bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of Adeno-Associated Virus on Adenovirus Replication and Gene Expression during Coinfection
Authors: Timpe Jennifer M. , Verrill Kristin C. , Trempe James P. 
Published: July 2006

ABSTRACT

Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the
E3
gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis.
Mutation of Dileucine-Like Motifs in the Human Immunodeficiency Virus Type 1 Capsid Disrupts Virus Assembly, Gag-Gag Interactions, Gag-Membrane Binding, and Virion Maturation
Authors: Joshi Anjali , Nagashima Kunio , Freed Eric O. 
Published: July 2006

ABSTRACT

The human immunodeficiency virus type 1 (HIV-1) Gag precursor protein Pr55
Gag
drives the assembly and release of virus-like particles in the infected cell. The capsid (CA) domain of Gag plays an important role in these processes by promoting Gag-Gag interactions during assembly. The C-terminal domain (CTD) of CA contains two dileucine-like motifs (L
189
/L
190
and I
201
/L
202
) implicated in regulating the localization of Gag to multivesicular bodies (MVBs). These dileucine-like motifs are located in the vicinity of the CTD dimer interface, a region of CA critical for Gag-Gag interactions during virus assembly and CA-CA interactions during core formation. To study the importance of the CA dileucine-like motifs in various aspects of HIV-1 replication, we introduced a series of mutations into these motifs in the context of a full-length, infectious HIV-1 molecular clone. CA mutants LL189,190AA and IL201,202AA were both severely impaired in virus particle production because of a variety of defects in the binding of Gag to membrane, Gag multimerization, and CA folding. In contrast to the model suggesting that the CA dileucine-like motifs regulate MVB targeting, the IL201,202AA mutation did not alter Gag localization to the MVB in either HeLa cells or macrophages. Revertants of single-amino-acid substitution mutants were obtained that no longer contained dileucine-like motifs but were nevertheless fully replication competent. The varied phenotypes of the mutants reported here provide novel insights into the interplay among Gag multimerization, membrane binding, virus assembly, CA dimerization, particle maturation, and virion infectivity.
Betaherpesvirus-Conserved Cytomegalovirus Tegument Protein ppUL32 (pp150) Controls Cytoplasmic Events during Virion Maturation
Authors: AuCoin David P. , Smith Geoffrey B. , Meiering Christopher D. , Mocarski Edward S. 
Published: July 2006

ABSTRACT
The
UL32 gene of human cytomegalovirus (CMV) encodes a prominent
betaherpesvirus-conserved virion tegument protein, called pp150 (basic
phosphoprotein/ppUL32), that accumulates within a cytoplasmic inclusion
adjacent to the nucleus at late times during infection. Using a UL32
deletion mutant (ΔUL32-BAC) (where BAC is bacterial artificial
chromosome), we demonstrate that pp150 is critical for virion
maturation in the cytoplasmic compartment. Cotransfection of a pp150
expression plasmid with ΔUL32-BAC DNA led to complementation of
the replication defect with focus formation due to secondary spread.
Deletion of the amino terminus of pp150 or disruption of the
betaherpesvirus conserved regions, CR1 and CR2, revealed these regions
to be critical for replication. In contrast, deletion of the carboxyl
terminus only partially compromised maturation while disruption of
glycosylation sites had no effect. An African green monkey CMV UL32
homolog complemented ΔUL32-BAC replication but murine CMV M32
failed to complement, consistent with evolutionary divergence of rodent
and primate cytomegaloviruses. Infection with ΔUL32-BAC showed
normal expression of all kinetic classes of viral genes and replication
of viral DNA, with accumulation of viral DNA-containing particles in
the cytoplasm; however, mutant virus did not spread to adjacent cells.
In contrast to this block in virion infectivity, cell-to-cell transfer
of pp65-containing particles was observed, suggesting that release of
dense bodies continued in the absence of pp150. These observations
demonstrate that pp150 is critical for virion egress, possibly at the
stage of final
envelopment.
Expression of the Jaagsiekte Sheep Retrovirus Envelope Glycoprotein Is Sufficient To Induce Lung Tumors in Sheep
Authors: Caporale Marco , Cousens Christina , Centorame Patrizia , Pinoni Chiara , De las Heras Marcelo , Palmarini Massimo 
Published: July 2006

