bluuebunny/crossref_metadata_2025 huggingface dataset:
Error-Prone Polymerase, DNA Polymerase IV, Is Responsible for Transient Hypermutation during Adaptive Mutation in
Escherichia coli
Authors: Tompkins Joshua D. , Nelson Jennifer L. , Hazel Jill C. , Leugers Stacy L. , Stumpf Jeffrey D. , Foster Patricia L.
Published: May 2003

The frequencies of nonselected mutations among adaptive Lac
+
revertants of
Escherichia coli
strains with and without the error-prone DNA polymerase IV (Pol IV) were compared. This frequency was more than sevenfold lower in the Pol IV-defective strain than in the wild-type strain. Thus, the mutations that occur during hypermutation are due to Pol IV.
Genomic Structure of the
Salmonella enterica
Serovar Typhimurium DT 64 Bacteriophage ST64T: Evidence for Modular Genetic Architecture
Authors: Mmolawa Princess T. , Schmieger Horst , Tucker Carly P. , Heuzenroeder Michael W.
Published: May 2003

The complete sequence of the double-stranded DNA genome of a serotype-converting temperate bacteriophage, ST64T, was determined. The 40,679-bp genomic sequence of ST64T has an overall GC content of 47.5% and was reminiscent of a number of lambdoid phages, in particular, P22. Inferred proteins of ST64T which exhibited a high degree of sequence similarity to P22 proteins (>90%) included the functional serotype conversion cassette, integrase, excisionase, Abc1, Abc2, early antitermination (gp24), NinD, NinH, NinZ, packaging (gp3 and gp2), head (with the exception of gp26, gp7, gp20, and gp16), and tail proteins. The putative immunity genes were highly related to those of
Salmonella enterica
serotype Typhimurium phage L, whereas the lysis genes were almost identical to those of
S. enterica
serovar Typhimurium PS3.
The Glycosyltransferase Gene Encoding the Enzyme Catalyzing the First Step of Mycothiol Biosynthesis (
mshA
)
Authors: Newton Gerald L. , Koledin Teresa , Gorovitz Batia , Rawat Mamta , Fahey Robert C. , Av-Gay Yossef
Published: May 2003

Mycothiol is the major thiol present in most actinomycetes and is produced from the pseudodisaccharide 1
d
-
myo
-inosityl 2-acetamido-2-deoxy-α-
d
-glucopyranoside (GlcNAc-Ins). A transposon mutant of
Mycobacterium smegmatis
shown to be GlcNAc-Ins and mycothiol deficient was sequenced to identify a putative glycosyltransferase gene designated
mshA
. The ortholog in
Mycobacterium tuberculosis
, Rv0486, was used to complement the mutant phenotype.
Synthesis and Localization of the
Salmonella
SPI-1 Type III Secretion Needle Complex Proteins PrgI and PrgJ
Authors: Sukhan Anand , Kubori Tomoko , Galán Jorge E.
Published: May 2003

An essential component of type III secretion systems (TTSS) is a supramolecular structure termed the needle complex. In
Salmonella enterica
, at least four proteins make up this structure: InvG, PrgH, PrgK, and PrgI. Another protein, PrgJ, is thought to play a role in the assembly of this structure, but its function is poorly understood. We have analyzed the expression and localization of PrgJ and the needle protein PrgI in different
S. enterica
serovar Typhimurium mutant strains. We found that the levels of PrgI and PrgJ were significantly reduced in a TTSS-deficient
invA
mutant strain and that the decreased levels were due to protein instability. In addition, we found that PrgJ, although associated with the needle complex in wild-type
S. enterica
serovar Typhimurium, was absent from needle complexes obtained from an
invJ
mutant strain, which exhibits very long needle substructures. We suggest that PrgJ is involved in capping the needle substructure of the needle complex.
Transplacental Transfer and Subsequent Neonate Utilization of Herpes Simplex Virus-Specific Immunity Are Resilient to Acute Maternal Stress
Authors: Yorty Jodi L. , Bonneau Robert H.
Published: May 2003

Neonates are severely compromised in the ability to generate an immune response to pathogens and thus rely heavily on maternally derived immunity that is acquired by transplacental and transmammary means. The passive transfer of maternal herpes simplex virus (HSV)-specific antibody is critical in determining the outcome of neonatal HSV infection. In adults, psychological stress alters immune responsiveness via the increased level of corticosterone that is produced as a result of hypothalamic-pituitary-adrenal axis activation. Although the behavioral and neuroendocrine effects of pre- and postnatal stress-induced increases in corticosterone are well documented, the effects of maternal stress on the efficacy of prenatally transferred and neonatally developed viral immunity has yet to be addressed. By using a well-established prenatal restraint-and-light stress mouse model, we investigated the effects of increased maternal corticosterone on the passive transfer of total and HSV-specific immunoglobulin G (IgG) antibody and subsequent neonatal susceptibility to HSV infection. Serum corticosterone levels in pregnant mice were significantly increased in response to restraint-and-light stress, and fetuses derived from these stressed mice had significantly elevated levels of corticosterone. Despite the increases in corticosterone, the passive transfer of total and HSV-specific IgG antibody persisted and, in turn, protected the neonate from systemic viral spread. Therefore, prenatal stress did not increase the susceptibility of neonates to HSV type 2-associated mortality. These findings demonstrate the resiliency of the passive transfer of protective HSV-specific immunity under conditions of acute psychological stress.
Human Immunodeficiency Virus Type 1 Entry Inhibitors Selected on Living Cells from a Library of Phage Chemokines
Authors: Hartley Oliver , Dorgham Karim , Perez-Bercoff Danielle , Cerini Fabrice , Heimann Anouk , Gaertner Hubert , Offord Robin E. , Pancino Gianfranco , Debré Patrice , Gorochov Guy
Published: May 2003

