bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transduction of Bone-Marrow-Derived Mesenchymal Stem Cells by Using Lentivirus Vectors Pseudotyped with Modified RD114 Envelope Glycoproteins
Authors: Zhang Xian-Yang , La Russa Vincent F. , Reiser Jakob 
Published: January 2004

ABSTRACT
Bone-marrow-derived mesenchymal stem cells (MSCs) have attracted considerable attention as tools for the systemic delivery of therapeutic proteins in vivo, and the ability to efficiently transfer genes of interest into such cells would create a number of therapeutic opportunities. We have designed and tested a series of human immunodeficiency virus type 1 (HIV-1)-based vectors and vectors based on the oncogenic murine stem cell virus to deliver and express transgenes in human MSCs. These vectors were pseudotyped with either the vesicular stomatitis virus G (VSV-G) glycoprotein (GP) or the feline endogenous virus RD114 envelope GP. Transduction efficiencies and transgene expression levels in MSCs were analyzed by quantitative flow cytometry and quantitative real-time PCR. While transduction efficiencies with virus particles pseudotyped with the VSV-G GP were found to be high, RD114 pseudotypes revealed transduction efficiencies that were 1 to 2 orders of magnitude below those observed with VSV-G pseudotypes. However, chimeric RD114 GPs, with the transmembrane and extracellular domains fused to the cytoplasmic domain derived from the amphotropic Moloney murine leukemia virus 4070A GP, revealed about 15-fold higher titers relative to the unmodified RD114 GP. The transduction efficiencies in human MSCs of HIV-1-based vectors pseudotyped with the chimeric RD114 GP were similar to those obtained with HIV-1 vectors pseudotyped with the VSV-G GP. Our results also indicate that RD114 pseudotypes were less toxic than VSV-G pseudotypes in human MSC progenitor assays. Taken together, these results suggest that lentivirus pseudotypes bearing alternative Env GPs provide efficient tools for ex vivo modification of human MSCs.
A Novel Fluorescence Resonance Energy Transfer Assay Demonstrates that the Human Immunodeficiency Virus Type 1 Pr55
Gag
I Domain Mediates Gag-Gag Interactions
Authors: Derdowski Aaron , Ding Lingmei , Spearman Paul 
Published: January 2004

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) assembly takes place at the plasma membrane of cells and is directed by the Pr55
Gag
polyprotein (Gag). One of the essential steps in the assembly process is the multimerization of Gag. We have developed a novel fluorescence resonance energy transfer (FRET) assay for the detection of protein-protein interactions between Gag molecules. We demonstrate that Gag multimerization takes place primarily on cellular membranes, with the majority of these interactions occurring on the plasma membrane. However, distinct sites of Gag-Gag interaction are also present at punctate intracellular locations. The I domain is a functional assembly domain within the nucleocapsid region of Gag that affects particle density, the subcellular localization of Gag, and the formation of detergent-resistant Gag protein complexes. Results from this study provide evidence that the I domain mediates Gag-Gag interactions. Using Gag-fluorescent protein fusion constructs that were previously shown to define the minimal I domain within HIV-1 Pr55
Gag
, we show by FRET techniques that protein-protein interactions are greatly diminished when Gag proteins lacking the I domain are expressed. Gag-Tsg101 interactions are also seen in living cells and result in a shift of Tsg101 to the plasma membrane. The results within this study provide direct evidence that the I domain mediates protein-protein interactions between Gag molecules. Furthermore, this study establishes FRET as a powerful tool for the detection of protein-protein interactions involved in retrovirus assembly.
Isolation of CD4-Independent Primary Human Immunodeficiency Virus Type 1 Isolates That Are Syncytium Inducing and Acutely Cytopathic for CD8+Lymphocytes
Authors: Zerhouni Bouchra , Nelson Julie A. E. , Saha Kunal 
Published: January 2004