ABSTRACT

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). The expression of the JSRV envelope (Env) alone is sufficient to transform a variety of cell lines in vitro and induce lung cancer in immunodeficient mice. In order to determine the role of the JSRV Env in OPA tumorigenesis in sheep, we derived a JSRV replication-defective virus (JS-RD) which expresses
env
under the control of its own long terminal repeat (LTR). JS-RD was produced by transiently transfecting 293T cells with a two plasmid system, involving (i) a packaging plasmid, with the putative JSRV packaging signal deleted, expressing the structural and enzymatic proteins Gag, Pro, and Pol, and (ii) a plasmid which expresses
env
in
trans
for JS-RD particles and provides the genomes necessary to deliver JSRV
env
upon infection. During the optimization of the JS-RD system we determined that both R-U5 (in the viral 5′ LTR) and the
env
region are important for JSRV particle production. Two independent experimental transmission studies were carried out with newborn lambs. Four of five lambs inoculated with JS-RD showed OPA lesions in the lungs at various times between 4 and 12 months postinoculation. Abundant expression of JSRV Env was detected in tumor cells of JS-RD-infected animals and PCR assays confirmed the presence of the deleted JS-RD genome. These data strongly suggest that the JSRV Env functions as a dominant oncoprotein in the natural immunocompetent host and that JSRV can induce OPA in the absence of viral spread.
NSm and 78-Kilodalton Proteins of Rift Valley Fever Virus Are Nonessential for Viral Replication in Cell Culture
Authors: Won Sungyong , Ikegami Tetsuro , Peters C. J. , Makino Shinji 
Published: July 2006

ABSTRACT
Rift Valley fever viruses carrying mutations of the M gene preglycoprotein region, one lacking NSm protein expression, one lacking 78-kDa protein expression, and one lacking expression of both proteins, were compared in cell culture. All of the mutants and their parent virus produced plaques with similar sizes and morphologies in Vero E6 cells and had similar growth kinetics in Vero, C6/36, and MRC5 cells, demonstrating that the NSm and 78-kDa proteins were not needed for the virus to replicate efficiently in cell culture. A competition-propagation assay revealed that the parental virus was slightly more fit than the mutant virus lacking expression of both proteins.
Virion-Associated Restriction Endonucleases of Chloroviruses
Authors: Agarkova Irina V. , Dunigan David D. , Van Etten James L. 
Published: July 2006

ABSTRACT

Chloroviruses are large, double-stranded-DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae. The prototype of the genus is
Paramecium bursaria
chlorella virus 1 (PBCV-1). Chlorovirus genomes contain various amounts of methylated nucleotides due to virus-encoded DNA methyltransferases (MTases); about 25% of the MTases are associated with companion DNA site-specific (restriction) endonucleases (REases). These enzymes constitute virally encoded restriction-modification (R/M) systems. Although several of the chlorovirus R/M systems are characterized, their biological functions are unknown. The PBCV-1 proteome reveals that two virus-encoded REases, but not their companion MTases, are virion associated, suggesting that viral REases might help degrade the host DNA early in infection. To test this hypothesis, host chromosomal DNA from PBCV-1-infected cells was examined by pulsed-field gel electrophoresis. Initiation of host chromosomal DNA degradation occurred within 5 min postinfection (p.i.). The DNA degradation was insensitive to protein synthesis inhibitors or UV inactivation of virus particles, consistent with the agent being a small protein associated with the virion. Nuclease activities, including those of the two predicted REases and an uncharacterized general nuclease(s), were detected in disrupted PBCV-1 particles. The general nuclease(s) degraded both host and viral DNAs in vitro, although the viral DNA was not degraded in vivo, suggesting differential intracellular trafficking of the virion-associated nucleases. Infection with chloroviruses lacking an R/M system(s) resulted in either delayed host chromosomal DNA degradation or no detectable host chromatin changes. These immediate-early events associated with chlorovirus infections may facilitate rapid switching of the host transcriptional apparatus to viral transcription, which begins within 5 to 10 min p.i.
Role of CCL5 (RANTES) in Viral Lung Disease
Authors: Culley Fiona J. , Pennycook Alasdair M. J. , Tregoning John S. , Dodd Jonathan S. , Walzl Gerhard , Wells Timothy N. , Hussell Tracy , Openshaw Peter J. M. 
Published: July 2006