The chemokine receptors CCR5 and CXCR4 are promising non-virus-encoded targets for human immunodeficiency virus (HIV) therapy. We describe a selection procedure to isolate mutant forms of RANTES (CCL5) with antiviral activity considerably in excess of that of the native chemokine. The phage-displayed library of randomly mutated and N-terminally extended variants was screened by using live CCR5-expressing cells, and two of the selected mutants, P1 and P2, were further characterized. Both were significantly more potent HIV inhibitors than RANTES, with P2 being the most active (50% inhibitory concentration of 600 pM in a viral coat-mediated cell fusion assay, complete protection of target cells against primary HIV type 1 strains at a concentration of 10 nM). P2 resembles AOP-RANTES in that it is a superagonist of CCR5 and potently induces receptor sequestration. P1, while less potent than P2, has the advantage of significantly reduced signaling activity via CCR5 (30% of that of RANTES). Additionally, both P1 and P2 exhibit not only significantly increased affinity for CCR5 but also enhanced receptor selectivity, retaining only trace levels of signaling activity via CCR1 and CCR3. The phage chemokine approach that was successfully applied here could be adapted to other chemokine-chemokine receptor systems and used to further improve the first-generation mutants reported in this paper.
Protection against Recurrent Ocular Herpes Simplex Virus Type 1 Disease after Therapeutic Vaccination of Latently Infected Mice
Authors: Richards C. M. , Case R. , Hirst T. R. , Hill T. J. , Williams N. A.
Published: May 2003

The potential of therapeutic vaccination of animals latently infected with herpes simplex virus type 1 (HSV-1) to enhance protective immunity to the virus and thereby reduce the incidence and severity of recurrent ocular disease was assessed in a mouse model. Mice latently infected with HSV-1 were vaccinated intranasally with a mixture of HSV-1 glycoproteins and recombinantEscherichia coliheat-labile enterotoxin B subunit (rEtxB) as an adjuvant. The systemic immune response induced was characterized by high levels of virus-specific immunoglobulin G1 (IgG1) in serum and very low levels of IgG2a. Mucosal immunity was demonstrated by high levels of IgA in eye and vaginal secretions. Proliferating T cells from lymph nodes of vaccinated animals produced higher levels of interleukin-10 (IL-10) than were produced by such cells from mock-vaccinated animals. This profile suggests that vaccination of latently infected mice modulates the Th1-dominated proinflammatory response usually induced upon infection. After reactivation of latent virus by UV irradiation, vaccinated mice showed reduced viral shedding in tears as well as a reduction in the incidence of recurrent herpetic corneal epithelial disease and stromal disease compared with mock-vaccinated mice. Moreover, vaccinated mice developing recurrent ocular disease showed less severe signs and a quicker recovery rate. Spread of virus to other areas close to the eye, such as the eyelid, was also significantly reduced. Encephalitis occurred in a small percentage (11%) of mock-vaccinated mice, but vaccinated animals were completely protected from such disease. The possible immune mechanisms involved in protection against recurrent ocular herpetic disease in therapeutically vaccinated animals are discussed.
Mechanisms of Avian Retroviral Host Range Extension
Authors: Rainey G. Jonah A. , Natonson Andrew , Maxfield Lori F. , Coffin John M.
Published: May 2003

Alpharetroviruses provide a useful system for the study of the molecular mechanisms of host range and receptor interaction. These viruses can be divided into subgroups based on diverse receptor usage due to variability within the two host range determining regions, hr1 and hr2, in their envelope glycoprotein SU (gp85). In previous work, our laboratory described selection from a subgroup B avian sarcoma-leukosis virus of an extended-host-range variant (LT/SI) with two adjacent amino acid substitutions in hr1. This virus retains its ability to use the subgroup BD receptor but can also infect QT6/BD cells, which bear a related subgroup E receptor (R. A. Taplitz and J. M. Coffin, J. Virol 71:7814-7819, 1997). Here, we report further analysis of this unusual variant. First, one (L154S) of the two substitutions is sufficient for host range extension, while the other (T155I) does not alter host range. Second, these mutations extend host range to non-avian cell types, including human, dog, cat, mouse, rat, and hamster. Third, interference experiments imply that the mutants interact efficiently with the subgroup BD receptor and possibly the related subgroup E receptor, but they have another means of entry that is not dependent on these interactions. Fourth, binding studies indicate that the mutant SU proteins retain the ability to interact as monomers with subgroup BD and BDE receptors but only bind the subgroup E receptor in the context of an Env trimer. Further, the mutant SU proteins bind well to chicken cells but do not bind any better than wild-type subgroup B to QT6 or human cells, even though the corresponding viruses are capable of infecting these cells.
Tomato Yellow Leaf Curl Sardinia Virus
Rep-Derived Resistance to Homologous and Heterologous Geminiviruses Occurs by Different Mechanisms and Is Overcome if Virus-Mediated Transgene Silencing Is Activated
Authors: Lucioli Alessandra , Noris Emanuela , Brunetti Angela , Tavazza Raffaela , Ruzza Valentino , Castillo Araceli G. , Bejarano Eduardo R. , Accotto Gian Paolo , Tavazza Mario
Published: May 2003

