bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations in Penicillin-Binding Protein (PBP) Genes and in Non-PBP Genes during Selection of Penicillin-Resistant
Streptococcus gordonii
Authors: Haenni Marisa , Moreillon Philippe
Published: November 2006

Penicillin resistance in
Streptococcus
spp. involves multiple mutations in both penicillin-binding proteins (PBPs) and non-PBP genes. Here, we studied the development of penicillin resistance in the oral commensal
Streptococcus gordonii
. Cyclic exposure of bacteria to twofold-increasing penicillin concentrations selected for a progressive 250- to 500-fold MIC increase (from 0.008 to between 2 and 4 μg/ml). The major MIC increase (≥35-fold) was related to non-PBP mutations, whereas PBP mutations accounted only for a 4- to 8-fold additional increase. PBP mutations occurred in class B PBPs 2X and 2B, which carry a transpeptidase domain, but not in class A PBP 1A, 1B, or 2A, which carry an additional transglycosylase domain. Therefore, we tested whether inactivation of class A PBPs affected resistance development in spite of the absence of mutations. Deletion of PBP 1A or 2A profoundly slowed down resistance development but only moderately affected resistance in already highly resistant mutants (MIC = 2 to 4 μg/ml). Thus, class A PBPs might facilitate early development of resistance by stabilizing penicillin-altered peptidoglycan via transglycosylation, whereas they might be less indispensable in highly resistant mutants which have reestablished a penicillin-insensitive cell wall-building machinery. The contribution of PBP and non-PBP mutations alone could be individualized in DNA transformation. Both PBP and non-PBP mutations conferred some level of intrinsic resistance, but combining the mutations synergized them to ensure high-level resistance (≥2 μg/ml). The results underline the complexity of penicillin resistance development and suggest that inhibition of transglycosylase might be an as yet underestimated way to interfere with early resistance development.
qnrA
in CTX-M-Producing
Escherichia coli
Isolates from France
Authors: Lavigne Jean-Philippe , Marchandin Hélène , Delmas Julien , Bouziges Nicole , Lecaillon Evelyne , Cavalie Laurent , Jean-Pierre Hélène , Bonnet Richard , Sotto Albert
Published: November 2006

By PCR, we screened for
qnr
genes 112 clinical isolates of extended-spectrum β-lactamase-producing
Escherichia coli
collected from hospitals in France during 2004. For the first time, 7.7% of CTX-M-producing
E. coli
isolates presented a plasmid-mediated resistance to quinolones. All strains harbored a
qnrA
gene located on a
sul1
-type class 1 integron with similar structure to the In
36
integron.
A
trans
-Golgi Network Resident Protein, golgin-97, Accumulates in Viral Factories and Incorporates into Virions during Poxvirus Infection
Authors: Alzhanova Dina , Hruby Dennis E.
Published: November 2006

Poxviruses are the only DNA viruses known to replicate and assemble in the cytoplasm of infected cells. Poxvirus morphogenesis is a complicated process in which four distinct infectious forms of the virus are produced: intracellular mature virus, intracellular enveloped virus, cell-associated enveloped virus, and extracellular enveloped virus. The source of primary membrane wrapping the intracellular mature virus, the first infectious form, is still unknown. Although the membrane was suggested to originate from the endoplasmic reticulum-Golgi intermediate compartment, none of the marker proteins from this or any other cell compartments has been found in the intracellular mature virus. Thus, it was hypothesized that the membrane is either extensively modified by the virus or synthesized de novo. In the work described here, we demonstrate that a host cell protein residing in the
trans
-Golgi network membrane, golgin-97, is transported to the sites of virus replication and assembly and becomes incorporated into the virions during poxvirus infection. Inside the virion, golgin-97 is associated with the insoluble core protein fraction. Being able to adopt a long rod-like structure, the protein apparently extends through the virion envelope and protrudes from its surface. Here we discuss the potential role and functions of golgin-97 in poxvirus replication and propose two working models.
Translational Inhibition and Increased Interferon Induction in Cells Infected with C Protein-Deficient Measles Virus
Authors: Nakatsu Yuichiro , Takeda Makoto , Ohno Shinji , Koga Ritsuko , Yanagi Yusuke
Published: November 2006

In addition to the phosphoprotein, the P gene of measles virus (MV) also encodes the V and C proteins by an RNA editing process and by alternative initiation of translation in a different reading frame, respectively. Although the MV C protein is required for efficient MV replication in vivo and in some cultured cells, its exact functions in virus infection are currently unclear. Here, we report that a recombinant MV lacking the C protein (MVΔC) grew poorly in a human cell line possessing the intact interferon (IFN) pathway and that this growth defect was associated with reduced viral translation and genome replication. The translational inhibition was correlated with phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2. Moreover, increased IFN induction was observed in MVΔC-infected cells. The NS1 protein of influenza virus, which binds to double-stranded RNA (dsRNA) and consequently inhibits IFN induction and dsRNA-dependent protein kinase activation, complemented the growth defect of MVΔC. These results indicate that the MV C protein inhibits IFN induction and modulates host antiviral responses, thereby ensuring MV growth in host cells.
Prevalence and Phylogeny of Kakugo Virus, a Novel Insect Picorna-Like Virus That Infects the Honeybee (
Apis mellifera
L.), under Various Colony Conditions
Authors: Fujiyuki Tomoko , Ohka Seii , Takeuchi Hideaki , Ono Masato , Nomoto Akio , Kubo Takeo
Published: November 2006