ABSTRACTPrevious studies have established the existence of CD4-independent simian immunodeficiency virus, human immunodeficiency virus type 2 (HIV-2), and laboratory strains of HIV-1. However, whether CD4-independent viruses may also exist in HIV-1-infected patients has remained unclear. We have recently reported the isolation of viruses from an AIDS patient that were able to infect CD8+cells independent of CD4, using CD8 as a receptor. Using a similar approach, here we examined viruses from 12 randomly selected patients (obtained from the AIDS Research and Reference Program, National Institutes of Health) for the presence of CD4-independent HIV-1. CD4-independent variants were isolated from infected CD8+cells from the viral quasispecies of 7 of 12 patients. The CD4-independent isolates were able to infect primary CD8+cells as well as a CD4−CD8+T-cell line. Soluble CD4 and blocking anti-CD4 or -CD8 antibody had no effect on infection of CD8+cells. Remarkably, two of the seven CD4-independent isolates, but not their parental bulk viruses, induced syncytia and caused acute death of infected CD8+cells. Some of the CD4-independent variants were also able to infect U87 cells that were negative for CD4, CD8, and common HIV coreceptors, suggesting a novel entry mechanism for these isolates. The CD4-independent isolates were derived from adults and children infected with subtypes A, B, and D. Although no common motif for CD4 independence was found, novel sequence changes were observed in critical areas of the envelopes of the CD4-independent viruses. These results demonstrate that HIV-1-infected patients can frequently harbor viruses that are able to mediate CD4-independent infection of CD8+cells. In addition, this study also provides evidence of primary HIV-1 variants that are syncytium inducing and acutely cytopathic for CD8+lymphocytes.
Heat Shock Protein 70 Is Related to Thermal Inhibition of Nuclear Export of the Influenza Virus Ribonucleoprotein Complex
Authors: Hirayama Etsuko , Atagi Hiromitsu , Hiraki Akihiro , Kim Jeman 
Published: January 2004

ABSTRACTThe influenza virus genome replicates and forms a viral ribonucleoprotein complex (vRNP) with nucleoprotein (NP) and RNA polymerases in the nuclei of host cells. vRNP is then exported into the cytoplasm for viral morphogenesis at the cell membrane. Matrix protein 1 (M1) and nonstructural protein 2/nuclear export protein (NS2/NEP) work in the nuclear export of vRNP by associating with it. It was previously reported that influenza virus production was inhibited in Madin-Darby canine kidney (MDCK) cells cultured at 41°C because nuclear export of vRNP was blocked by the dissociation of M1 from vRNP (A. Sakaguchi, E. Hirayama, A. Hiraki, Y. Ishida, and J. Kim, Virology306:244-253, 2003). Previous data also suggested that a certain protein(s) synthesized only at 41°C inhibited the association of M1 with vRNP. The potential of heat shock protein 70 (HSP70) as a candidate obstructive protein was investigated. Induction of HSP70 by prostaglandin A1 (PGA1) at 37°C caused the suppression of virus production. The nuclear export of viral proteins was inhibited by PGA1, and M1 was not associated with vRNP, indicating that HSP70 prevents M1 from binding to vRNP. An immunoprecipitation assay showed that HSP70 was bound to vRNP, suggesting that the interaction of HSP70 with vRNP is the reason for the dissociation of M1. Moreover, NS2 accumulated in the nucleoli of host cells cultured at 41°C, showing that the export of NS2 was also disturbed at 41°C. However, NS2 was exported normally from the nucleus, irrespective of PGA1 treatment at 37°C, suggesting that HSP70 does not influence NS2.
Critical Residues for GTP Methylation and Formation of the Covalent m
7
GMP-Enzyme Intermediate in the Capping Enzyme Domain of
Bamboo Mosaic Virus
Authors: Huang Yih-Leh , Han Yu-Tsung , Chang Ya-Ting , Hsu Yau-Heiu , Meng Menghsiao 
Published: January 2004