The replication-associated protein (Rep) of geminiviruses is involved in several biological processes brought about by the presence of distinct functional domains. Recently, we have exploited the multifunctional character of the
Tomato yellow leaf curl Sardinia virus
(TYLCSV) Rep to develop a molecular interference strategy to impair TYLCSV infection. We showed that transgenic expression of its N-terminal 210 amino acids (Rep-210) confers resistance to the homologous virus by inhibiting viral transcription and replication. We have now used biochemical and transgenic approaches to carry out a fuller investigation of the molecular resistance mechanisms in transgenic plants expressing Rep-210. We show that Rep-210 confers resistance through two distinct molecular mechanisms, depending on the challenging virus. Resistance to the homologous virus is achieved by the ability of Rep-210 to tightly inhibit C1 gene transcription, while that to heterologous virus is due to the interacting property of the Rep-210 oligomerization domain. Furthermore, we present evidence that in Rep-210-expressing plants, the duration of resistance is related to the ability of the challenging virus to shut off transgene expression by a posttranscriptional homology-dependent gene silencing mechanism. A model of Rep-210-mediated geminivirus resistance that takes transgene- and virus-mediated mechanisms into account is proposed.
Cloning of an Avian Adeno-Associated Virus (AAAV) and Generation of Recombinant AAAV Particles
Authors: Bossis Ioannis , Chiorini John A.
Published: May 2003

Recent studies have proposed that adeno-associated viruses (AAVs) are not evolutionarily linked to other mammalian autonomous parvoviruses but are more closely linked to the autonomous parvoviruses of birds. To better understand the relationship between primate and avian AAVs (AAAVs), we cloned and sequenced the genome of an AAAV (ATCC VR-865) and generated recombinant AAAV particles. The genome of AAAV is 4,694 nucleotides in length and has organization similar to that of other AAVs. The entire genome of AAAV displays 56 to 65% identity at the nucleotide level with the other known AAVs. The AAAV genome has inverted terminal repeats of 142 nucleotides, with the first 122 forming the characteristic T-shaped palindromic structure. The putative Rep-binding element consists of a tandem (GAGY)
4
repeat, and the putative terminal resolution site (
trs
), CCGGT/CG, contains a single nucleotide substitution relative to the AAV
2
trs
. The Rep open reading frame of AAAV displays 50 to 54% identity at the amino acid level with the other AAVs, with most of the diversity clustered at the carboxyl and amino termini. Comparison of the capsid proteins of AAAV and the primate dependoviruses indicate that divergent regions are localized to surface-exposed loops. Despite these sequence differences, we were able to produce recombinant AAAV particles carrying a
lacZ
reporter gene by cotransfection in 293T cells and were able to examine transduction efficiency in both chicken primary cells and several cell lines. Our findings indicate that AAAV is the most divergent AAV described to date but maintains all the characteristics unique to the genera of dependovirus.
Turnover of
env
Variable Region 1 and 2 Genotypes in Subjects with Late-Stage Human Immunodeficiency Virus Type 1 Infection
Authors: Kitrinos Kathryn M. , Hoffman Noah G. , Nelson Julie A. E. , Swanstrom Ronald
Published: May 2003

The
env
gene of human immunodeficiency virus type 1 (HIV-1) includes some of the most genetically diverse regions of the viral genome, which are called variable regions 1 through 5 (V1 through V5). We have developed a heteroduplex tracking assay to detect changes in variable regions 1 and 2 of
env
(V1/V2-HTA). Using sequences from two molecular clones as probes, we have studied the nature of longitudinal virus population changes in a cohort of HIV-1-infected subjects. Viral sequences present in 21 subjects with late-stage HIV-1 infection were initially screened for stability of the virus population by V1/V2-HTA. The virus populations at entry comprised an average of five coexisting V1/V2 genotypic variants (as identified by HTA). Eight of the 21 subjects were examined in detail because of the dynamic behavior of their
env
variants over an approximately 9-month period. In each of these cases we detected a single discrete transition of V1/V2 genotypes based on monthly sampling. The major V1/V2 genotypes (those present at >10% abundance) from the eight subjects were cloned and sequenced to define the nature of V1/V2 variability associated with a discrete transition. Based on a comparison of V1/V2 genotypic variants present at entry with the newly emerged variants we categorized the newly emerged variants into two groups: variants without length differences and variants with length differences. Variants without length differences had fewer nucleotide substitutions, with the changes biased to either V1 or V2, suggestive of recent evolutionary events. Variants with length differences included ones with larger numbers of changes that were distributed, suggestive of recall of older genotypes. Most length differences were located in domains where the codon motif AVT (V = A, G, C) had become enriched and fixed. Finally, recombination events were detected in two subjects, one of which resulted in the reassortment of V1 and V2 regions. We suggest that turnover in V1/V2 populations was largely driven by selection on either V1 or V2 and that escape was accomplished either through changes focused in the region under selection or by the appearance of a highly divergent variant.
Follicular Dendritic Cell Dedifferentiation by Treatment with an Inhibitor of the Lymphotoxin Pathway Dramatically Reduces Scrapie Susceptibility
Authors: Mabbott Neil A. , Young Janice , McConnell Irene , Bruce Moira E.
Published: May 2003