We previously identified a novel insect picorna-like virus, termed Kakugo virus (KV), from the brains of aggressive worker honeybees that had counterattacked a giant hornet. To survey the prevalence of KV in worker populations engaged in various labors, we quantified KV genomic RNA. KV was detected specifically from aggressive workers in some colonies, while it was also detected from other worker populations in other colonies where the amount of KV detected in the workers was relatively high, suggesting that KV can infect various worker populations in the honeybee colonies. To investigate whether the KV strains detected were identical, phylogenetic analysis was performed. There was less than a 2% difference in the RNA-dependent RNA polymerase (RdRp) sequences between KV strains from aggressive workers and those from other worker populations, suggesting that all of the viruses detected were virtually the same KV. We also found that some of the KV-infected colonies were parasitized by
Varroa
mites, and the sequences of the KV strains detected from the mites were the same as those detected from the workers of the same colonies, suggesting that the mites mediate KV prevalence in the honeybee colonies. KV strains had approximately 6% and 15% sequence differences in the RdRp region from deformed wing virus and Varroa destructor virus 1, respectively, suggesting that KV represents a viral strain closely related to, but distinct from, these two viruses.
A Helper-Dependent Capsid-Modified Adenovirus Vector Expressing Adeno-Associated Virus Rep78 Mediates Site-Specific Integration of a 27-Kilobase Transgene Cassette
Authors: Wang Hongjie , Lieber André
Published: November 2006

Random integration of viral gene therapy vectors and subsequent activation or disruption of cellular genes poses safety risks. Major efforts in the field are aimed toward targeting vector integration to specific sites in the host genome. The adeno-associated virus (AAV) Rep78 protein is able to target AAV integration to a specific site on human chromosome 19, called AAVS1. We studied whether this ability could be harnessed to achieve site-specific integration of a 27-kb transgene cassette into a model cell line for human hematopoietic cells (Mo7e). To deliver
rep78
and the transgene to Mo7e cells, we used helper-dependent adenovirus (Ad) vectors containing Ad serotype 35 fiber knob domains (HD-Ad). An HD-Ad vector containing the
rep78
gene under the control of the globin locus control region (LCR) (Ad.LCR-rep78) conferred Rep78 expression on Mo7e cells. Upon coinfection of Ad.LCR-rep78 with an HD-Ad vector containing a 27-kb globin-LCR-green fluorescent protein (GFP) transgene cassette flanked by AAV inverted terminal repeats (ITRs) (Ad.AAV-LCR-GFP), transduced cells were cloned and expanded (without selection pressure), and vector integration was analyzed in clones with more than 30% GFP-positive cells. Vector integration into the AAVS1 region was seen in 30% of analyzed integration sites, and GFP expression from these integrants was stable over time. Of the remaining integration sites, 25% were within the genomic globin LCR. In almost 90% of sites, transgene integration occurred via the Ad ITR. This indicates that rescue of the AAV ITR-flanked transgene cassette from Ad.AAV-LCR-GFP is not required for Rep78-mediated integration into AAVS1 and that free ends within the vector genome can be created by breaks within the Ad ITRs, whose structure is apparently recognized by cellular “nicking” enzymes. The finding that 55% of all analyzed integration sites were either within the AAVS1 or globin LCR region demonstrates that a high frequency of targeted integration of a large transgene cassette can be achieved in human hematopoietic stem cell lines.
Mimivirus Giant Particles Incorporate a Large Fraction of Anonymous and Unique Gene Products
Authors: Renesto Patricia , Abergel Chantal , Decloquement Philippe , Moinier Danielle , Azza Saïd , Ogata Hiroyuki , Fourquet Patrick , Gorvel Jean-Pierre , Claverie Jean-Michel
Published: November 2006

Acanthamoeba polyphaga mimivirus
is the largest known virus in both particle size and genome complexity. Its 1.2-Mb genome encodes 911 proteins, among which only 298 have predicted functions. The composition of purified isolated virions was analyzed by using a combined electrophoresis/mass spectrometry approach allowing the identification of 114 proteins. Besides the expected major structural components, the viral particle packages 12 proteins unambiguously associated with transcriptional machinery, 3 proteins associated with DNA repair, and 2 topoisomerases. Other main functional categories represented in the virion include oxidative pathways and protein modification. More than half of the identified virion-associated proteins correspond to anonymous genes of unknown function, including 45 “ORFans.” As demonstrated by both Western blotting and immunogold staining, some of these “ORFans,” which lack any convincing similarity in the sequence databases, are endowed with antigenic properties. Thus, anonymous and unique genes constituting the majority of the mimivirus gene complement encode bona fide proteins that are likely to participate in well-integrated processes.
Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1
Authors: Hakki Morgan , Marshall Emily E. , De Niro Katherine L. , Geballe Adam P.
Published: November 2006