ABSTRACT

Open reading frame 1 of
Bamboo mosaic virus
(BaMV), a
Potexvirus
in the alphavirus-like superfamily, encodes a 155-kDa replicase responsible for the formation of the 5′ cap structure and replication of the viral RNA genome. The N-terminal domain of the viral replicase functions as an mRNA capping enzyme, which exhibits both GTP methyltransferase and
S
-adenosylmethionine (AdoMet)-dependent guanylyltransferase activities. We mutated each of the four conserved amino acids among the capping enzymes of members within alphavirus-like superfamily and a dozen of other residues to gain insight into the structure-function relationship of the viral enzyme. The mutant enzymes were purified and subsequently characterized. H68A, the mutant enzyme bearing a substitution at the conserved histidine residue, has an ∼10-fold increase in GTP methyltransferase activity but completely loses the ability to form the covalent m
7
GMP-enzyme intermediate. High-pressure liquid chromatography analysis confirmed the production of m
7
GTP by the GTP methyltransferase activity of H68A. Furthermore, the produced m
7
GTP sustained the formation of the m
7
GMP-enzyme intermediate for the wild-type enzyme in the presence of
S
-adenosylhomocysteine (AdoHcy), suggesting that the previously observed AdoMet-dependent guanylation of the enzyme using GTP results from reactions of GTP methylation and subsequently guanylation of the enzyme using m
7
GTP. Mutations occurred at the other three conserved residues (D122, R125, and Y213), and H66 resulted in abolition of activities for both GTP methylation and formation of the covalent m
7
GMP-enzyme intermediate. Mutations of amino acids such as K121, C234, D310, W312, R316, K344, W406, and K409 decreased both activities by various degrees, and the extents of mutational effects follow similar trends. The affinity to AdoMet of the various BaMV capping enzymes, except H68A, was found in good correlations with not only the magnitude of GTP methyltransferase activity but also the capability of forming the m
7
GMP-enzyme intermediate. Taken together with the AdoHcy dependence of guanylation of the enzyme using m
7
GTP, a basic working mechanism, with the contents of critical roles played by the binding of AdoMet/AdoHcy, of the BaMV capping enzyme is proposed and discussed.
Quinoline Derivatives Are Therapeutic Candidates for Transmissible Spongiform Encephalopathies
Authors: Murakami-Kubo Ikuko , Doh-ura Katsumi , Ishikawa Kensuke , Kawatake Satoshi , Sasaki Kensuke , Kira Jun-ichi , Ohta Shigeru , Iwaki Toru 
Published: January 2004

ABSTRACT

We previously reported that quinacrine inhibited the formation of an abnormal prion protein (PrPres), a key molecule in the pathogenesis of transmissible spongiform encephalopathy, or prion disease, in scrapie-infected neuroblastoma cells. To elucidate the structural aspects of its inhibiting action, various chemicals with a quinoline ring were screened in the present study. Assays of the scrapie-infected neuroblastoma cells revealed that chemicals with a side chain containing a quinuclidine ring at the 4 position of a quinoline ring (represented by quinine) inhibited the PrPres formation at a 50% inhibitory dose ranging from 10
−1
to 10
1
μM. On the other hand, chemicals with a side chain at the 2 position of a quinoline ring (represented by 2,2′-biquinoline) more effectively inhibited the PrPres formation at a 50% inhibitory dose ranging from 10
−3
to 10
−1
μM. A metabolic labeling study revealed that the action of quinine or biquinoline was not due to any alteration in the biosynthesis or turnover of normal prion protein, whereas surface plasmon resonance analysis showed a strong binding affinity of biquinoline with a recombinant prion protein. In vivo studies revealed that 4-week intraventricular infusion of quinine or biquinoline was effective in prolonging the incubation period in experimental mouse models of intracerebral infection. The findings suggest that quinoline derivatives with a nitrogen-containing side chain have the potential of both inhibiting PrPres formation in vitro and prolonging the incubation period of infected animals. These chemicals are new candidates for therapeutic drugs for use in the treatment of transmissible spongiform encephalopathies.
Fusion Proteins Consisting of Human Immunodeficiency Virus Type 1 Integrase and the Designed Polydactyl Zinc Finger Protein E2C Direct Integration of Viral DNA into Specific Sites
Authors: Tan Wenjie , Zhu Kai , Segal David J. , Barbas Carlos F. , Chow Samson A. 
Published: January 2004

