P131N) RNA to wild-type levels. The expression of P2 and P3 also complemented the replication of this mutant RNA, although very inefficiently. Complementation was not observed in reactions that contained P2 alone, 2BC, or 2C. Based on these results, we propose that RNA replication complexes are initially formed with the primary cleavage products of P23 (i.e., P2 and P3 or 2A and 2BCP3), and that 2A and 2BCP3 are preferentially used in this process.
Nonneutralizing Antibodies to the CD4-Binding Site on the gp120 Subunit of Human Immunodeficiency Virus Type 1 Do Not Interfere with the Activity of a Neutralizing Antibody against the Same Site
Authors: Herrera Carolina , Spenlehauer Catherine , Fung Michael S. , Burton Dennis R. , Beddows Simon , Moore John P.
Published: December 2002

We have investigated whether nonneutralizing monoclonal antibodies (MAbs) to the gp120 subunit of the envelope glycoprotein (Env) complex of human immunodeficiency virus type 1 (HIV-1) can interfere with HIV-1 neutralization by another anti-gp120 MAb. We used neutralizing (b12) and nonneutralizing (205-42-15, 204-43-1, 205-46-9) MAbs to the epitope cluster overlapping the CD4-binding site (CD4BS) on gp120. All the MAbs, neutralizing or otherwise, cross-competed for binding to monomeric gp120, indicating the close topological proximity of their epitopes. However, the nonneutralizing CD4BS MAbs did not interfere with the neutralization activity of MAb b12. In contrast, in a binding assay using oligomeric Env expressed on the surface of Env-transfected cells, the nonneutralizing MAbs did partially compete with b12 for Env binding. The surface of Env-transfected cells contains two categories of binding site for CD4BS MAbs. One type of site is recognized by both b12 and nonneutralizing CD4BS MAbs; the other is recognized by only b12. Binding assays for Env-gp120 interactions based on the use of monomeric gp120 or Env-transfected cells do not predict the outcome of HIV-1 neutralization assays, and they should therefore be used only with caution when gauging the properties of anti-Env MAbs.
Identification and Characterization of Avian Retroviruses in Chicken Embryo-Derived Yellow Fever Vaccines: Investigation of Transmission to Vaccine Recipients
Authors: Hussain Althaf I. , Johnson Jeffrey A. , da Silva Freire Marcos , Heneine Walid
Published: December 2002

All currently licensed yellow fever (YF) vaccines are propagated in chicken embryos. Recent studies of chick cell-derived measles and mumps vaccines show evidence of two types of retrovirus particles, the endogenous avian retrovirus (EAV) and the endogenous avian leukosis virus (ALV-E), which originate from the chicken embryonic fibroblast substrates. In this study, we investigated substrate-derived avian retrovirus contamination in YF vaccines currently produced by three manufacturers (YF-vax [Connaught Laboratories], Stamaril [Aventis], and YF-FIOCRUZ [FIOCRUZ-Bio-Manguinhos]). Testing for reverse transcriptase (RT) activity was not possible because of assay inhibition. However, Western blot analysis of virus pellets with anti-ALV RT antiserum detected three distinct RT proteins in all vaccines, indicating that more than one source is responsible for the RTs present in the vaccines. PCR analysis of both chicken substrate DNA and particle-associated RNA from the YF vaccines showed no evidence of the long terminal repeat sequences of exogenous ALV subgroups A to D in any of the vaccines. In contrast, both ALV-E and EAV particle-associated RNA were detected at equivalent titers in each vaccine by RT-PCR. Quantitative real-time RT-PCR revealed 61,600, 348,000, and 1,665,000 ALV-E RNA copies per dose of Stamaril, YF-FIOCRUZ, and YF-vax vaccines, respectively.
ev
locus-specific PCR testing of the vaccine-associated chicken substrate DNA was positive both for the nondefective
ev
-12 locus in two vaccines and for the defective
ev
-1 locus in all three vaccines. Both intact and
ev
-1
pol
sequences were also identified in the particle-associated RNA. To investigate the risks of transmission, serum samples from 43 YF vaccine recipients were studied. None of the samples were seropositive by an ALV-E-based Western blot assay or had detectable EAV or ALV-E RNA sequences by RT-PCR. YF vaccines produced by the three manufacturers all have particles containing EAV genomes and various levels of defective or nondefective ALV-E sequences. The absence of evidence of infection with ALV-E or EAV in 43 YF vaccine recipients suggests low risks for transmission of these viruses, further supporting the safety of these vaccines.
Human Immunodeficiency Virus Type 1 Vif Is Efficiently Packaged into Virions during Productive but Not Chronic Infection
Authors: Kao Sandra , Akari Hirofumi , Khan Mohammad A. , Dettenhofer Markus , Yu Xiao-Fang , Strebel Klaus
Published: December 2002