The human cytomegalovirus (HCMV) TRS1 and IRS1 genes block the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) and the consequent shutoff of cellular protein synthesis that occur during infection with vaccinia virus (VV) deleted of the double-stranded RNA binding protein gene E3L (VVΔE3L). To further define the underlying mechanism, we first evaluated the effect of pTRS1 on protein kinase R (PKR), the double-stranded RNA (dsRNA)-dependent eIF2α kinase. Immunoblot analyses revealed that pTRS1 expression in the context of a VVΔE3L recombinant decreased levels of PKR in the cytoplasm and increased its levels in the nucleus of infected cells, an effect not seen with wild-type VV or a VVΔE3L recombinant virus expressing E3L. This effect of pTRS1 was confirmed by visualizing the nuclear relocalization of PKR-EGFP expressed by transient transfection. PKR present in both the nuclear and cytoplasmic fractions was nonphosphorylated, indicating that it was unactivated when TRS1 was present. PKR also accumulated in the nucleus during HCMV infection as determined by indirect immunofluorescence and immunoblot analysis. Binding assays revealed that pTRS1 interacted with PKR in mammalian cells and in vitro. This interaction required the same carboxy-terminal region of pTRS1 that is necessary to rescue VVΔE3L replication in HeLa cells. The carboxy terminus of pIRS1 was also required for rescue of VVΔE3L and for mediating an interaction of pIRS1 with PKR. These results suggest that these HCMV genes directly interact with PKR and inhibit its activation by sequestering it in the nucleus, away from both its activator, cytoplasmic dsRNA, and its substrate, eIF2α.
Preferential Translation of Vesicular Stomatitis Virus mRNAs Is Conferred by Transcription from the Viral Genome
Authors: Whitlow Zackary W. , Connor John H. , Lyles Douglas S.
Published: November 2006

Host protein synthesis is inhibited in cells infected with vesicular stomatitis virus (VSV). It has been proposed that viral mRNAs are subjected to the same inhibition but are predominantly translated because of their abundance. To compare translation efficiencies of viral and host mRNAs during infection, we used an enhanced green fluorescent protein (EGFP) reporter expressed from a recombinant virus or from the host nucleus in stably transfected cells. Translation efficiency of host-derived EGFP mRNA was reduced more than threefold at eight hours postinfection, while viral-derived mRNA was translated around sevenfold more efficiently than host-derived EGFP mRNA in VSV-infected cells. To test whether mRNAs transcribed in the cytoplasm are resistant to shutoff of translation during VSV infection, HeLa cells were infected with a recombinant simian virus 5 (rSV5) that expressed GFP. Cells were then superinfected with VSV or mock superinfected. GFP mRNA transcribed by rSV5 was not resistant to translation inhibition during superinfection with VSV, indicating that transcription in the cytoplasm is not sufficient for preventing translation inhibition. To determine if
cis
-acting sequences in untranslated regions (UTRs) were involved in preferential translation of VSV mRNAs, we constructed EGFP reporters with VSV or control UTRs and measured the translation efficiency in mock-infected and VSV-infected cells. The presence of VSV UTRs did not affect mRNA translation efficiency in mock- or VSV-infected cells, indicating that VSV mRNAs do not contain
cis
-acting sequences that influence translation. However, we found that when EGFP mRNAs transcribed by VSV or by the host were translated in vitro, VSV-derived EGFP mRNA was translated 22 times more efficiently than host-derived EGFP mRNA. This indicated that VSV mRNAs do contain
cis
-acting structural elements (that are not sequence based), which enhance translation efficiency of viral mRNAs.
A Full-Length cDNA Infectious Clone of North American Type 1 Porcine Reproductive and Respiratory Syndrome Virus: Expression of Green Fluorescent Protein in the Nsp2 Region
Authors: Fang Ying , Rowland Raymond R. R. , Roof Michael , Lunney Joan K. , Christopher-Hennings Jane , Nelson Eric A.
Published: November 2006

The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States.
Hemagglutinin (HA) Proteins from H1 and H3 Serotypes of Influenza A Viruses Require Different Antigen Designs for the Induction of Optimal Protective Antibody Responses as Studied by Codon-Optimized HA DNA Vaccines
Authors: Wang Shixia , Taaffe Jessica , Parker Christopher , Solórzano Alicia , Cao Hong , García-Sastre Adolfo , Lu Shan
Published: November 2006

Effective antibody responses provide crucial immunity against influenza virus infection. The hemagglutinin (HA) protein is the major target of protective antibody responses induced by viral infection and by vaccination with both inactivated and live-attenuated flu vaccines, but knowledge about the optimal designs of protective HA antigens from different flu serotypes is still limited. In this study, we have significantly improved the immunogenicity of HA-expressing DNA vaccines by using codon-optimized HA sequences for either an H1 serotype (A/NewCal/20/99) or an H3 serotype (A/Panama/2007/99) human influenza A virus and then used these constructs as model antigens to identify the optimal HA antigen designs to elicit high-level protective antibody responses. Two forms of HA antigen, a wild-type, full-length HA and a secreted form with transmembrane (TM) domain-truncated HA, were produced. Both forms of HA DNA vaccines, from either H1 or H3 serotypes, were able to elicit high levels of HA-specific immunoglobulin G responses in immunized rabbits as measured by enzyme-linked immunosorbent assay. Interestingly, the abilities of H1 HA and H3 HA antigens to elicit hemagglutination inhibition (HI) and neutralizing antibody (NAb) responses differ. For the H1 HA antigens, the full-length HA induced significantly higher HI and NAb responses than did the TM-truncated HA. For the H3 HA antigen, both the full-length HA and TM-truncated HA induced high levels of HI and NAb responses. These data indicate that H1 and H3 antigens have different expression requirements for the induction of an optimal protective antibody response and that the structure integrity of HA antigens is critical for eliciting type-specific protective antibody responses. Our findings will have an important impact on future subunit-based flu vaccine development.
Crystal Structure of the West Nile Virus Envelope Glycoprotein
Authors: Nybakken Grant E. , Nelson Christopher A. , Chen Beverly R. , Diamond Michael S. , Fremont Daved H.
Published: November 2006