Transmissible spongiform encephalopathies (TSEs) may be acquired peripherally, in which case infectivity usually accumulates in lymphoid tissues before dissemination to the nervous system. Studies of mouse scrapie models have shown that mature follicular dendritic cells (FDCs), expressing the host prion protein (PrPc), are critical for replication of infection in lymphoid tissues and subsequent neuroinvasion. Since FDCs require lymphotoxin signals from B lymphocytes to maintain their differentiated state, blockade of this stimulation with a lymphotoxin β receptor-immunoglobulin fusion protein (LTβR-Ig) leads to their temporary dedifferentiation. Here, a single treatment with LTβR-Ig before intraperitoneal scrapie inoculation blocked the early accumulation of infectivity and disease-specific PrP (PrPSc) within the spleen and substantially reduced disease susceptibility. These effects coincided with an absence of FDCs in the spleen for ca. 28 days after treatment. Although the period of FDC dedifferentiation was extended to at least 49 days by consecutive LTβR-Ig treatments, this had little added protective benefit after injection with a moderate dose of scrapie. We also demonstrate that mature FDCs are critical for the transmission of scrapie from the gastrointestinal tract. Treatment with LTβR-Ig before oral scrapie inoculation blocked PrPScaccumulation in Peyer's patches and mesenteric lymph nodes and prevented neuroinvasion. However, treatment 14 days after oral inoculation did not affect survival time or susceptibility, suggesting that infectivity may have already spread to the peripheral nervous system. Although manipulation of FDCs may offer a potential approach for early intervention in peripherally acquired TSEs, these data suggest that the duration of the treatment window may vary widely depending on the route of exposure.
Comprehensive Analysis of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific Gamma Interferon-Secreting CD8+T Cells in Primary HIV-1 Infection
Authors: Cao Jianhong , McNevin John , Holte Sarah , Fink Lisa , Corey Lawrence , McElrath M. Juliana
Published: May 2003

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+T cells provide an important defense in controlling HIV-1 replication, particularly following acquisition of infection. To delineate the breadth and potency of these responses in patients upon initial presentation and before treatment, we determined the fine specificities and frequencies of gamma interferon (IFN-γ)-secreting CD8+T cells recognizing all HIV-1 proteins in patients with primary infection. In these subjects, the earliest detected responses were directed predominantly against Nef, Tat, Vpr, and Env. Tat- and Vpr-specific CD8+T cells accounted for the greatest frequencies of mean IFN-γ spot-forming cells (SFC). Nef-specific responses (10 of 21) were more commonly detected. A mean of 2.3 epitopes were recognized with various avidities per subject, and the number increased with the duration of infection (R= 0.47,P= 0.031). The mean frequency of CD8+T cells (985 SFC/106peripheral blood mononuclear cells) correlated with the number of epitopes recognized (R= 0.84,P< 0.0001) and the number of HLA-restricting alleles (R= 0.79,P< 0.0001). Neither the total SFC frequencies nor the number of epitopes recognized correlated with the concurrent plasma viral load. Seventeen novel epitopes were identified, four of which were restricted to HLA alleles (A23 and B72) that are common among African descendents. Thus, primary HIV-1 infection induces strong CD8+-T-cell immunity whose specificities broaden over time, but their frequencies and breadth do not correlate with HIV-1 containment when examined concurrently. Many novel epitopes, particularly directed to Nef, Tat, and Env, and frequently with unique HLA restrictions, merit further consideration in vaccine design.
The Differential Ability of HLA B*5701+Long-Term Nonprogressors and Progressors To Restrict Human Immunodeficiency Virus Replication Is Not Caused by Loss of Recognition of Autologous ViralgagSequences
Authors: Migueles Stephen A. , Laborico Alisha C. , Imamichi Hiromi , Shupert W. Lesley , Royce Cassandra , McLaughlin Mary , Ehler Linda , Metcalf Julia , Liu Shuying , Hallahan Claire W. , Connors Mark
Published: May 2003

Although the HLA B*5701 class I allele is highly overrepresented among human immunodeficiency virus (HIV)-infected long-term nonprogressors (LTNPs), it is also present at the expected frequency (11%) in patients with progressive HIV infection. Whether B57+progressors lack restriction of viral replication because of escape from recognition of highly immunodominant B57-restrictedgagepitopes by CD8+T cells remains unknown. In this report, we investigate the association between restriction of virus replication and recognition of autologous virus sequences in 27 B*57+patients (10 LTNPs and 17 progressors). Amplification and direct sequencing of single molecules of viral cDNA or proviral DNA revealed low frequencies of genetic variations in these regions ofgag. Furthermore, CD8+T-cell recognition of autologous viral variants was preserved in most cases. In two patients, responses to autologous viral variants were not demonstrable at one epitope. By using a novel technique to isolate primary CD4+T cells expressing autologous viral gene products, it was found that 1 to 13% of CD8+T cells were able to respond to these cells by gamma interferon production. In conclusion, escape-conferring mutations occur infrequently within immunodominant B57-restrictedgagepitopes and are not the primary mechanism of virus evasion from immune control in B*5701+HIV-infected patients. Qualitative features of the virus-specific CD8+T-cell response not measured by current assays remain the most likely determinants of the differential abilities of HLA B*5701+LTNPs and progressors to restrict virus replication.
Fusion Deficiency Induced by Mutations at the Dimer Interface in the Newcastle Disease Virus Hemagglutinin-Neuraminidase Is due to a Temperature-Dependent Defect in Receptor Binding
Authors: Corey Elizabeth A. , Mirza Anne M. , Levandowsky Elizabeth , Iorio Ronald M.
Published: May 2003