Packaging of the human immunodeficiency virus type 1 Vif protein into virus particles is mediated through an interaction with viral genomic RNA and results in the association of Vif with the nucleoprotein complex. Despite the specificity of this process, calculations of the amount of Vif packaged have produced vastly different results. Here, we compared the efficiency of packaging of Vif into virions derived from acutely and chronically infected H9 cells. We found that Vif was efficiently packaged into virions from acutely infected cells (60 to 100 copies per virion), while packaging into virions from chronically infected H9 cells was near the limit of detection (four to six copies of Vif per virion). Superinfection by an exogenous Vif-defective virus did not rescue packaging of endogenous Vif expressed in the chronically infected culture. In contrast, exogenous Vif expressed by superinfection of wild-type virus was readily packaged (30 to 40 copies per virion). Biochemical analyses suggest that the differences in the relative packaging efficiencies were not due to gross differences in the steady-state distribution of Vif in chronically or acutely infected cells but are likely due to differences in the relative rates of de novo synthesis of Vif. Despite its low packaging efficiency, endogenously expressed Vif was sufficient to direct the production of viruses with almost wild-type infectivity. The results from our study provide novel insights into the biochemical properties of Vif and offer an explanation for the reported differences regarding Vif packaging.
Determinants of the Host Range Restriction of Replication of Bovine Parainfluenza Virus Type 3 in Rhesus Monkeys Are Polygenic
Authors: Skiadopoulos Mario H. , Schmidt Alexander C. , Riggs Jeffrey M. , Surman Sonja R. , Elkins William R. , St. Claire Marisa , Collins Peter L. , Murphy Brian R.
Published: December 2002

The Kansas strain of bovine parainfluenza virus type 3 (BPIV3) is 100- to 1,000-fold restricted in replication in the respiratory tracts of nonhuman primates compared to human PIV3 (HPIV3), an important pathogen of infants and young children. BPIV3 is also restricted in replication in human infants and children, yet it is immunogenic and is currently being evaluated in clinical trials as a vaccine candidate to protect against illness caused by HPIV3. We have examined the genetic basis for the host range attenuation phenotype of BPIV3 by exchanging each open reading frame (ORF) of a recombinant wild-type HPIV3 with the analogous ORF from BPIV3, with the caveats that the multiple ORFs of the P gene were exchanged as a single unit and that the HN and F genes were exchanged as a single unit. Recombinant chimeric bovine-human PIV3s were recovered from cDNA, and the levels of viral replication in vitro and in the respiratory tract of rhesus monkeys were determined. Recombinant chimeric HPIV3s bearing the BPIV3 N or P ORF were highly attenuated in the upper and lower respiratory tracts of monkeys, whereas those bearing the BPIV3 M or L ORF or the F and HN genes were only moderately attenuated. This indicates that the genetic determinants of the host range restriction of replication of BPIV3 for primates are polygenic, with the major determinants being the N and P ORFs. Monkeys immunized with these bovine-human chimeric viruses, including the more highly attenuated ones, developed higher levels of HPIV3 hemagglutination-inhibiting serum antibodies than did monkeys immunized with BPIV3 and were protected from challenge with wild-type HPIV3. Furthermore, host range determinants could be combined with attenuating point mutations to achieve an increased level of attenuation. Thus, chimeric recombinant bovine-human PIV3 viruses that manifest different levels of attenuation in rhesus monkeys are available for evaluation as vaccine candidates to protect infants from the severe lower respiratory tract disease caused by HPIV3.
An Attenuating Mutation in nsP1 of the Sindbis-Group Virus S.A.AR86 Accelerates Nonstructural Protein Processing and Up-Regulates Viral 26S RNA Synthesis
Authors: Heise Mark T. , White Laura J. , Simpson Dennis A. , Leonard Christopher , Bernard Kristen A. , Meeker Rick B. , Johnston Robert E.
Published: December 2002

The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.
The Stability of the Duplex between Sense and Antisense Transcription-Regulating Sequences Is a Crucial Factor in Arterivirus Subgenomic mRNA Synthesis
Authors: Pasternak Alexander O. , van den Born Erwin , Spaan Willy J. M. , Snijder Eric J.
Published: December 2002