The envelope glycoprotein (E) of West Nile virus (WNV) undergoes a conformational rearrangement triggered by low pH that results in a class II fusion event required for viral entry. Herein we present the 3.0-Å crystal structure of the ectodomain of WNV E, which reveals insights into the flavivirus life cycle. We found that WNV E adopts a three-domain architecture that is shared by the E proteins from dengue and tick-borne encephalitis viruses and forms a rod-shaped configuration similar to that observed in immature flavivirus particles. Interestingly, the single N-linked glycosylation site on WNV E is displaced by a novel α-helix, which could potentially alter lectin-mediated attachment. The localization of histidines within the hinge regions of E implicates these residues in pH-induced conformational transitions. Most strikingly, the WNV E ectodomain crystallized as a monomer, in contrast to other flavivirus E proteins, which have crystallized as antiparallel dimers. WNV E assembles in a crystalline lattice of perpendicular molecules, with the fusion loop of one E protein buried in a hydrophobic pocket at the DI-DIII interface of another. Dimeric E proteins pack their fusion loops into analogous pockets at the dimer interface. We speculate that E proteins could pivot around the fusion loop-pocket junction, allowing virion conformational transitions while minimizing fusion loop exposure.
Double-Stranded RNA Binding of Influenza B Virus Nonstructural NS1 Protein Inhibits Protein Kinase R but Is Not Essential To Antagonize Production of Alpha/Beta Interferon
Authors: Dauber Bianca , Schneider Jana , Wolff Thorsten
Published: November 2006

Expression of alpha/beta interferon (IFN-α/β) in virus-infected vertebrate cells is a key event in the establishment of a sustained antiviral response, which is triggered by double-stranded RNA (dsRNA) produced during viral replication. These antiviral cytokines initiate the expression of cellular proteins with activities that limit the replication and spread of the invading viruses. Within this response, the dsRNA-dependent protein kinase R (PKR) that is expressed at constitutive levels and upregulated by IFN-α/β acts as an important antiviral effector that can block the cellular translational machinery. We previously demonstrated that efficient replication of influenza B virus depends on the viral dsRNA-binding NS1 protein that inhibits the transcriptional activation of IFN-α/β genes. Here we tested the postulate that the viral NS1 protein counteracts antiviral responses through sequestering intracellular dsRNA by analyzing a collection of recombinant influenza B viruses. As expected, viruses expressing dsRNA-binding-defective NS1 proteins were strongly attenuated for replication in IFN-competent hosts. Interestingly, these virus mutants failed to prevent activation of PKR but could effectively limit IFN induction. Conversely, a mutant virus expressing the N-terminal dsRNA-binding domain of NS1 prevented PKR activation, but not IFN induction, suggesting an important role for the NS1 C-terminal part in silencing the activation route of IFN-α/β genes. Thus, our findings indicate an unexpected mechanistic dichotomy of the influenza B virus NS1 protein in the suppression of antiviral responses, which involves at least one activity that is largely separable from dsRNA binding.
Encapsidation Determinants Located Downstream of the Major Splice Donor in the Maedi-Visna Virus Leader Region
Authors: Bjarnadottir Helga , Gudmundsson Bjarki , Gudnason Janus , Jonsson Jon J.
Published: November 2006

We investigated the role of the 5′-untranslated region between the primer binding site and the
gag
initiation codon in ovine lentivirus maedi-visna virus (MVV) genomic RNA encapsidation. We identified five computer-predicted stem-loops, three of which were highly conserved in primary sequence and structure. One stable 83-nucleotide (nt) stem-loop (SL4) was not conserved in the primary sequence, but phylogenetic analysis revealed several base pair covariations. The deletion of individual stem-loops did not markedly affect the relative encapsidation efficiency (REE). Only one mutant, carrying a disruption of a 31-nt stem-loop (SL5), had 58% REE in fetal ovine synovial (FOS) cells. A 168-nt deletion (Δ3MSD) downstream of the major splice donor (MSD) which removed three stem-loops, including SL5, resulted in 24% and 20% REE in FOS and 293T cells, respectively. A 100-nt deletion (Δ5MSD) upstream of the MSD resulted in 15-fold lower cellular genomic RNA levels than the wild-type levels in 293T cells. The Δ5MSD mutant and a double mutant (DM) (Δ5MSD and Δ3MSD) did not express detectable levels of virion proteins in 293T cells. In contrast, the region deleted in Δ5MSD was dispensable in FOS cells, and the DM had the same REE as the Δ3MSD virus. Thus, the region upstream of the MSD contains sequences critical for RNA and protein expression in a cell type-specific fashion. Our results indicate that MVV encapsidation determinants are located downstream of the MSD. These results provide comparative insight into lentiviral encapsidation and can be utilized in the design of MVV-based gene transfer vectors.
Replication of Murine Hepatitis Virus Is Regulated by Papain-Like Proteinase 1 Processing of Nonstructural Proteins 1, 2, and 3
Authors: Graham Rachel L. , Denison Mark R.
Published: November 2006