The tetrameric paramyxovirus hemagglutinin-neuraminidase (HN) protein mediates attachment to sialic acid-containing receptors as well as cleavage of the same moiety via its neuraminidase (NA) activity. The X-ray crystallographic structure of an HN dimer from Newcastle disease virus (NDV) suggests that a single site in two different conformations mediates both of these activities. This conformational change is predicted to involve an alteration in the association between monomers in each HN dimer and to be part of a series of changes in the structure of HN that link its recognition of receptors to the activation of the other viral surface glycoprotein, the fusion protein. To explore the importance of the dimer interface to HN function, we performed a site-directed mutational analysis of residues in a domain defined by residues 218 to 226 at the most membrane-proximal part of the dimer interface in the globular head. Proteins carrying substitutions for residues F220, S222, and L224 in this domain were fusion deficient. However, this fusion deficiency was not due to a direct effect of the mutations on fusion. Rather, the fusion defect was due to a severely impaired ability to mediate receptor recognition at 37°C, a phenotype that is not attributable to a change in NA activity. Since each of these mutated proteins efficiently mediated attachment in the cold, it was also not due to an inherent inability of the mutated proteins to recognize receptors. Instead, the interface mutations acted by weakening the interaction between HN and its receptor(s). The phenotype of these mutants correlates with the disruption of intermonomer subunit interactions.
Sequence and Structure of Human Rhinoviruses Reveal the Basis of Receptor Discrimination
Authors: Vlasak Marketa , Blomqvist Soile , Hovi Tapani , Hewat Elizabeth , Blaas Dieter
Published: May 2003

The sequences of the capsid protein VP1 of all minor receptor group human rhinoviruses were determined. A phylogenetic analysis revealed that minor group HRVs were not more related to each other than to the nine major group HRVs whose sequences are known. Examination of the surface exposed amino acid residues of HRV1A and HRV2, whose X-ray structures are available, and that of three-dimensional models computed for the remaining eight minor group HRVs indicated a pattern of positively charged residues within the region, which, in HRV2, was shown to be the binding site of the very-low-density lipoprotein (VLDL) receptor. A lysine in the HI loop of VP1 (K224 in HRV2) is strictly conserved within the minor group. It lies in the middle of the footprint of a single repeat of the VLDL receptor on HRV2. Major group virus serotypes exhibit mostly negative charges at the corresponding positions and do not bind the negatively charged VLDL receptor, presumably because of charge repulsion.
Targeting of the Human Immunodeficiency Virus Type 1 Envelope to thetrans-Golgi Network through Binding to TIP47 Is Required for Env Incorporation into Virions and Infectivity
Authors: Blot Guillaume , Janvier Katy , Le Panse Sophie , Benarous Richard , Berlioz-Torrent Clarisse
Published: May 2003

Here, we report that human immunodeficiency virus type 1 (HIV-1) Env glycoprotein is located mainly in thetrans-Golgi network (TGN) due to determinants present in the cytoplasmic domain of the transmembrane gp41 glycoprotein (TMgp41). Internalization assays demonstrated that Env present at the cell surface returns to the TGN. We found that the cytoplasmic domain of TMgp41 binds to TIP47, a protein required for the transport of mannose-6-phosphate receptors from endosomes to the TGN. Overexpression of a mutant of TIP47 affected the transport of Env from endosomes to the TGN. Retrograde transport of Env to the TGN requires a Y802W803diaromatic motif present in the TMgp41 cytoplasmic domain. Mutation of this motif abolished both targeting to the TGN as well as interaction with TIP47. These data support the view that binding of TIP47 to HIV-1 Env facilitates its delivery to the TGN. Lastly, we show that virus mutated in the Y802W803motif is poorly infectious and presents a defect in Env incorporation, supporting a model in which retrograde transport of Env is implicated in the optimization of fully infectious HIV-1 production.
Human Cytomegalovirus Infection Inhibits Tumor Necrosis Factor Alpha (TNF-α) Signaling by Targeting the 55-Kilodalton TNF-α Receptor
Authors: Baillie J. , Sahlender D. A. , Sinclair J. H.
Published: May 2003

Infection with human cytomegalovirus (HCMV) results in complex interactions between viral and cellular factors which perturb many cellular functions. HCMV is known to target the cell cycle, cellular transcription, and immunoregulation, and it is believed that this optimizes the cellular environment for viral DNA replication during productive infection or during carriage in the latently infected host. Here, we show that HCMV infection also prevents external signaling to the cell by disrupting the function of TNFRI, the 55-kDa receptor for tumor necrosis factor alpha (TNF-α), one of the receptors for a potent cytokine involved in eliciting a wide spectrum of cellular responses, including antiviral responses. HCMV infection of fully permissive differentiated monocytic cell lines and U373 cells resulted in a reduction in cell surface expression of TNFRI. The reduction appeared to be due to relocalization of TNFRI from the cell surface and was reflected in the elimination of TNF-α-induced Jun kinase activity. Analysis of specific phases of infection suggested that viral early gene products were responsible for this relocalization. However, a mutant HCMV in which all viral gene products known to be involved in down-regulation of major histocompatibility complex (MHC) class I were deleted still resulted in relocalization of TNFRI. Consequently, TNFRI relocalization by HCMV appears to be mediated by a novel viral early function not involved in down-regulation of cell surface MHC class I expression. We suggest that upon infection, HCMV isolates the cell from host-mediated signals, forcing the cell to respond only to virus-specific signals which optimize the cell for virus production and effect proviral responses from bystander cells.
Generation of Transduction-Competent Retroviral Vectors by Infection with a Single Hybrid Vaccinia Virus
Authors: Konetschny Christian , Holzer Georg W. , Urban Carsten , Hämmerle Thomas , Mayrhofer Josef , Falkner Falko G.
Published: May 2003