ABSTRACTCCL5/RANTES is a key proinflammatory chemokine produced by virus-infected epithelial cells and present in respiratory secretions of asthmatics. To examine the role of CCL5 in viral lung disease, we measured its production during primary respiratory syncytial virus (RSV) infection and during secondary infection after sensitizing vaccination that induces Th2-mediated eosinophilia. A first peak of CCL5 mRNA and protein production was seen at 18 to 24 h of RSV infection, before significant lymphocyte recruitment occurred. Treatment in vivo with Met-RANTES (a competitive chemokine receptor blocker) throughout primary infection decreased CD4+and CD8+cell recruitment and increased viral replication. In RSV-infected, sensitized mice with eosinophilic disease, CCL5 production was further augmented; Met-RANTES treatment again reduced inflammatory cell recruitment and local cytokine production. A second wave of CCL5 production occurred on day 7, attributable to newly recruited T cells. Paradoxically, mice treated with Met-RANTES during primary infection demonstrated increased cellular infiltration during reinfection. We therefore show that RSV induces CCL5 production in the lung and this causes the recruitment of RSV-specific cells, including those making additional CCL5. If this action is blocked with Met-RANTES, inflammation decreases and viral clearance is delayed. However, the exact effects of chemokine modulation depend critically on time of administration, a factor that may potentially complicate the use of chemokine blockers in inflammatory diseases.
New Antiviral Target Revealed by the Hexameric Structure of Mouse Hepatitis Virus Nonstructural Protein nsp15
Authors: Xu Xiaoling , Zhai Yujia , Sun Fei , Lou Zhiyong , Su Dan , Xu Yuanyuan , Zhang Rongguang , Joachimiak Andrzej , Zhang Xuejun C. , Bartlam Mark , Rao Zihe 
Published: July 2006

ABSTRACT
The unique coronavirus transcription/replication machinery comprised of multiple virus-encoded nonstructural proteins (nsp) plays a vital role during initial and intermediate phases of the viral life cycle. The crystal structure of mouse hepatitis virus strain A59 (MHV-A59) nsp15 is reported at 2.15-Å resolution. nsp15 is an XendoU endoribonuclease and is the first one from this family to have its structure unveiled. The MHV-A59 nsp15 monomer structure has a novel protein fold. Two nsp15 trimers form a back-to-back hexamer that is believed to be the functional unit. The structure reveals the catalytic site including the highly conserved residues His262, His277, and Lys317, which is supported by mutagenesis analysis. Gel filtration and enzyme activity assays confirmed that the hexamer is the active form for nsp15 and demonstrate the specificity of nsp15 for uridylate. The high sequence conservation of nsp15 in coronaviruses, including that of severe acute respiratory syndrome, suggests that this protein may provide a new target for the design of antiviral therapeutics.
Amino Acid Residues in the N-Terminal Region of the PA Subunit of Influenza A Virus RNA Polymerase Play a Critical Role in Protein Stability, Endonuclease Activity, Cap Binding, and Virion RNA Promoter Binding
Authors: Hara Koyu , Schmidt Florian I. , Crow Mandy , Brownlee George G. 
Published: July 2006

