bluuebunny/crossref_metadata_2025_split huggingface dataset:
Efficient Generation and Amplification of High-Capacity Adeno-Associated Virus/Adenovirus Hybrid Vectors
Authors: Gonçalves Manuel A. F. V. , van der Velde Ietje , Janssen Josephine M. , Maassen Bram T. H. , Heemskerk Evert H. , Opstelten Dirk-Jan E. , Knaän-Shanzer Shoshan , Valerio Dinko , de Vries Antoine A. F. 
Published: October 2002

ABSTRACT

Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in
trans
both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/
loxP
-mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation.
Intrapatient Diversity and Its Correlation with Viral Setpoint in Human Immunodeficiency Virus Type 1 CRF02_A/G-IbNG Infection
Authors: Mani Indu , Gilbert Peter , Sankalé Jean-Louis , Eisen Geoffrey , Mboup Souleymane , Kanki Phyllis J. 
Published: October 2002

ABSTRACT

The human immunodeficiency virus type 1 (HIV-1) viral setpoint during the disease-free interval has been strongly associated with future risk of disease progression. An awareness of the correlation between viral setpoint and HIV-1 genetic evolution over time is important in the understanding of viral dynamics and infection. We examined genetic diversity in HIV-1 CRF02_A/G-IbNG-infected seroincident women in Dakar, Senegal; determined whether a viral setpoint kinetic pattern existed for CRF02_A/G-IbNG during the disease-free interval; and correlated viral load level and diversity. Samples were drawn during the disease-free interval from consenting CRF02_A/G-IbNG-infected, antiretroviral therapy-naïve female commercial sex workers in Dakar, Senegal. Based on sequential plasma RNA values, low and high viral setpoint groups were established. Intrapatient diversity and divergence over time was determined from earlier and later time point DNA samples from each person. Most individuals followed the viral setpoint paradigm. For each 1|−|log
10
copy/ml of plasma increase in viral load, intrapatient diversity increased by 1.4% (
P
= 0.028). A greater diversification rate was observed in the high viral setpoint group than in the low viral setpoint group (
P
= 0.01). Greater nucleotide (
P
= 0.015) and amino acid (
P
= 0.048) divergences and a greater nucleotide divergence rate (
P
= 0.03) were found in the high viral setpoint group. There was no difference between the groups in the ratio of the number of nonsynonymous substitutions per nonsynonymous site to the number of synonymous substitutions per synonymous site. The greater intrapatient diversity, divergence, and diversification rates observed in the high viral setpoint group supports the notion that diversity is driven by cycles of viral replication resulting in accumulated mutations. Recognizing diversity potential based on viral load levels in individuals may inform the design of vaccines and therapies.
Mutations Conferring Resistance to Neutralization by a Soluble Form of the Neurotrophin Receptor (p75NTR) Map outside of the Known Antigenic Sites of the Rabies Virus Glycoprotein
Authors: Langevin Christelle , Tuffereau Christine 
Published: October 2002

ABSTRACTThe neurotrophin receptor (p75NTR) serves as a receptor for rabies virus (RV). We expressed and purified a soluble chimera consisting of the p75NTR ectodomain fused to the human immunoglobulin G1 (IgG1) Fc fragment (p75-Fc). Although p75-Fc interacts with RV, the infectivity of RV did not decrease significantly when it was incubated in the presence of the soluble receptor alone. However, when it was subsequently incubated with an antihuman IgG directed against the Fc fragment of p75-Fc, the infectivity of RV was significantly lowered (>90%), whereas incubation with antihuman IgG alone had no effect. We then selected eight independent RV mutants that were not neutralized by p75-Fc and antihuman IgG (srr[soluble receptor resistant] mutants). Each mutant carried a single mutation in the glycoprotein gene leading to one amino acid substitution in the protein. A total of four different substitutions were found. Two of the mutations were located at position 318 (phenylalanine replaced by a serine or a valine residue), and two were located at position 352 (histidine replaced by a tyrosine or an arginine residue). All of the mutations prevented the interaction with p75NTR as either a soluble or a membrane-anchored form. Two mutants (F318S) and (H352R) resulted in the formation of small plaques on BSR cells, probably due to the slower maturation of the glycoprotein. Immunoprecipitation, immunofluorescence, and neutralization assays showed that the four mutated glycoproteins still interacted with representative anti-RV glycoprotein monoclonal antibodies (MAbs), indicating that p75NTR binds outside of the known RV glycoprotein antigenic sites.
Complementation Analysis of the Flavivirus Kunjin NS3 and NS5 Proteins Defines the Minimal Regions Essential for Formation of a Replication Complex and Shows a Requirement of NS3 in
cis
for Virus Assembly
Authors: Liu Wen Jun , Sedlak Petra L. , Kondratieva Natasha , Khromykh Alexander A. 
Published: October 2002