Recombinant vaccinia viruses that express defective retroviral vectors upon a single infection event in normal host cells were constructed. The
gag-pol
and envelope genes and a retroviral vector unit were inserted as vaccinia virus promoter-controlled transcription units at three separate loci. The triple recombinant virus was used to infect such diverse cell types as monkey and rabbit kidney, human lung, and primary chicken cells, resulting in the production of transduction-competent defective retroviral vectors. Infection of Chinese hamster ovary cells, which are nonpermissive for vaccinia virus replication, also resulted in production of retroviral vectors and secondary permanent transduction of the host cells. Since vaccinia virus supports the expression of cytotoxic proteins, the vesicular stomatitis virus G glycoprotein could be chosen as the envelope allowing a broad host range of transduction. Functionality of particles was monitored by expression of the green fluorescent protein in transduced 3T3 cell clones. This is the first description of a single chimeric virus encoding and releasing functional retroviral vectors, providing proof of principle of the new concept. No replication-competent retrovirus was detectable by sensitive reverse transcriptase assays. Since vaccinia virus has a broad host range, is extremely robust, and can be obtained at high titers and safe nonreplicating vaccinia virus strains are available, the hybrid system may open new perspectives for gene delivery.
Intraventricular Brain Injection of Adeno-Associated Virus Type 1 (AAV1) in Neonatal Mice Results in Complementary Patterns of Neuronal Transduction to AAV2 and Total Long-Term Correction of Storage Lesions in the Brains of β-Glucuronidase-Deficient Mice
Authors: Passini Marco A. , Watson Deborah J. , Vite Charles H. , Landsburg Daniel J. , Feigenbaum Alyson L. , Wolfe John H.
Published: May 2003

Inherited metabolic disorders that affect the central nervous system typically result in pathology throughout the brain; thus, gene therapy strategies need to achieve widespread delivery. We previously found that although intraventricular injection of the neonatal mouse brain with adeno-associated virus serotype 2 (AAV2) results in dispersed gene delivery, many brain structures were poorly transduced. This limitation may be overcome by using different AAV serotypes because the capsid proteins use different cellular receptors for entry, which may allow enhanced global targeting of the brain. We tested this with AAV1 and AAV5 vectors. AAV5 showed very limited brain transduction after neonatal injection, even though it has different transduction patterns than AAV2 in adult brain injections. In contrast, AAV1 vectors, which have not been tested in the brain, showed robust widespread transduction. Complementary patterns of transduction between AAV1 and AAV2 were established and maintained in the adult brain after neonatal injection. In the majority of structures, AAV1 transduced many more cells than AAV2. Both vectors transduced mostly neurons, indicating that differential expression of receptors on the surfaces of neurons occurs in the developing brain. The number of cells positive for a vector-encoded secreted enzyme (β-glucuronidase) was notably greater and more widespread in AAV1-injected brains. A comprehensive analysis of AAV1-treated brains from β-glucuronidase-deficient mice (mucopolysaccharidosis type VII) showed complete reversal of pathology in all areas of the brain for at least 1 year, demonstrating that the combination of this serotype and experimental strategy is therapeutically effective for treating global neurometabolic disorders.
Interactions of Rotavirus VP4 Spike Protein with the Endosomal Protein Rab5 and the Prenylated Rab Acceptor PRA1
Authors: Enouf Vincent , Chwetzoff Serge , Trugnan Germain , Cohen Jean
Published: May 2003

Rotavirus spike protein VP4 is implicated in several important functions, such as cell attachment, penetration, hemagglutination, neutralization, virulence, and host range. It is present at the plasma membrane and colocalizes with the cytoskeleton in infected cells. We looked for cellular partners responsible for the localization of VP4 by two-hybrid screening of a monkey CV1 cell cDNA library. In the screen we isolated repeatedly three cDNAs encoding either two isoforms (a and c) of Rab5 protein or the prenylated Rab acceptor (PRA1). The small GTPase Rab5 is a molecule regulating the vesicular traffic and the motility of early endosomes along microtubules. Rab5 interacts with a large number of effectors, in particular with PRA1. Interactions of VP4 with both partners, Rab5 and PRA1, were confirmed by coimmunoprecipitation from infected- or transfected-cell lysates. Interaction of Rab5 and PRA1 was restricted to free VP4, since neither triple-layered particles nor NSP4-VP4-VP7 heterotrimeric complexes could be coprecipitated. Site-directed and deletion mutants of VP4 were used to map a VP4 domain(s) interacting with Rab5 or PRA1. Of the 10 mutants tested, 2 interacted exclusively with a single partner. In contrast, the domain extending from amino acids 560 to 722 of VP4 is essential for both interactions. These results suggest that Rab5 and PRA1 may be involved in the localization and trafficking of VP4 in infected cells.
Charged Assembly Helix Motif in Murine Leukemia Virus Capsid: an Important Region for Virus Assembly and Particle Size Determination
Authors: Cheslock Sara Rasmussen , Poon Dexter T. K. , Fu William , Rhodes Terence D. , Henderson Louis E. , Nagashima Kunio , McGrath Connor F. , Hu Wei-Shau
Published: May 2003

We have identified a region near the C terminus of capsid (CA) of murine leukemia virus (MLV) that contains many charged residues. This motif is conserved in various lengths in most MLV-like viruses. One exception is that spleen necrosis virus (SNV) does not contain a well-defined domain of charged residues. When 33 amino acids of the MLV motif were deleted to mimic SNV CA, the resulting mutant produced drastically reduced amounts of virions and the virions were noninfectious. Furthermore, these viruses had abnormal sizes, often contained punctate structures resembling those in the cell cytoplasm, and packaged both ribosomal and viral RNA. When 11 or 15 amino acids were deleted to modify the MLV CA to resemble those from other gammaretroviruses, the deletion mutants produced virions at levels comparable to those of the wild-type virus and were able to complete one round of virus replication without detectable defects. We generated 10 more mutants that displayed either the wild-type or mutant phenotype. The distribution of the wild-type or mutant phenotype did not directly correlate with the number of amino acids deleted, suggesting that the function of the motif is determined not simply by its length but also by its structure. Structural modeling of the wild-type and mutant proteins suggested that this region forms α-helices; thus, we termed this motif the “charged assembly helix.” This is the first description of the charged assembly helix motif in MLV CA and demonstration of its role in virus budding and assembly.
Basic Residues of the Helix Six Domain of Influenza Virus M1 Involved in Nuclear Translocation of M1 Can Be Replaced by PTAP and YPDL Late Assembly Domain Motifs
Authors: Hui Eric Ka-Wai , Barman Subrata , Yang Tae Yong , Nayak Debi P.
Published: May 2003