Coronaviruses are positive-strand RNA viruses that translate their genome RNA into polyproteins that are co- and posttranslationally processed into intermediate and mature replicase nonstructural proteins (nsps). In murine hepatitis virus (MHV), nsps 1, 2, and 3 are processed by two papain-like proteinase activities within nsp3 (PLP1 and PLP2) to yield nsp1, an nsp2-3 intermediate, and mature nsp2 and nsp3. To determine the role in replication of processing between nsp2 and nsp3 at cleavage site 2 (CS2) and PLP1 proteinase activity, mutations were engineered into the MHV genome at CS2, at CS1 and CS2, and at the PLP1 catalytic site, alone and in combination. Mutant viruses with abolished cleavage at CS2 were delayed in growth and RNA synthesis but grew to wild-type titers of >107PFU/ml. Mutant viruses with deletion of both CS1 and CS2 exhibited both a delay in growth and a decrease in peak viral titer to ∼104PFU/ml. Inactivation of PLP1 catalytic residues resulted in a mutant virus that did not process at either CS1 or CS2 and was severely debilitated in growth, achieving only 102PFU/ml. However, when both CS1 and CS2 were deleted in the presence of inactivated PLP1, the growth of the resulting mutant virus was partially compensated, comparable to that of the CS1 and CS2 deletion mutant. These results demonstrate that interactions of PLP1 with CS1 and CS2 are critical for protein processing and suggest that the interactions play specific roles in regulation of the functions of nsp1, 2, and 3 in viral RNA synthesis.
Cryoelectron Microscopy of Protein IX-Modified Adenoviruses Suggests a New Position for the C Terminus of Protein IX
Authors: Marsh Michael P. , Campos Samuel K. , Baker Matthew L. , Chen Christopher Y. , Chiu Wah , Barry Michael A.
Published: November 2006

Recombinant human adenovirus is a useful gene delivery vector for clinical gene therapy. Minor capsid protein IX of adenovirus has been of recent interest since multiple studies have shown that modifications can be made to its C terminus to alter viral tropism or add molecular tags and/or reporter proteins. We examined the structure of an engineered adenovirus displaying the enhanced green fluorescent protein (EGFP) fused to the C terminus of protein IX. Cryoelectron microscopy and reconstruction localized the C-terminal EGFP fusion between the H2 hexon and the H4 hexon, positioned between adjacent facets, directly above the density previously assigned as protein IIIa. The original assignment of IIIa was based largely on indirect evidence, and the data presented herein support the reassignment of the IIIa density as protein IX.
Structurally Mapping the Diverse Phenotype of Adeno-Associated Virus Serotype 4
Authors: Govindasamy Lakshmanan , Padron Eric , McKenna Robert , Muzyczka Nicholas , Kaludov Nikola , Chiorini John A. , Agbandje-McKenna Mavis
Published: November 2006

The adeno-associated viruses (AAVs) can package and deliver foreign DNA into cells for corrective gene delivery applications. The AAV serotypes have distinct cell binding, transduction, and antigenic characteristics that have been shown to be dictated by the capsid viral protein (VP) sequence. To understand the contribution of capsid structure to these properties, we have determined the crystal structure of AAV serotype 4 (AAV4), one of the most diverse serotypes with respect to capsid protein sequence and antigenic reactivity. Structural comparison of AAV4 to AAV2 shows conservation of the core β strands (βB to βI) and helical (αA) secondary structure elements, which also exist in all other known parvovirus structures. However, surface loop variations (I to IX), some containing compensating structural insertions and deletions in adjacent regions, result in local topological differences on the capsid surface. These include AAV4 having a deeper twofold depression, wider and rounder protrusions surrounding the threefold axes, and a different topology at the top of the fivefold channel from that of AAV2. Also, the previously observed “valleys” between the threefold protrusions, containing AAV2's heparin binding residues, are narrower in AAV4. The observed differences in loop topologies at subunit interfaces are consistent with the inability of AAV2 and AAV4 VPs to combine for mosaic capsid formation in efforts to engineer novel tropisms. Significantly, all of the surface loop variations are associated with amino acids reported to affect receptor recognition, transduction, and anticapsid antibody reactivity for AAV2. This observation suggests that these capsid regions may also play similar roles in the other AAV serotypes.
Live Attenuated Influenza Virus Expressing Human Interleukin-2 Reveals Increased Immunogenic Potential in Young and Aged Hosts
Authors: Ferko Boris , Kittel Christian , Romanova Julia , Sereinig Sabine , Katinger Hermann , Egorov Andrej
Published: November 2006