Subgenomic mRNAs of nidoviruses (arteriviruses and coronaviruses) are composed of a common leader sequence and a “body” part of variable size, which are derived from the 5′- and 3′-proximal part of the genome, respectively. Leader-to-body joining has been proposed to occur during minus-strand RNA synthesis and to involve transfer of the nascent RNA strand from one site in the template to another. This discontinuous step in subgenomic RNA synthesis is guided by short transcription-regulating sequences (TRSs) that are present at both these template sites (leader TRS and body TRS). Sense-antisense base pairing between the leader TRS in the plus strand and the body TRS complement in the minus strand is crucial for strand transfer. Here we show that extending the leader TRS-body TRS duplex beyond its wild-type length dramatically enhanced the subgenomic mRNA synthesis of the arterivirus
Equine arteritis virus
(EAV). Generally, the relative amount of a subgenomic mRNA correlated with the calculated stability of the corresponding leader TRS-body TRS duplex. In addition, various leader TRS mutations induced the generation of minor subgenomic RNA species that were not detected upon infection with wild-type EAV. The synthesis of these RNA species involved leader-body junction events at sites that bear only limited resemblance to the canonical TRS. However, with the mutant leader TRS, but not with the wild-type leader TRS, these sequences could form a duplex that was stable enough to direct subgenomic RNA synthesis, again demonstrating that the stability of the leader TRS-body TRS duplex is a crucial factor in arterivirus subgenomic mRNA synthesis.
Characterization of the Genomic Promoter of the Prototypic Arenavirus Lymphocytic Choriomeningitis Virus
Authors: Perez Mar , de la Torre Juan Carlos
Published: December 2002

The genome of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) consists of two negative-sense, single-strand RNA segments designated L and S. Arenavirus genomes exhibit high sequence conservation at their 3′ ends. All arenavirus genomes examined to date have a conserved terminal sequence element (3′-terminal 20 nucleotides [nt]) thought to be a highly conserved viral promoter. Terminal complementarity between the 5′ and 3′ ends of the L and S RNAs predicts the formation of a thermodynamically stable panhandle structure that could contribute to the control of RNA synthesis. We investigated these issues by using a transcription- and replication-competent minireplicon system. A series of overlapping deletions spanning the 3′-terminal 20-nt region of an LCMV minigenome (MG) was generated, and the mutant MGs were analyzed for their activity as templates for RNA synthesis by the LCMV polymerase. The minimal LCMV genomic promoter was found to be contained within the 3′-terminal 19 nt. Substitution of C for G at the last 3′-end nucleotide position in the MG resulted in nondetection of RNA transcription or replication, whereas the addition of a C at the 3′ end did not have any significant affect on RNA synthesis mediated by the LCMV polymerase. All other mutations introduced within the 3′-terminal 19 nt of the MG resulted in undetectable levels of promoter activity. Deletions and nucleotide substitutions within the MG 5′ end that disrupted terminal complementarity abolished chloramphenicol acetyltransferase expression and RNA synthesis mediated by the LCMV polymerase. These findings indicate that both sequence specificity within the 3′-terminal 19 nt and the integrity of the predicted panhandle structure appear to be required for efficient RNA synthesis mediated by the LCMV polymerase.
Evidence of the Coevolution of Antigenicity and Host Cell Tropism of Foot-and-Mouth Disease Virus In Vivo
Authors: Tami Cecilia , Taboga Oscar , Berinstein Analía , Núñez José I. , Palma Eduardo L. , Domingo Esteban , Sobrino Francisco , Carrillo Elisa
Published: December 2002