ABSTRACT
The RNA-dependent RNA polymerase of influenza virus is a heterotrimer formed by the PB1, PB2, and PA subunits. Although PA is known to be required for polymerase activity, its precise role is still unclear. Here, we investigated the function of the N-terminal region of PA. Protease digestion of purified recombinant influenza virus A/PR/8/34 PA initially suggested that its N-terminal region is folded into a 25-kDa domain. We then systematically introduced point mutations into evolutionarily conserved amino acids in the N-terminal region of influenza virus A/WSN/33. Most alanine-scanning mutations between residues L109 and F117 caused PA degradation, mediated by a proteasome-ubiquitin pathway, and as a consequence interfered with polymerase activity. Three further PA mutations, K102A, D108A, and K134A, were investigated in detail. Mutation K102A caused a general decrease both in transcription and replication in vivo, whereas mutations D108A and K134A selectively inhibited transcription. Both the D108A and K134A mutations completely inhibited endonuclease activity in vitro, explaining their selective defect in transcription. K102A, on the other hand, resulted in a significant decrease in both cap binding and viral RNA promoter-binding activity and consequently inhibited both transcription and replication. These results suggest that the N-terminal region of PA is involved in multiple functions of the polymerase, including protein stability, endonuclease activity, cap binding, and promoter binding.
Insights into the Selective Pressures Restricting
Pelargonium Flower Break Virus
Genome Variability: Evidence for Host Adaptation
Authors: Rico Patricia , Ivars Pilar , Elena Santiago F. , Hernández Carmen 
Published: July 2006

ABSTRACT

The molecular diversity of
Pelargonium flower break virus
(PFBV) was assessed using a collection of isolates from different geographical origins, hosts, and collecting times. The genomic region examined was 1,828 nucleotides (nt) long and comprised the coding sequences for the movement (p7 and p12) and the coat (CP) proteins, as well as flanking segments including the entire 3′ untranslated region (3′ UTR). Some constraints limiting viral heterogeneity could be inferred from sequence analyses, such as the conservation of the amino acid sequences of p7 and of the shell domain of the CP, the maintenance of a leucine zipper motif in p12, and the preservation of a particular folding in the 3′ UTR. A remarkable covariation, involving five specific amino acid sites, was found in the CP of isolates largely propagated in the local lesion host
Chenopodium quinoa
and in the progeny of a PFBV variant subjected to serial passages in this host. Concomitant with this covariation, up to 30 nucleotide substitutions in a 1,428-nt region of the viral RNA could be attributable to
C. quinoa
-specific adaptation, representing one of the most outstanding cases of host-driven genome variation for a plant virus. Globally, the results indicate that the selective pressures exerted by the host play a critical role in shaping PFBV populations and that these populations are likely being selected for at both protein and RNA levels.
Distinct Domains of the Influenza A Virus M2 Protein Cytoplasmic Tail Mediate Binding to the M1 Protein and Facilitate Infectious Virus Production
Authors: McCown Matthew F. , Pekosz Andrew 
Published: July 2006

ABSTRACT
The cytoplasmic tail of the influenza A virus M2 protein is highly conserved among influenza A virus isolates. The cytoplasmic tail appears to be dispensable with respect to the ion channel activity associated with the protein but important for virus morphology and the production of infectious virus particles. Using reverse genetics and transcomplementation assays, we demonstrate that the M2 protein cytoplasmic tail is a crucial mediator of infectious virus production. Truncations of the M2 cytoplasmic tail result in a drastic decrease in infectious virus titers, a reduction in the amount of packaged viral RNA, a decrease in budding events, and a reduction in budding efficiency. The M1 protein binds to the M2 cytoplasmic tail, but the M1 binding site is distinct from the sequences that affect infectious virus particle formation. Influenza A virus strains A/Udorn/72 and A/WSN/33 differ in their requirements for M2 cytoplasmic tail sequences, and this requirement maps to the M1 protein. We conclude that the M2 protein is required for the formation of infectious virus particles, implicating the protein as important for influenza A virus assembly in addition to its well-documented role during virus entry and uncoating.
Supramolecular Architecture of Severe Acute Respiratory Syndrome Coronavirus Revealed by Electron Cryomicroscopy
Authors: Neuman Benjamin W. , Adair Brian D. , Yoshioka Craig , Quispe Joel D. , Orca Gretchen , Kuhn Peter , Milligan Ronald A. , Yeager Mark , Buchmeier Michael J. 
Published: July 2006