ABSTRACT
In order to establish a productive infection, a retrovirus must integrate the cDNA of its RNA genome into the host cell chromosome. While this critical process makes retroviruses an attractive vector for gene delivery, the nonspecific nature of integration presents inherent hazards and variations in gene expression. One approach to alleviating the problem involves fusing retroviral integrase to a sequence-specific DNA-binding protein that targets a defined chromosomal site. We prepared proteins consisting of wild-type or truncated human immunodeficiency virus type 1 (HIV-1) integrase fused to the synthetic polydactyl zinc finger protein E2C. The purified fusion proteins bound specifically to the 18-bp E2C recognition sequence as analyzed by DNase I footprinting. The fusion proteins were catalytically active and biased integration of retroviral DNA near the E2C-binding site in vitro. The distribution was asymmetric, and the major integration hot spots were localized within a 20-bp region upstream of the C-rich strand of the E2C recognition sequence. Integration bias was not observed with target plasmids bearing a mutated E2C-binding site or when HIV-1 integrase and E2C were added to the reaction as separate proteins. The results demonstrate that the integrase-E2C fusion proteins offer an efficient approach and a versatile framework for directing the integration of retroviral DNA into a predetermined DNA site.
The Pseudorabies Virus US3 Protein Is a Component of Primary and of Mature Virions
Authors: Granzow Harald , Klupp Barbara G. , Mettenleiter Thomas C. 
Published: January 2004

ABSTRACT
Herpesviruses acquire a primary envelope by budding of capsids at the inner leaflet of the nuclear membrane. They then traverse into the cytoplasm after fusion of the primary envelope with the outer leaflet of the nuclear membrane. In the alphaherpesvirus pseudorabies virus (PrV), the latter process is impaired when the US3 protein is absent. Acquisition of final tegument and envelope occurs in the cytoplasm. Besides the capsid components, only the UL31 and UL34 gene products of PrV have unequivocally been shown to be part of primary enveloped virions, whereas they lack several tegument proteins present in mature virions (reviewed by T. C. Mettenleiter, J. Virol. 76:1537-1547, 2002). Using immunoelectron microscopy, we show that the US3 protein is present in primary enveloped as well as in mature virions. It is also detectable in intracytoplasmic inclusions produced in the absence of other viral tegument components or envelope-associated glycoproteins. In particular, inclusions formed in the absence of the inner tegument protein UL37 contained the US3 protein. Thus, the US3 protein is a tegument component of both forms of enveloped alphaherpes virions. We hypothesize that US3 protein in primary virions modulates deenvelopment at the outer leaflet of the nuclear membrane and is either lost from primary virions during nuclear egress and subsequently reacquired early during tegumentation or is retained during transit of the nucleocapsid through the nuclear membrane.
Cytotoxic-T-Lymphocyte Human Papillomavirus Type 16 E5 Peptide with CpG-Oligodeoxynucleotide Can Eliminate Tumor Growth in C57BL/6 Mice
Authors: Chen Yi-Fang , Lin Chih-Wei , Tsao Yeou-Ping , Chen Show-Li 
Published: January 2004

ABSTRACTPreviously, we identified human papillomavirus type 16 (HPV-16) E5 as a tumor rejection antigen that can induce cytotoxic T lymphocytes (CTLs) to protect against tumor growth (D. W. Liu et al., J. Virol.74:9083-9089, 2000). In the present study, we further mapped the CTL epitope of E5 protein by analyzing E5-specific CD8+gamma interferon-positive (IFN-γ+) double-positive cells in C57BL/6 mice with flow cytometry. The results showed the region spanning amino acids 25 to 33 (VCLLIRPLL) contained the potential Db-restricted CTL epitope. Subsequently, to determine whether peptide E5 25-33-based vaccination could induce E5-specific CTL activity, syngeneic animals received E5 25-33 emulsified with either CpG oligodeoxynucleotide (CpG ODN 1826) or Freund's adjuvant, and the growth of the tumors was monitored. The results showed that although both adjuvants induced E5-specific CD8+IFN-γ+T cells and eradicated E5-containing tumor growth, CpG ODN was found to stimulate stronger CTL response than Freund's adjuvant. We also compared the immune response of the effector/memory/recall phase induced by E5 25-33 peptide or by E5 protein that was synthesized in vivo by adenovirus-based E5 gene delivery. E5 25-33 peptide plus CpG ODN was shown to be a superior vaccine compared to the adenovirus-based E5 gene. Interestingly, their chronological patterns of immune response were similar, suggesting that E5 25-33 is a major CTL peptide of E5 protein.
Cell-Dependent Role for the Poliovirus 3′ Noncoding Region in Positive-Strand RNA Synthesis
Authors: Brown David M. , Kauder Steven E. , Cornell Christopher T. , Jang Gwendolyn M. , Racaniello Vincent R. , Semler Bert L. 
Published: January 2004