Despite the reported efficacy of commercially available influenza virus vaccines, a considerable proportion of the human population does not respond well to vaccination. In an attempt to improve the immunogenicity of live influenza vaccines, an attenuated, cold-adapted (ca) influenza A virus expressing human interleukin-2 (IL-2) from the NS gene was generated. Intranasal immunization of young adult and aged mice with the IL-2-expressing virus resulted in markedly enhanced mucosal and cellular immune responses compared to those of mice immunized with the nonrecombinantcaparent strain. Interestingly, the mucosal immunoglobulin A (IgA) and CD8+T-cell responses in the respiratory compartment could be restored in aged mice primed with the IL-2-expressing virus to magnitudes similar to those in young adult mice. The immunomodulating effect of locally expressed IL-2 also gave rise to a systemic CD8+T-cell and distant urogenital IgA response in young adult mice, but this effect was less distinct in aged mice. Importantly, only mice immunized with the recombinant IL-2 virus were completely protected from a pathogenic wild-type virus challenge and revealed a stronger onset of virus-specific CD8+T-cell recall response. Our findings emphasize the potential of reverse genetics to improve the efficacy of live influenza vaccines, thus rendering them more suitable for high-risk age groups.
Hepatitis C Virus Entry Requires a Critical Postinternalization Step and Delivery to Early Endosomes via Clathrin-Coated Vesicles
Authors: Meertens Laurent , Bertaux Claire , Dragic Tatjana
Published: November 2006

Hepatitis C virus (HCV) is a major human pathogen associated with life-threatening liver disease. Entry into hepatocytes requires CD81 and a putative second receptor. In this study, we elucidated the postreceptor attachment stages of HCV entry using HCV pseudoparticles (HCVpp) as a model system. By means of dominant-negative mutants and short interfering RNAs of various cellular proteins, we showed that HCVpp enter via clathrin-coated vesicles and require delivery to early but not to late endosomes. However, the kinetics of HCV envelope glycoprotein-mediated fusion are delayed compared to those of other viruses that enter in early endosomes. Entry of HCVpp can be efficiently blocked by bafilomycin A1, which neutralizes the pH in early endosomes and impairs progression of endocytosis beyond this stage. However, low-pH exposure of bafilomycin A1-treated target cells does not induce entry of HCVpp at the plasma membrane or in the early stages of endocytosis. These observations indicate that, subsequent to internalization, HCVpp entry necessitates additional, low-pH-dependent interactions, modifications, or trafficking, and that these events are irreversibly disrupted by bafilomycin A1 treatment.
Genetic and Neutralization Properties of Subtype C Human Immunodeficiency Virus Type 1 Molecular
env
Clones from Acute and Early Heterosexually Acquired Infections in Southern Africa
Authors: Li Ming , Salazar-Gonzalez Jesus F. , Derdeyn Cynthia A. , Morris Lynn , Williamson Carolyn , Robinson James E. , Decker Julie M. , Li Yingying , Salazar Maria G. , Polonis Victoria R. , Mlisana Koleka , Karim Salim Abdool , Hong Kunxue , Greene Kelli M. , Bilska Miroslawa , Zhou Jintao , Allen Susan , Chomba Elwyn , Mulenga Joseph , Vwalika Cheswa , Gao Feng , Zhang Ming , Korber Bette T. M. , Hunter Eric , Hahn Beatrice H. , Montefiori David C.
Published: November 2006

A standard panel of subtype C human immunodeficiency virus type 1 (HIV-1) Env-pseudotyped viruses was created by cloning, sequencing, and characterizing functional gp160 genes from 18 acute and early heterosexually acquired infections in South Africa and Zambia. In general, the gp120 region of these clones was shorter (most evident in V1 and V4) and less glycosylated compared to newly transmitted subtype B viruses, and it was underglycosylated but no different in length compared to chronic subtype C viruses. The gp120s also exhibited low amino acid sequence variability (12%) in V3 and high variability (39%) immediately downstream of V3, a feature shared with newly transmitted subtype B viruses and chronic viruses of both subtypes. When tested as Env-pseudotyped viruses in a luciferase reporter gene assay, all clones possessed an R5 phenotype and resembled primary isolates in their sensitivity to neutralization by HIV-1-positive plasmas. Results obtained with a multisubtype plasma panel suggested partial subtype preference in the neutralizing antibody response to infection. The clones were typical of subtype C in that all were resistant to 2G12 (associated with loss of N-glycosylation at position 295) and most were resistant to 2F5, but all were sensitive to 4E10 and many were sensitive to immunoglobulin G1b12. Finally, conserved neutralization epitopes in the CD4-induced coreceptor binding domain of gp120 were poorly accessible and were difficult to induce and stabilize with soluble CD4 on Env-pseudotyped viruses. These results illustrate key genetic and antigenic properties of subtype C HIV-1 that might impact the design and testing of candidate vaccines. A subset of these gp160 clones are suitable for use as reference reagents to facilitate standardized assessments of vaccine-elicited neutralizing antibody responses.
Characterization of
White Bream Virus
Reveals a Novel Genetic Cluster of Nidoviruses
Authors: Schütze Heike , Ulferts Rachel , Schelle Barbara , Bayer Sonja , Granzow Harald , Hoffmann Bernd , Mettenleiter Thomas C. , Ziebuhr John
Published: November 2006

The order
Nidovirales
comprises viruses from the families
Coronaviridae
(genera
Coronavirus
and
Torovirus
),
Roniviridae
(genus
Okavirus
), and
Arteriviridae
(genus
Arterivirus
). In this study, we characterized
White bream virus
(WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5′ leader sequence that is identical to the 5′-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.
NSAIDs and Cancer Prevention: Targets Downstream of COX-2
Authors: Cha Yong I. , DuBois Raymond N.
Published: November 2006