ABSTRACT

We have previously reported successful
trans
-complementation of defective Kunjin virus genomic RNAs with a range of large lethal deletions in the nonstructural genes NS1, NS3, and NS5 (A. A. Khromykh et al., J. Virol.
74:
3253-3263, 2000). In this study we have mapped further the minimal region in the NS5 gene essential for efficient
trans
-complementation of genome-length RNAs in repBHK cells to the first 316 of the 905 codons. To allow amplification and easy detection of complemented defective RNAs with deletions apparently affecting virus assembly, we have developed a dual replicon complementation system. In this system defective replicon RNAs with a deletion(s) in the nonstructural genes also encoded the puromycin resistance gene (PAC gene) and the reporter gene for β-galactosidase (β-Gal). Complementation of these defective replicon RNAs in repBHK cells resulted in expression of PAC and β-Gal which allowed establishment of cell lines stably producing replicating defective RNAs by selection with puromycin and comparison of replication efficiencies of complemented defective RNAs by β-Gal assay. Using this system we demonstrated that deletions in the C-terminal 434 codons of NS3 (codons 178 to 611) were complemented for RNA replication, while any deletions in the first 178 codons were not. None of the genome-length RNAs containing deletions in NS3 shown to be complementable for RNA replication produced secreted defective viruses during complementation in repBHK cells. In contrast, structural proteins produced from these complemented defective RNAs were able to package helper replicon RNA. The results define minimal regions in the NS3 and NS5 genes essential for the formation of complementable replication complex and show a requirement of NS3 in
cis
for virus assembly.
The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro
Authors: Lu Bin , Ma Chien-Hui , Brazas Robert , Jin Hong 
Published: October 2002

ABSTRACT

The phosphoprotein (P protein) of respiratory syncytial virus (RSV) is a key component of the viral RNA-dependent RNA polymerase complex. The protein is constitutively phosphorylated at the two clusters of serine residues (116, 117, and 119 [116/117/119] and 232 and 237 [232/237]). To examine the role of phosphorylation of the RSV P protein in virus replication, these five serine residues were altered to eliminate their phosphorylation potential, and the mutant proteins were analyzed for their functions with a minigenome assay. The reporter gene expression was reduced by 20% when all five phosphorylation sites were eliminated. Mutants with knockout mutations at two phosphorylation sites (S232A/S237A [PP2]) and at five phosphorylation sites (S116L/S117R/S119L/S232A/S237A [PP5]) were introduced into the infectious RSV A2 strain. Immunoprecipitation of
33
P
i
-labeled infected cells showed that P protein phosphorylation was reduced by 80% for rA2-PP2 and 95% for rA2-PP5. The interaction between the nucleocapsid (N) protein and P protein was reduced in rA2-PP2- and rA2-PP5-infected cells by 30 and 60%, respectively. Although the two recombinant viruses replicated well in Vero cells, rA2-PP2 and, to a greater extent, rA2-PP5, replicated poorly in HEp-2 cells. Virus budding from the infected HEp-2 cells was affected by dephosphorylation of P protein, because the majority of rA2-PP5 remained cell associated. In addition, rA2-PP5 was also more attenuated than rA2-PP2 in replication in the respiratory tracts of mice and cotton rats. Thus, our data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro, phosphorylation of P protein is required for efficient virus replication in vitro and in vivo.
Identification of Conserved and Variable Structures in the Human Immunodeficiency Virus gp120 Glycoprotein of Importance for CXCR4 Binding
Authors: Basmaciogullari Stéphane , Babcock Gregory J. , Van Ryk Donald , Wojtowicz Woj , Sodroski Joseph 
Published: October 2002