In this work we analyze the antigenic properties and the stability in cell culture of virus mutants recovered upon challenge of peptide-vaccinated cattle with foot-and-mouth disease virus (FMDV) C3 Arg85. Previously, we showed that a significant proportion of 29 lesions analyzed (41%) contained viruses with single amino acid replacements (R141G, L144P, or L147P) within a major antigenic site located at the G-H loop of VP1, known to participate also in interactions with integrin receptors. Here we document that no replacements at this site were found in viruses from 12 lesions developed in six control animals upon challenge with FMDV C3 Arg85. Sera from unprotected, vaccinated animals exhibited poor neutralization titers against mutants recovered from them. Sequence analyses of the viruses recovered upon 10 serial passages in BHK-21 and FBK-2 cells in the presence of preimmune (nonneutralizing) sera revealed that mutants reverted to the parental sequence, suggesting an effect of the amino acid replacements in the interaction of the viruses with cells. Parallel passages in the presence of subneutralizing concentrations of immune homologous sera resulted in the maintenance of mutations R141G and L147P, while mutation L144P reverted to the C3 Arg85 sequence. Reactivity with a panel of FMDV type C-specific monoclonal antibodies indicated that mutant viruses showed altered antigenicity. These results suggest that the selective pressure exerted by host humoral immune response can play a role in both the selection and stability of antigenic FMDV variants and that such variants can manifest alterations in cell tropism.
A Truncated Form of Nef Selected during Pathogenic Reversion of Simian Immunodeficiency Virus SIVmac239Δnef Increases Viral Replication
Authors: Chakrabarti Lisa A. , Metzner Karin J. , Ivanovic Tijana , Cheng Hua , Louis-Virelizier Jean , Connor Ruth I. , Cheng-Mayer Cecilia
Published: December 2002

The live, attenuated vaccine simian immunodeficiency virus SIVmac239Δnef efficiently protects rhesus macaques against infection with wild-type SIVmac but occasionally causes CD4+T-cell depletion and progression to simian AIDS (SAIDS). Virus recovered from a vaccinated macaque (Rh1490) that progressed to SAIDS had acquired an additional deletion in thenefgene, resulting in a frameshift that restored the originalnefopen reading frame (R. I. Connor, D. C. Montefiori, J. M. Binley, J. P. Moore, S. Bonhoeffer, A. Gettie, E. A. Fenamore, K. E. Sheridan, D. D. Ho, P. J. Dailey, and P. A. Marx, J. Virol. 72:7501-7509, 1998). Intravenous inoculation of the Rh1490 viral isolate into four naive rhesus macaques induced CD4+T-cell depletion and disease in three out of four animals within 2 years, indicating a restoration of virulence. A DNA fragment encompassing the truncatednefgene amplified from the Rh1490 isolate was inserted into the genetic backbone of SIVmac239. The resulting clone, SIVmac239-Δ2nef, expressed a Nef protein of approximately 23 kDa, while the original SIVmac239Δnef clone expressed a shorter protein of 8 kDa. The revertant form of Nef did not cause downregulation of CD4, CD3, or major histocompatibility complex class I. The infectivity of SIVmac239-Δ2nef was similar to that of SIVmac239Δnef in single-cycle assays using indicator cell lines. In contrast, SIVmac239-Δ2nef replicated more efficiently than SIVmac239Δnef in peripheral blood mononuclear cell (PBMC) cultures infected under unstimulated conditions. The p27 Gag antigen levels in SIVmac239-Δ2nef-infected cultures were still lower than those obtained with wild-type SIVmac239, consistent with a partial recovery of Nef function. The transcriptional activity of long terminal repeat (LTR)-luciferase constructs containing thenefdeletions did not differ markedly from that of wild-type LTR. Introduction of a premature stop codon within Nef-Δ2 abolished the replicative advantage in PBMCs, demonstrating that the Nef-Δ2 protein, rather than the structure of the U3 region of the LTR, was responsible for the increase in viral replication. Taken together, these results show that SIV with a deletion in thenefgene can revert to virulence and that expression of a form ofnefwith multiple deletions may contribute to this process by increasing viral replication.
Negative Regulation of Mitotic Promoting Factor by the Checkpoint Kinase Chk1 in Simian Virus 40 Lytic Infection
Authors: Okubo Eiji , Lehman John M. , Friedrich Thomas D.
Published: December 2002

Lytic infection of African green monkey kidney (CV-1) cells by simian virus 40 (SV40) is characterized by stimulation of DNA synthesis leading to bypass of mitosis and replication of cellular and viral DNA beyond a 4C DNA content. To define mechanisms underlying the absence of mitosis, the expression levels of upstream regulatory molecules of mitosis-promoting factor (MPF) were compared in parallel synchronized cultures of SV40-infected and uninfected CV-1 cells. The DNA replication/damage checkpoint kinase Chk1 was phosphorylated in both uninfected and SV40-infected cultures arrested at G
1
/S by mimosine, consistent with checkpoint activation. Following release of uninfected cultures from G
1
/S, Chk1 phosphorylation was lost even though Chk1 protein levels were retained. In contrast, G
1
/S-released SV40-infected cultures exhibited dephosphorylation of Chk1 in S phase, followed by an increase in Chk1 phosphorylation coinciding with entry of infected cells into >G
2
. Inhibitors of Chk1, UCN-01 and caffeine, induced mitosis and abnormal nuclear condensation and increased the protein kinase activity of MPF in SV40-infected CV-1 cells. These results demonstrate that SV40 lytic infection triggers components of a DNA damage checkpoint pathway. In addition, chemical inhibition of Chk1 activity suggests that Chk1 contributes to the absence of mitosis during SV40 lytic infection.
Microarray Analysis of Host Cell Gene Transcription in Response to Varicella-Zoster Virus Infection of Human T Cells and Fibroblasts In Vitro and SCIDhu Skin Xenografts In Vivo
Authors: Jones Jeremy O. , Arvin Ann M.
Published: December 2002