ABSTRACT
Coronavirus particles are enveloped and pleomorphic and are thus refractory to crystallization and symmetry-assisted reconstruction. A novel methodology of single-particle image analysis was applied to selected virus features to obtain a detailed model of the oligomeric state and spatial relationships among viral structural proteins. Two-dimensional images of the S, M, and N structural proteins of severe acute respiratory syndrome coronavirus and two other coronaviruses were refined to a resolution of ∼4 nm. Proteins near the viral membrane were arranged in overlapping lattices surrounding a disordered core. Trimeric glycoprotein spikes were in register with four underlying ribonucleoprotein densities. However, the spikes were dispensable for ribonucleoprotein lattice formation. The ribonucleoprotein particles displayed coiled shapes when released from the viral membrane. Our results contribute to the understanding of the assembly pathway used by coronaviruses and other pleomorphic viruses and provide the first detailed view of coronavirus ultrastructure.
Transposon-Assisted Cloning and Traceless Mutagenesis of Adenoviruses: Development of a Novel Vector Based on SpeciesD
Authors: Ruzsics Zsolt , Wagner Markus , Osterlehner Andrea , Cook Jonathan , Koszinowski Ulrich , Burgert Hans-Gerhard 
Published: July 2006

ABSTRACT
Until
recently, adenovirus (Ad)-mediated gene therapy was almost exclusively
based on human Ad type 5 (Ad5). Preexisting immunity and the limited,
coxsackievirus and adenovirus receptor-dependent tropism of Ad5
stimulated attempts to exploit the natural diversity in tropism of the
other 50 known human Ad serotypes. Aiming in particular at
immunotherapy and vaccination, we have screened representative
serotypes from different Ad species for their ability to infect
dendritic cells. Ad19a, an Ad from species D, was selected for
development as a new vector for vaccination and cancer gene therapy. To
clone and manipulate its genome, we have developed a novel methodology,
coined “exposon mutagenesis,” that allows the rapid and
precise introduction of virtually any genetic alteration (deletions,
point mutations, or insertions) into recombinant Ad bacterial
artificial chromosomes. The versatility of the system was exemplified
by deleting the E3 region of Ad19a, by specifically knocking out
expression of a species-specific E3 gene, E3/49K, and by reinserting
E3/49K into an E3 null Ad19a mutant. The technology requires only
limited sequence information and is applicable to other Ad species.
Therefore, it should be extremely valuable for the analysis of gene
functions from any Ad species. In addition, a basic,
replication-defective E1- and E3-deleted Ad19a vector expressing GFP
(Ad19aGFP) was generated. This new vector based on species D Ads
exhibits a very promising tropism for lymphoid and muscle cells and
shows great potential as an alternative vector for transduction of cell
types that are resistant to or only poorly transduced by conventional
Ad5-based
vectors.
Eclipse Phase of Herpes Simplex Virus Type 1 Infection: Efficient Dynein-Mediated Capsid Transport without the Small Capsid Protein VP26
Authors: Döhner Katinka , Radtke Kerstin , Schmidt Simone , Sodeik Beate 
Published: July 2006

ABSTRACT
Cytoplasmic dynein,together with its cofactor dynactin, transports incoming herpes simplex virus type 1 (HSV-1) capsids along microtubules (MT) to the MT-organizing center (MTOC). From the MTOC, capsids move further to the nuclear pore, where the viral genome is released into the nucleoplasm. The small capsid protein VP26 can interact with the dynein light chains Tctex1 (DYNLT1) and rp3 (DYNLT3) and may recruit dynein to the capsid. Therefore, we analyzed nuclear targeting of incoming HSV1-ΔVP26 capsids devoid of VP26 and of HSV1-GFPVP26 capsids expressing a GFPVP26 fusion instead of VP26. To compare the cell entry of different strains, we characterized the inocula with respect to infectivity, viral genome content, protein composition, and particle composition. Preparations with a low particle-to-PFU ratio showed efficient nuclear targeting and were considered to be of higher quality than those containing many defective particles, which were unable to induce plaque formation. When cells were infected with HSV-1 wild type, HSV1-ΔVP26, or HSV1-GFPVP26, viral capsids were transported along MT to the nucleus. Moreover, when dynein function was inhibited by overexpression of the dynactin subunit dynamitin, fewer capsids of HSV-1 wild type, HSV1-ΔVP26, and HSV1-GFPVP26 arrived at the nucleus. Thus, even in the absence of the potential viral dynein receptor VP26, HSV-1 used MT and dynein for efficient nuclear targeting. These data suggest that besides VP26, HSV-1 encodes other receptors for dynein or dynactin.