ABSTRACTCD4 and the chemokine receptors, CXCR4 and CCR5, serve as receptors for human immunodeficiency virus type 1 (HIV-1). Binding of the HIV-1 gp120 envelope glycoprotein to the chemokine receptors normally requires prior interaction with CD4. Mapping the determinants on gp120 for the low-affinity interaction with CXCR4 has been difficult due to the nonspecific binding of this viral glycoprotein to cell surfaces. Here we examine the binding of a panel of gp120 mutants to paramagnetic proteoliposomes displaying CXCR4 on their surfaces. We show that the gp120 β19 strand and third variable (V3) loop contain residues important for CXCR4 interaction. Basic residues from both elements, as well as a conserved hydrophobic residue at the V3 tip, contribute to CXCR4 binding. Removal of the gp120 V1/V2 variable loops allows the envelope glycoprotein to bind CXCR4 in a CD4-independent manner. These results indicate that although some variable gp120 residues contribute to the specific binding to CCR5 or CXCR4, gp120 elements common to CXCR4- or CCR5-using strains are involved in the interaction with both coreceptors.
Characterization of Moloney Murine Leukemia Virus p12 Mutants Blocked during Early Events of Infection
Authors: Yuan Bing , Fassati Ariberto , Yueh Andrew , Goff Stephen P. 
Published: October 2002

ABSTRACT

Mutations affecting either the N- or C-terminal regions of the Gag protein p12 block replication of Moloney murine leukemia virus (M-MuLV). Viruses carrying mutations in this portion of
gag
can mediate the assembly and release of virions but are unable to successfully carry out the early phase of the M-MuLV life cycle. Wild-type and mutant viruses were found to synthesize similar levels of linear viral DNA in both cytoplasmic and nuclear fractions, and there were no significant differences in either the density or sedimentation of the viral protein-nucleic acid complexes. Analysis of the termini of the linear viral DNAs showed that the 3′ ends of the mutant viral DNA were processed normally by the integrase. Further, the preintegration complexes extracted from the cytoplasm of cells infected with the mutant viruses were competent for integration into target DNA in vitro. Nevertheless, no circular viral DNAs were detected in cells infected by the mutants, and functional proviruses were not formed. These results suggest that p12 has an unexpected role in the early phase of the life cycle and is needed after viral DNA synthesis to deliver the incoming DNA to the correct location and in the appropriate state to permit either circularization or integration of the viral DNA in vivo.
The Mason-Pfizer Monkey Virus Internal Scaffold Domain Enables In Vitro Assembly of Human Immunodeficiency Virus Type 1 Gag
Authors: Sakalian Michael , Dittmer Stephanie S. , Gandy A. Dustin , Rapp Nathan D. , Zábranský Aleš , Hunter Eric 
Published: October 2002

ABSTRACT
The Mason-Pfizer monkey virus (M-PMV) Gag protein possesses the ability to assemble into an immature capsid when synthesized in a reticulocyte lysate translation system. In contrast, the human immunodeficiency virus (HIV) Gag protein is incapable of assembly in parallel assays. To enable the assembly of HIV Gag, we have combined or inserted regions of M-PMV Gag into HIV Gag. By both biochemical and morphological criteria, several of these chimeric Gag molecules are capable of assembly into immature capsid-like structures in this in vitro system. Chimeric species containing large regions of M-PMV Gag fused to HIV Gag sequences failed to assemble, while species consisting of only the M-PMV p12 region, and its internal scaffold domain (ISD), fused to HIV Gag were capable of assembly, albeit at reduced kinetics compared to M-PMV Gag. The ability of the ISD to induce assembly of HIV Gag, which normally assembles at the plasma membrane, suggests a common requirement for a concentrating factor in retrovirus assembly. Despite the dramatic effect of the ISD on chimera assembly, the function of HIV Gag domains in that process was found to remain essential, since an assembly-defective mutant of HIV CA, M185A, abolished assembly when introduced into the chimera. This continued requirement for HIV Gag domain function in the assembly of chimeric molecules will allow this in vitro system to be used for the analysis of potential inhibitors of HIV immature particle assembly.
Identification, Localization, and Regulation of Expression of the UL24 Protein of Herpes Simplex Virus Type 1
Authors: Pearson Angela , Coen Donald M. 
Published: October 2002