During primary infection, varicella-zoster virus (VZV) is spread via lymphocytes to skin, where it induces a rash and establishes latency in sensory ganglia. A live, attenuated varicella vaccine (vOka) was generated by using the VZV Oka strain (pOka), but the molecular basis for vOka attenuation remains unknown. Little is known concerning the effects of wild-type or attenuated VZV on cellular gene regulation in the host cells that are critical for pathogenesis. In this study, transcriptional profiles of primary human T cells and fibroblasts infected with VZV in cell culture were determined by using 40,000-spot human cDNA microarrays. Cellular gene transcription in human skin xenografts in SCID mice that were infected with VZV in vivo was also evaluated. The profiles of cellular gene transcripts that were induced or inhibited in infected human foreskin fibroblasts (HFFs), T cells, and skin in response to pOka and vOka infection were similar. However, significant alterations in cellular gene regulation were observed among the three differentiated human cell types that were examined, suggesting specific differences in the biological consequences of VZV infection related to the target cell. Changes in cellular gene transcription detected by microarray analysis were confirmed for selected genes by quantitative real-time reverse transcription-PCR analysis of VZV-infected cells. Interestingly, the transcription of caspase 8 was found to be decreased in infected T cells but not in HFFs or skin, which may signify a tissue-specific antiapoptosis mechanism. The use of microarrays to demonstrate differences in effects on host cell genes in primary, biologically relevant cell types provides background information for experiments to link these various response phenotypes with mechanisms of VZV pathogenesis that are important for the natural course of human infection.
Cytoplasmic Tail of Moloney Murine Leukemia Virus Envelope Protein Influences the Conformation of the Extracellular Domain: Implications for Mechanism of Action of the R Peptide
Authors: Aguilar Hector C. , Anderson W. French , Cannon Paula M.
Published: December 2002

The envelope (Env) protein of Moloney murine leukemia virus (MoMuLV) is a homotrimeric complex whose monomers consist of linked surface (SU) and transmembrane (TM) proteins cleaved from a precursor protein by a cellular protease. In addition, a significant fraction of virion-associated TM is further processed by the viral protease to remove the C-terminal 16 amino acids of the cytoplasmic domain, the R peptide. This cleavage greatly enhances the fusogenicity of the protein and is necessary for the formation of a fully functional Env protein complex. We have previously proposed that R peptide cleavage enhances fusogenicity by altering the conformation of the ectodomain of the protein (Y. Zhao et al., J. Virol. 72:5392-5398, 1998). Using a series of truncation and point mutants of MoMuLV Env, we now provide direct biochemical and immunological evidence that the cytoplasmic tail and the membrane-spanning region of Env can influence the overall structure of the ectodomain of the protein and alter the strength of the SU-TM interaction. The R-peptide-truncated form of the protein, in particular, exhibits a markedly different conformation than the full-length protein.
The Herpes Simplex Virus Type 2 R1 Protein Kinase (ICP10 PK) Functions as a Dominant Regulator of Apoptosis in Hippocampal Neurons Involving Activation of the ERK Survival Pathway and Upregulation of the Antiapoptotic Protein Bag-1
Authors: Perkins D. , Pereira E. F. R. , Aurelian L.
Published: December 2002