Influenza type A virus matrix (M1) protein possesses multiple functional motifs in the helix 6 (H6) domain (amino acids 91 to 105), including nuclear localization signal (NLS) (101-RKLKR-105) involved in translocating M1 from the cytoplasm into the nucleus. To determine the role of the NLS motif in the influenza virus life cycle, we mutated these and the neighboring sequences by site-directed mutagenesis, and influenza virus mutants were generated by reverse genetics. Our results show that infectious viruses were rescued by reverse genetics from all single alanine mutations of amino acids in the H6 domain and the neighboring region except in three positions (K104A and R105A within the NLS motif and E106A in loop 6 outside the NLS motif). Among the rescued mutant viruses, R101A and R105K exhibited reduced growth and small-plaque morphology, and all other mutant viruses showed the wild-type phenotype. On the other hand, three single mutations (K104A, K105A, and E106A) and three double mutations (R101A/K102A, K104A/K105A, and K102A/R105A) failed to generate infectious virus. Deletion (ΔYRKL) or mutation (4A) of YRKL also abolished generation of infectious virus. However, replacement of the YRKL motif with PTAP or YPDL as well as insertion of PTAP after 4A mutation yielded infectious viruses with the wild-type phenotype. Furthermore, mutant M1 proteins (R101A/K102A, ΔYRKL, 4A, PTAP, 4A+PTAP, and YPDL) when expressed alone from cloned cDNAs were only cytoplasmic, whereas the wild-type M1 expressed alone was both nuclear and cytoplasmic as expected. These results show that the nuclear translocation function provided by the positively charged residues within the NLS motif does not play a critical role in influenza virus replication. Furthermore, these sequences of H6 domain can be replaced by late (L) domain motifs and therefore may provide a function similar to that of the L domains of other negative-strand RNA and retroviruses.
A Domain in the C-Terminal Region of Latency-Associated Nuclear Antigen 1 of Kaposi's Sarcoma-Associated Herpesvirus Affects Transcriptional Activation and Binding to Nuclear Heterochromatin
Authors: Viejo-Borbolla Abel , Kati Emrah , Sheldon Julie A. , Nathan Kavita , Mattsson Karin , Szekely Laszlo , Schulz Thomas F.
Published: May 2003

The latency-associated nuclear antigen 1 (LANA-1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for the maintenance and replication of viral episomal DNA. The binding sites for nuclear heterochromatin and transcriptional repressor complexes are located in an amino-terminal region of LANA-1, whereas those for viral episomal DNA, p53, pRB, and members of the BRD/fsh family of nuclear proteins are located in its carboxy-terminal domain. LANA-1 activates or represses several cellular and viral promoters. In this report we show that a domain of 15 amino acids (amino acids 1129 to 1143), located close to the carboxy-terminal end of LANA-1, is required for the interaction of LANA-1 with nuclear heterochromatin or nuclear matrix, and for the ability of LANA-1 to activate the Epstein-Barr virus Cp promoter. LANA-1 proteins that are tightly associated with nuclear heterochromatin or matrix differ in molecular weight from LANA-1 proteins that can be dissociated from the nuclear matrix by high-salt buffers, suggesting that posttranslational modifications may determine the association of LANA-1 with nuclear heterochromatin or matrix.
Promyelocytic Leukemia Protein Mediates Interferon-Based Anti-Herpes Simplex Virus 1 Effects
Authors: Chee Ana V. , Lopez Pascal , Pandolfi Pier Paolo , Roizman Bernard
Published: May 2003

Herpes simplex virus (HSV) 1 disaggregates the nuclear domain 10 (ND10) nuclear structures and disperses its organizing promyelocytic leukemia protein (PML). An earlier report showed that ectopic overexpression of PML precludes the disaggregation of ND10 but has no effect on viral replication. PML has been reported to mediate the effects of interferon (IFN) and viral mutants lacking ICP0 (Δα0 mutants). To test the hypothesis that HSV disaggregates ND10 structures and disperses PML to preclude IFN-mediated antiviral effects, we tested the accumulation of viral proteins and virus yields from murine PML+/+and PML−/−cells mock treated or exposed to IFN-α, IFN-γ, or both and infected with the wild-type or Δα0 mutant virus. We report the following results. (i) The levels of growth of wild-type and mutant viruses and of accumulation of viral proteins were not significantly different in untreated PML+/+and PML−/−cells. (ii) Major effects of IFN-α and -γ were observed in PML+/+cells infected with the Δα0 mutant virus, and more minor effects were observed in cells infected with the wild-type virus. The effects of the IFNs on either wild-type or the mutant virus in PML−/−cells were minimal. (iii) The mixture of IFN-α and -γ was more effective than either IFN alone, but again, the effect was more drastic in PML+/+cells than in PML−/−cells. We concluded that the anti-HSV state induced by exogenous IFN is mediated by PML and that the virus targets the ND10 structures and disseminates PML in order to preclude the establishment of the antiviral state induced by IFNs.
Activated Notch1 Inhibits p53-Induced Apoptosis and Sustains Transformation by Human Papillomavirus Type 16 E6 and E7 Oncogenes through a PI3K-PKB/Akt-Dependent Pathway
Authors: Nair Pradip , Somasundaram Kumaravel , Krishna Sudhir
Published: May 2003