ABSTRACT
We previously reported the isolation of a mutant poliovirus lacking the entire genomic RNA 3′ noncoding region. Infection of HeLa cell monolayers with this deletion mutant revealed only a minor defect in the levels of viral RNA replication. To further analyze the consequences of the genomic 3′ noncoding region deletion, we examined viral RNA replication in a neuroblastoma cell line, SK-N-SH cells. The minor genomic RNA replication defect in HeLa cells was significantly exacerbated in the SK-N-SH cells, resulting in a decreased capacity for mutant virus growth. Analysis of the nature of the RNA replication deficiency revealed that deleting the poliovirus genomic 3′ noncoding region resulted in a positive-strand RNA synthesis defect. The RNA replication deficiency in SK-N-SH cells was not due to a major defect in viral translation or viral protein processing. Neurovirulence of the mutant virus was determined in a transgenic mouse line expressing the human poliovirus receptor. Greater than 1,000 times more mutant virus was required to paralyze 50% of inoculated mice, compared to that with wild-type virus. These data suggest that, together with a cellular factor(s) that is limiting in neuronal cells, the poliovirus 3′ noncoding region is involved in positive-strand synthesis during genome replication.
A
cis
-Acting Replication Element in the Sequence Encoding the NS5B RNA-Dependent RNA Polymerase Is Required for Hepatitis C Virus RNA Replication
Authors: You Shihyun , Stump Decherd D. , Branch Andrea D. , Rice Charles M. 
Published: January 2004

ABSTRACT

RNA structures play key roles in the replication of RNA viruses. Sequence alignment software, thermodynamic RNA folding programs, and classical comparative phylogenetic analysis were used to build models of six RNA elements in the coding region of the hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B. The importance of five of these elements was evaluated by site-directed mutagenesis of a subgenomic HCV replicon. Mutations disrupting one of the predicted stem-loop structures, designated 5BSL3.2, blocked RNA replication, implicating it as an essential
cis
-acting replication element (CRE). 5BSL3.2 is about 50 bases in length and is part of a larger predicted cruciform structure (5BSL3). As confirmed by RNA structure probing, 5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop. Mutational analysis and structure probing were used to explore the importance of these features. Primary sequences in the loops were shown to be important for HCV RNA replication, and the upper helix appears to serve as an essential scaffold that helps maintain the overall RNA structure. Unlike certain picornavirus CREs, whose function is position independent, 5BSL3.2 function appears to be context dependent. Understanding the role of 5BSL3.2 and determining how this new CRE functions in the context of previously identified elements at the 5′ and 3′ ends of the RNA genome should provide new insights into HCV RNA replication.
Quantification of the DNA Cleavage and Packaging Proteins U
L
15 and U
L
28 in A and B Capsids of Herpes Simplex Virus Type 1
Authors: Beard Philippa M. , Duffy Carol , Baines Joel D. 
Published: January 2004

ABSTRACT

The proteins produced by the herpes simplex virus type 1 (HSV-1) genes U
L
15 and U
L
28 are believed to form part of the terminase enzyme, a protein complex essential for the cleavage of newly synthesized, concatameric herpesvirus DNA and the packaging of the resultant genome lengths into preformed capsids. This work describes the purification of recombinant forms of pU
L
15 and pU
L
28, which allowed the calculation of the average number of copies of each protein in A and B capsids and in capsids lacking the putative portal encoded by U
L
6. On average, 1.0 (±0.29 [standard deviation]) copies of pU
L
15 and 2.4 (±0.97) copies of pU
L
28 were present in B capsids, 1.2 (±0.72) copies of pU
L
15 and 1.5 (±0.86) copies of pU
L
28 were found in mutant capsids lacking the putative portal protein pU
L
6, and approximately 12.0 (±5.63) copies of pU
L
15 and 0.6 (±0.32) copies of pU
L
28 were present in each A capsid. These results suggest that the packaging machine is partly comprised of approximately 12 copies of pU
L
15, as found in A capsids, with wild-type B and mutant U
L
6(−) capsids containing an incomplete complement of cleavage and packaging proteins. These results are consistent with observations that B capsids form by default in the absence of packaging machinery in vitro and in vivo. In contrast, A capsids may be the result of initiated but aborted attempts at DNA packaging, resulting in the retention of at least part of the DNA packaging machinery.