ABSTRACT

The
UL24
gene of herpes simplex virus type 1 is conserved across many herpesviruses, but its protein product has not been identified. We expressed the
UL24
gene in insect cells from a recombinant baculovirus and used the resulting protein to raise a rat antiserum. In immunoblotting experiments, this antiserum recognized a 30-kDa protein in lysates from infected cells. The identity of this species as UL24 was confirmed by using a virus encoding a truncated form of UL24. On the basis of biochemical fractionation of infected cells, UL24 appeared to be predominantly nucleus associated, especially at later times in infection. Although certain
UL24
transcripts exhibit early kinetics, UL24 protein accumulated at later times in infection and levels were decreased sixfold in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid; thus, UL24 was expressed with leaky-late kinetics. The entire
UL24
open reading frame is encoded by mRNAs with two different 5′ ends. A mutation that eliminates the more abundant transcripts that originate at the first transcription start site resulted in a 10-fold reduction in the level of UL24 expressed but did not eliminate expression. Thus, the less abundant transcripts originating at the second transcription start site can evidently be translated, although transcripts originating at the first start site appear to be the major contributors to the expression of UL24. We conclude that UL24 is a bona fide herpes simplex virus type 1 protein that associates primarily with nuclei and whose expression is regulated at multiple levels.
Characterization of Two New Structural Glycoproteins, GP
3
and GP
4
, of Equine Arteritis Virus
Authors: Wieringa Roeland , de Vries Antoine A. F. , Raamsman Martin J. B. , Rottier Peter J. M. 
Published: October 2002

ABSTRACT

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family
Arteriviridae
of the order
Nidovirales
. Four envelope proteins have hitherto been identified in EAV particles: the predominant membrane proteins M and G
L
, the unglycosylated small envelope protein E, and the nonabundant membrane glycoprotein G
S
. In this study, we established that the products of EAV open reading frame 3 (ORF3) and ORF4 (designated GP
3
and GP
4
, respectively) are also minor structural glycoproteins. The proteins were first characterized by various analyses after in vitro translation of RNA transcripts in a rabbit reticulocyte lysate in the presence and absence of microsomal membranes. We subsequently expressed ORF3 and -4 in baby hamster kidney cells by using the vaccinia virus expression system and, finally, analyzed the GP
3
and GP
4
proteins synthesized in EAV-infected cells. The results showed that GP
4
is a class I integral membrane protein of 28 kDa with three functional N-glycosylation sites and with little, if any, of its carboxy terminus exposed. Both after independent expression and in EAV-infected cells, the protein localizes in the endoplasmic reticulum (ER), as demonstrated biochemically by analysis of its oligosaccharide side chains and as visualized directly by immunofluorescence studies. GP
3
, on the other hand, is a heavily glycosylated protein whose hydrophobic amino terminus is not cleaved off. It is an integral membrane protein anchored by either or both of its hydrophobic terminal domains and with no parts detectably exposed cytoplasmically. Also, GP
3
localizes in the ER when expressed independently and in the context of an EAV infection. Only a small fraction of the GP
3
and GP
4
proteins synthesized in infected cells ends up in virions. Most, but not all, of the oligosaccharides of these virion glycoproteins are biochemically mature. Our results bring the number of EAV envelope proteins to six.
The Genes Encoding the gCIII Complex of Human Cytomegalovirus Exist in Highly Diverse Combinations in Clinical Isolates
Authors: Rasmussen Lucy , Geissler Aimee , Cowan Catherine , Chase Amanda , Winters Mark 
Published: October 2002