Activated Notch1 (AcN1) alleles cooperate with oncogenes from DNA tumor viruses in transformation of epithelial cells. AcN1 signaling has pleiotropic effects, and suggested oncogenic roles include driving proliferation through cyclin D1 or the generation of resistance to apoptosis on matrix withdrawal through a phosphatidylinositol 3-kinase (PI3K)-PKB/Akt-dependent pathway. Here, we extend the antiapoptotic role for AcN1 by showing inhibition of p53-induced apoptosis and transactivation. Chemical inhibitors of the PI3K pathway block AcN1-induced inhibition of p53-dependent apoptosis and nuclear localization of Hdm2. We show that expression of wild-type p53 does not inhibit synergistic transformation by AcN1 and human papillomavirus E6 and E7 oncogenes. We suggest that activation of Notch signaling may serve as an additional mechanism to inhibit wild-type p53 function in papillomavirus-associated neoplasia.
Mutagenesis versus Inhibition in the Efficiency of Extinction of Foot-and-Mouth Disease Virus
Authors: Pariente Nonia , Airaksinen Antero , Domingo Esteban
Published: May 2003

RNA viruses replicate near the error threshold for maintenance of genetic information, and an increase in mutation frequency during replication may drive RNA viruses to extinction in a process termed lethal mutagenesis. This report addresses the efficiency of extinction (versus escape from extinction) of foot-and-mouth disease virus (FMDV) by combinations of the mutagenic base analog 5-fluorouracil (FU) and the antiviral inhibitors guanidine hydrochloride (G) and heparin (H). Selection of G- or H-resistant, extinction-escape mutants occurred with low-fitness virus only in the absence of FU and with high-fitness virus with some mutagen-inhibitor combinations tested. The combination of FU, G, and H prevented selection of extinction-escape mutants in all cases examined, and extinction of high-fitness FMDV could not be achieved by equivalent inhibitory activity exerted by the nonmutagenic agents. The G-resistant phenotype was mapped in nonstructural protein 2C by introducing the relevant mutations in infectious cDNA clones. Decreases in FMDV infectivity were accompanied by modest decreases in the intracellular and extracellular levels of FMDV RNA, maximal intracellular concentrations of FU triphosphate, and a decrease in the intracellular concentrations of UTP. In addition to indicating a key participation of mutagenesis in virus extinction, the results suggest that picornaviruses provide versatile experimental systems to approach the problem of extinction failure associated with inhibitor-escape mutants during treatments based on enhanced mutagenesis.
transActivity of the Norovirus Camberwell Proteinase and Cleavage of the N-Terminal Protein Encoded by ORF1
Authors: Seah Ee Ling , Marshall John A. , Wright Peter J.
Published: May 2003

The virus-encoded proteinase of Camberwell virus, a genogroup 2 norovirus, was synthesized inEscherichia coli. The purified proteinase had correct N and C termini and showedtransactivity in cell-free assays.transactivity was also demonstrated in COS cells transfected with constructs encoding either the proteinase or a proteinase-polymerase fusion. The N-terminal protein of ORF1 was cleaved in COS cells, possibly at the site E194/S.
Functional morphology of the temporal region in the Rhynchocephalia
Authors: Wu Xiao-Chun
Published: May 2003

Study of the functional morphology of the temporal region of the Rhynchocephalia by means of the analysis of jaw musculature and bone structures in living lepidosaurians suggests that the streptostyly of the quadrate is a derived state in the Lepidosauria and that its origin may have been coupled with the loss of the M. pterygoideus atypicus. Further, this analysis supports the view that the retention of a fixed quadrate in early rhynchocephalians with an incomplete lower temporal bar may have been required for precise tooth occlusion and suggests that a solid lower temporal bar in later rhynchocephalians may have served as a brace to support the lateral side of the quadrate condyle and thus prevent it from twisting anteriorly rather than posteriorly during the jaw cycle. It is hypothesized that a common ancestor shared by the Rhynchocephalia and Squamata had a skull with an incomplete lower temporal bar but a rigid quadrate. It is also hypothesized that the presence of the anterior portion of the M. pterygoideus (MPT) is a primitive state, and that the size of the pterygoid flange may have been a good indicator of the presence of the anterior portion of the MPT in fossil reptiles. As in Sphenodon, if the mandible is able to move anteroposteriorly, it must retract first during the jaw cycle in those reptiles in which a pterygoid flange is retained.
Archaeovenator hamiltonensis, a new varanopid (Synapsida: Eupelycosauria) from the Upper Carboniferous of Kansas
Authors: Reisz Robert R , Dilkes David W
Published: May 2003

Study of a nearly complete skeleton of a small amniote, preserved as part and counterpart in Upper Carboniferous limestone slabs of the Hamilton Quarry, Kansas, indicates that it is a new genus and species of varanopid synapsid. Archaeovenator hamiltonensis gen., and sp. nov. is the oldest known varanopid. Phylogenetic analysis indicates that it is the sister taxon to all other known varanopids. Discovery of this new varanopid is important because it improves our knowledge of the terrestrial fauna of the Hamilton quarry, expands the known diversity of Late Carboniferous basal synapsids, and extends the fossil record of Varanopidae deeper within the Stephanian. Varanopidae has the longest fossil record (Late Carboniferous  upper Middle Permian) of all known clades of basal synapsids.