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) can trigger or block apoptosis in a cell type-dependent manner. We have recently shown that the protein kinase activity of the large subunit of the HSV-2 ribonucleotide reductase (R1) protein (ICP10 PK) blocks apoptosis in cultured hippocampal neurons by activating the extracellular signal-regulated kinase (ERK) survival pathway (Perkins et al., J. Virol. 76:1435-1449, 2002). The present studies were designed to better elucidate the mechanism of ICP10 PK-induced neuroprotection and determine whether HSV-1 has similar activity. The data indicate that apoptosis inhibition by ICP10 PK involves a c-Raf-1-dependent mechanism and induction of the antiapoptotic protein Bag-1 by the activated ERK survival pathway. Also associated with neuroprotection by ICP10 PK are increased activation/stability of the transcription factor CREB and stabilization of the antiapoptotic protein Bcl-2. HSV-1 and the ICP10 PK-deleted HSV-2 mutant ICP10ΔPK activate JNK, c-Jun, and ATF-2, induce the proapoptotic protein BAD, and trigger apoptosis in hippocampal neurons. c-Jun activation and apoptosis are inhibited in hippocampal cultures infected with HSV-1 in the presence of the JNK inhibitor SP600125, suggesting that JNK/c-Jun activation is required for HSV-1-induced apoptosis. Ectopically delivered ICP10 PK (but not its PK-negative mutant p139) inhibits apoptosis triggered by HSV-1 or ICP10ΔPK. Collectively, the data indicate that ICP10 PK-induced activation of the ERK survival pathway results in Bag-1 upregulation and overrides the proapoptotic JNK/c-Jun signal induced by other viral proteins.
Viability of a Drug-Resistant Human Immunodeficiency Virus Type 1 Protease Variant: Structural Insights for Better Antiviral Therapy
Authors: Prabu-Jeyabalan Moses , Nalivaika Ellen A. , King Nancy M. , Schiffer Celia A.
Published: December 2002

Under the selective pressure of protease inhibitor therapy, patients infected with human immunodeficiency virus (HIV) often develop drug-resistant HIV strains. One of the first drug-resistant mutations to arise in the protease, particularly in patients receiving indinavir or ritonavir treatment, is V82A, which compromises the binding of these and other inhibitors but allows the virus to remain viable. To probe this drug resistance, we solved the crystal structures of three natural substrates and two commercial drugs in complex with an inactive drug-resistant mutant (D25N/V82A) HIV-1 protease. Through structural analysis and comparison of the protein-ligand interactions, we found that Val82 interacts more closely with the drugs than with the natural substrate peptides. The V82A mutation compromises these interactions with the drugs while not greatly affecting the substrate interactions, which is consistent with previously published kinetic data. Coupled with our earlier observations, these findings suggest that future inhibitor design may reduce the probability of the appearance of drug-resistant mutations by targeting residues that are essential for substrate recognition.
TRAF1 Is a Critical Regulator of JNK Signaling by the TRAF-Binding Domain of the Epstein-Barr Virus-Encoded Latent Infection Membrane Protein 1 but Not CD40
Authors: Eliopoulos Aristides G. , Waites Elyse R. , Blake Sarah M. S. , Davies Clare , Murray Paul , Young Lawrence S.
Published: December 2002

The oncogenic Epstein-Barr virus (EBV)-encoded latent infection membrane protein 1 (LMP1) mimics a constitutive active tumor necrosis factor (TNF) family receptor in its ability to recruit TNF receptor-associated factors (TRAFs) and TNF receptor-associated death domain protein (TRADD) in a ligand-independent manner. As a result, LMP1 constitutively engages signaling pathways, such as the JNK and p38 mitogen-activated protein kinases (MAPK), the transcription factor NF-κB, and the JAK/STAT cascade, and these activities may explain many of its pleiotropic effects on cell phenotype, growth, and transformation. In this study we demonstrate the ability of the TRAF-binding domain of LMP1 to signal on the JNK/AP-1 axis in a cell type- dependent manner that critically involves TRAF1 and TRAF2. Thus, expression of this LMP1 domain in TRAF1-positive lymphoma cells promotes significant JNK activation, which is blocked by dominant-negative TRAF2 but not TRAF5. However, TRAF1 is absent in many established epithelial cell lines and primary nasopharyngeal carcinoma (NPC) biopsy specimens. In these cells, JNK activation by the TRAF-binding domain of LMP1 depends on the reconstitution of TRAF1 expression. The critical role of TRAF1 in the regulation of TRAF2-dependent JNK signaling is particular to the TRAF-binding domain of LMP1, since a homologous region in the cytoplasmic tail of CD40 or the TRADD-interacting domain of LMP1 signal on the JNK axis independently of TRAF1 status. These data further dissect the signaling components used by LMP1 and identify a novel role for TRAF1 as a modulator of oncogenic signals.