ABSTRACT
The UL74 (glycoprotein O [gO])-UL75 (gH)-UL115 (gL) complex of human cytomegalovirus (CMV), known as the gCIII complex, is likely to play an important role in the life cycle of the virus. The gH and gL proteins have been associated with biological activities, such as the induction of virus-neutralizing antibody, cell-virus fusion, and cell-to-cell spread of the virus. The sequences of the two gH gene variants, readily recognizable by restriction endonuclease polymorphism, are well conserved among clinical isolates, but nothing is known about the sequence variability of the gL and gO genes. Sequencing of the full-length gL and gO genes was performed with 22 to 39 clinical isolates, as well as with laboratory strains AD169, Towne, and Toledo, to determine phylogenetically based variants of the genes. The sequence information provided the basis for identifying gL and gO variants by restriction endonuclease polymorphism. The predicted gL amino acid sequences varied less than 2% among the isolates, but the variability of gO among the isolates approached 45%. The variants of the genes coding for gCIII in laboratory strains Towne, AD169, and Toledo were different from those in most clinical isolates. When clinical isolates from different patient populations with various degrees of symptomatic CMV disease were surveyed, the gO1 variant occurred almost exclusively with the gH1 variant. The gL2 variant occurred with a significantly lower frequency in the gH1 variant group. There were no configurations of the gCIII complex that were specifically associated with symptomatic CMV disease or human immunodeficiency virus serologic status. The potential for the gCIII complex to exist in diverse genetic combinations in clinical isolates points to a new aspect that must be considered in studies of the significance of CMV strain variability.
Mutational Analysis of the Uukuniemi Virus (
Bunyaviridae
Family) Promoter Reveals Two Elements of Functional Importance
Authors: Flick Ramon , Elgh Fredrik , Pettersson Ralf F. 
Published: October 2002

ABSTRACT

We have performed an extensive mutational analysis of the proposed promoter region of the phlebovirus Uukuniemi (UUK), a member of the
Bunyaviridae
family. This was achieved by using a recently developed RNA polymerase I (Pol I)-driven reverse genetics system (R. Flick and R. F. Pettersson, J. Virol.
75:
1643-1655, 2001). Chimeric cDNAs containing the coding region for the reporter chloramphenicol acetyltransferase (CAT) in an antisense orientation were flanked by the 5′- and 3′-terminal nontranslated regions of the UUK virus-sense RNA (vRNA) derived from the medium-sized (M) RNA segment. The chimeric cDNAs (Pol I expression cassettes) were cloned between the murine Pol I promoter and terminator, and the plasmids were transfected into BHK-21 cells. CAT activity was determined after cotransfection with viral expression plasmids encoding the RNA-dependent RNA polymerase (L) and the nucleoprotein (N) or, alternatively, after superinfection with UUK virus helper virus. Using oligonucleotide-directed mutagenesis, single point mutations (substitutions, deletions, and insertions) were introduced into the viral promoter region. Differences in CAT activities were interpreted to reflect the efficiency of mRNA transcription from the mutated promoter and the influence on RNA replication. Analysis of 109 mutants allowed us to define two important regulatory regions within the proximal promoter region (site A, positions 3 to 5 and
2
to
4
; site B, positions 8 and
8
, where underlined nucleotides refer to positions in the vRNA 3′ end). Complementary double nucleotide exchanges in the proximal promoter region, which maintained the possibility for base pairing between the 5′ and 3′ ends, demonstrated that nucleotides in the two described regions are essential for viral polymerase recognition in a base-specific manner. Thus, mere preservation of panhandle base pairing between the 5′ and 3′ ends is not sufficient for promoter activity. In conclusion, we have been able to demonstrate that both ends of the M RNA segment build up the promoter region and are involved in the specific recognition by the viral polymerase.