Preclinical and clinical studies have clearly shown a benefit of nonsteroidal anti-inflammatory drug (NSAID) use in reducing cancer risk. However, the adverse gastrointestinal and cardiovascular side effects associated with NSAIDs and COX-2 selective inhibitors (coxibs) have provoked more scrutiny of the precise role of specific downstream mediators in the prostaglandin (PG) signaling cascade. NSAIDs and coxibs inhibit PG biosynthesis. One of the PGs produced at high levels in the tumor microenvironment is PGE2, which is thought to play a major role in cancer progression. Thus, a better understanding of PGE2 signaling could enable identification of novel and safer therapeutic targets downstream of the cyclooxygenase enzymes. We review the emerging molecular mechanisms by which COX-2–derived PGE2 is involved in cancer progression and delineate potential opportunities for development of novel pharmacologic approaches utilizing this pathway.
The Amt/MEP/Rh Family: Structure of AmtB and the Mechanism of Ammonia Gas Conduction
Authors: Khademi Shahram , Stroud Robert M.
Published: November 2006

The atomic structures of the first members of the Amt/MEP/Rh family show that they are 11-crossing membrane proteins that form trimers in the membrane. Each monomer supports a hydrophobic channel that conducts NH3but not any water or ions. The reprotonation of NH3on the receiving side raises the pH on that side in the absence of metabolism of NH3, and there is no transfer of protons through the protein.
Identification of Carboxypeptidase of Glutamate Like-B as a Candidate Suppressor in Cell Growth and Metastasis in Human Hepatocellular Carcinoma
Authors: Zhang Pingping , Chan David Wai , Zhu YiYi , Li Jin Jun , Ng Irene Oi-Lin , Wan Dafang , Gu Jianren
Published: November 2006

Purpose: We have previously done large-scale cDNA transfection screening on human hepatocellular carcinoma (HCC) cells and have identified 3,806 cDNA genes that possess the ability of either stimulating or inhibiting cell growth. In this study, we characterized one of these growth suppressor genes, carboxypeptidase of glutamate like-B (CPGL-B), in HCC.

Experimental Design: Semiquantitative reverse-transcription PCR was used to examine the expression levels of CPGL-B. The cellular localization and functions of CPGL-B were investigated by enforced expression of CPGL-B in HCC cells.

Results: From our previous cDNA transfection screening, we identified a gene named CPGL and its isoform, CPGL-B. With computational analysis, CPGL was located at chromosome 18q22.3 and was a homologue of peptidase family M20. CPGL was expressed in all adult and fetal tissues, whereas its isoform, CPGL-B, lacking exons 3 and 4, was expressed in all fetal tissues but only in liver and placenta of adult tissues. In HCC, CPGL-B was frequently underexpressed (35 of 90, 38.9%) in tumorous tissues compared with the corresponding nontumorous livers. Intriguingly, the underexpression was significantly associated with the presence of venous invasion (P = 0.018) and tumor microsatellite formation (P = 0.004). Stable transfection of CPGL-B in SMMC7721 HCC cells showed significant inhibition in cell viability, colony formation, cell invasion, and tumor formation in nude mice. CPGL-B also down-regulated CXCR3, matrix metalloproteinase 11, and CD44s, which are involved in cell growth and cell migration.

Conclusions: These findings suggest that the frequent underexpression of CPGL-B may be associated with cell growth and metastasis of HCC.
Activation of mammalian target of rapamycin and the p70 S6 kinase by arsenic trioxide in BCR-ABL–expressing cells
Authors: Yoon Patrick , Giafis Nick , Smith Jessica , Mears Heather , Katsoulidis Efstratios , Sassano Antonella , Altman Jessica , Redig Amanda J. , Tallman Martin S. , Platanias Leonidas C.
Published: November 2006

Arsenic trioxide (As2O3) exhibits important antitumor activities in vitro and in vivo, but the precise mechanisms by which it induces its effects are not known. We provide evidence that during treatment of BCR-ABL–expressing cells with As2O3, there is activation of a cellular pathway involving the p70 S6 kinase (p70S6K). Our data show that p70S6K is rapidly phosphorylated on Thr421 and Ser424 and is activated in an As2O3-inducible manner. The mammalian target of rapamycin (mTOR) is also phosphorylated/activated in an As2O3-inducible manner, and its activity is required for downstream engagement of p70S6K. p70S6K subsequently phosphorylates the S6 ribosomal protein on Ser235/Ser236 and Ser240/Ser244 to promote initiation of mRNA translation. Treatment of chronic myelogenous leukemia–derived cell lines with As2O3 also results in phosphorylation of the 4E-BP1 repressor of mRNA translation on Thr37/Thr46 and Thr70, sites required for its deactivation and its dissociation from the eukaryotic initiation factor 4E complex to allow cap-dependent mRNA translation. In studies to determine the functional relevance of this pathway, we found that inhibition of mTOR and downstream cascades enhances induction of apoptosis by As2O3. Consistent with this, the mTOR inhibitor rapamycin strongly potentiated As2O3-mediated suppression of primitive leukemic progenitors from the bone marrow of chronic myelogenous leukemia patients. Altogether, our data show that the mTOR/p70S6K pathway is activated in a negative feedback regulatory manner in response to As2O3 in BCR-ABL–transformed cells and plays a key regulatory role in the induction of anti-leukemic responses. [Mol Cancer Ther 2006;5(11):2815–23]
Color and Genomic Ancestry in Brazilians: A Study with Forensic Microsatellites
Authors: Pimenta Juliana R. , Zuccherato Luciana W. , Debes Adriana A. , Maselli Luciana , Soares Rosângela P. , Moura-Neto Rodrigo S. , Rocha Jorge , Bydlowski Sergio P. , Pena Sérgio D.J.
Published: November 2006

The population of Brazil, formed by extensive admixture between Amerindians, Europeans and Africans, is one of the most variable in the world. We have recently published a study that used ancestry-informative markers to conclude that in Brazil, at an individual level, color, as determined by physical evaluation, was a poor predictor of genomic ancestry, estimated by molecular markers. To corroborate these findings we undertook the present investigation based on data from 12 commercially available forensic microsatellites that were utilized to estimate the personal genomic origin for each of 752 individuals from the city of São Paulo, belonging to different Brazilian color categories (275 Whites, 192 Intermediates and 285 Blacks). The genotypes permitted the calculation of a personal likelihood-ratio estimator of African or European ancestry. Although the 12 marker set proved capable of discriminating between European and African individuals, we observed very significant overlaps among the three color categories of Brazilians. This was confirmed quantitatively using a Bayesian analysis of population structure that did not demonstrate significant genetic differentiation between the three color groups. These results corroborate and validate our previous conclusions using ancestry-informative markers that in Brazil at the individual level there is significant dissociation of color and genomic ancestry.
Pattern of Lymph Node Metastases in Patients with Squamous Cell Carcinoma of the Thoracic Esophagus Who Underwent Three-Field Lymphadenectomy
Authors: Li Hecheng , Zhang Yawei , Cai Hui , Xiang Jiaqing
Published: November 2006

Background/Aims: Lymph nodes in patients with squamous cell carcinoma of the thoracic esophagus might be involved with metastases at cervical, mediastinal, and abdominal sites. The range of lymph node dissection is still controversial. The pattern of lymph node metastasis and factors that are correlated with lymph node metastasis affect the surgical procedure of lymph node dissection. The purpose of the present study was to explore the pattern of lymph node metastasis and factors that are correlated with lymph node metastasis in patients with esophageal cancer who underwent three-field lymphadenectomy. Methods: Lymph node metastases in 230 patients who underwent radical esophagectomy with three-field lymphadenectomy were analyzed. The metastatic sites of lymph nodes were correlated with tumor location by chi-square test. Logistic regression was used to analyze clinicopathological factors related to lymph node metastasis. Results:Lymph node metastases were found in 133 of the 230 patients (57.8%). The average number of resected lymph nodes was 25.3 ± 11.4 (range 11–71). The proportions of lymph node metastases were 41.6, 19.44, and 8.3% in neck, thoracic mediastinum, and abdominal cavity, respectively, for patients with upper thoracic esophageal carcinomas, 33.3, 34.7, and 14%, respectively, in those with middle thoracic esophageal carcinomas, and 36.4, 34.1, and 43.2%, respectively, for patients with lower thoracic esophageal carcinomas. We did not observe any significant difference in lymph node metastatic rates among upper, middle, and lower thoracic carcinomas for cervical or thoracic nodes. The difference in lymph node metastatic rates for nodes in the abdominal cavity was significant among upper, middle, and lower thoracic carcinomas. The lower thoracic esophageal cancers were more likely to metastasize to the abdominal cavity than tumors at other thoracic sites. A logistic regression model showed that depth of tumor invasion and lymphatic vessel invasion were factors influencing lymph node metastases. Conclusions: Based on our data, cervical and mediastinal node dissection should be performed independent of the tumor location. Abdominal node dissection should be conducted more vigorously for lower thoracic esophageal cancers than for cancers at other locations. Patients with deeper tumor invasion or lymphatic vessel invasion were more likely to develop lymph node metastases.
Extravascular Lung Water Measured Using Single Transpulmonary Thermodilution Reflects Perioperative Pulmonary Edema Induced by Esophagectomy
Authors: Sato Y. , Motoyama S. , Maruyama K. , Okuyama M. , Hayashi K. , Nakae H. , Tajimi K. , Ogawa J.
Published: November 2006

Pulmonary edema is the most frequent postoperative complication following esophagectomy for thoracic esophageal cancer. We enrolled 23 patients who underwent esophagectomy with extended lymph node dissection for thoracic esophageal cancer in a prospective observational clinical trial. We used the PiCCO device to measure extravascular lung water with the aim of determining whether it correlates with the respiratory index and whether it is predictive of pulmonary complications. Based on constant criteria, the tracheal tubes of 11 patients were removed on the morning of postoperative day 1 (extubation group), while 12 patients remained intubated (intubation group). These two groups significantly differed in that all patients in the extubation group recovered without any pulmonary complications, whereas 4 patients (33%) in the intubation group developed pulmonary complications. The extravascular lung water measured using PiCCO correlated significantly with the respiratory index. In the intubation group, both extravascular lung water and respiratory index were elevated 12 h after surgery and were even higher 24 h after surgery. The extravascular lung water measured using PiCCO reflects the level of postoperative pulmonary edema and predicts the pulmonary complications induced by esophagectomy with extended lymph node dissection.