bluuebunny/crossref_metadata_2025_split huggingface dataset:
Herpes Simplex Virus Type 1/Adeno-Associated Virus
rep
+
Hybrid Amplicon Vector Improves the Stability of Transgene Expression in Human Cells by Site-Specific Integration
Authors: Wang Y. , Camp S. M. , Niwano M. , Shen X. , Bakowska J. C. , Breakefield X. O. , Allen P. D. 
Published: July 2002

ABSTRACT

Herpes simplex virus type 1 (HSV-1) amplicon vectors are promising gene delivery tools, but their utility in gene therapy has been impeded to some extent by their inability to achieve stable transgene expression. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). A
rep
−
HSV/AAV hybrid amplicon vector was made by inserting a transgene cassette flanked with AAV ITRs into an HSV-1 amplicon backbone, and a
rep
+
HSV/AAV hybrid amplicon was made by inserting
rep68
/
78
outside the
rep
−
vector 3′ AAV ITR sequence. Both vectors also had a pair of
loxP
sites flanking the ITRs. The resulting hybrid amplicon vectors were successfully packaged and compared to a standard amplicon vector for stable transduction frequency (STF) in human 293 and Gli36 cell lines and primary myoblasts. The
rep
+
, but not the
rep
−
, hybrid vector improved STF in all three types of cells; 84% of Gli36 and 40% of 293 stable clones transduced by the
rep
+
hybrid vector integrated the transgene into the AAVS1 site. Due to the difficulty in expanding primary myoblasts, we did not assess site-specific integration in these cells. A strategy to attempt further improvement of STF by “deconcatenating” the hybrid amplicon DNA via Cre-
loxP
recombination was tested, but it did not increase STF. These data demonstrate that introducing the integrating elements of AAV into HSV-1 amplicon vectors can significantly improve their ability to achieve stable gene transduction by conferring the AAV-like capability of site-specific genomic integration in dividing cells.
RNA 3′ Readthrough of Oncoretrovirus and Lentivirus: Implications for Vector Safety and Efficacy
Authors: Zaiss Anne-Kathrin , Son Sodany , Chang Lung-Ji 
Published: July 2002

ABSTRACT
The expression of reporter genes driven by the same human elongation factor 1α (EF1α) promoter in murine leukemia virus (MLV)- and human immunodeficiency virus type 1 (HIV-1)-based vectors was studied in either transfected or virally transduced cells. The HIV-1 vectors consistently expressed 3 to 10 times higher activity than the MLV vectors at both the RNA and protein levels. The difference was not attributable to transcriptional interference, alternative enhancer/silencer, or differential EF1α intron splicing. Based on nuclear run-on assays, both vectors exhibited similar EF1α transcriptional activity. The reduced RNA levels of MLV vectors could not be explained by the decrease in RNA half-lives. Southern analysis of proviral DNA indicated that both HIV-1 and MLV vectors efficiently propagated the EF1α intron in the transduced cells. To decipher the discrepancy in transgene expression between MLV and HIV-1 vectors, the role of RNA 3′-end processing was examined using a sensitive Cre/lox reporter assay. The results showed that MLV vectors, but not HIV-1 vectors, displayed high frequencies of readthrough of the 3′ polyadenylation signal. Interestingly, the polyadenylation signal of a self-inactivating (SIN) HIV-1 vector was as leaky as that of the MLV vectors, suggesting a potential risk of oncogene activation by the lentiviral SIN vectors. Together, our results suggest that an efficient polyadenylation signal would improve both the efficacy and the safety of these vectors.
Characterization of Varicella-Zoster Virus Gene 21 and 29 Proteins in Infected Cells
Authors: Cohrs Randall J. , Wischer Jeanne , Essman Carrie , Gilden Donald H. 
Published: July 2002

ABSTRACT

Varicella-zoster virus (VZV) transcription is limited in latently infected human ganglia. Note that much of the transcriptional capacity of the virus genome has not been analyzed in detail; to date, only VZV genes mapping to open reading frames (ORFs) 4, 21, 29, 62, and 63 have been detected. ORF 62 encodes the major immediate-early virus transcription transactivator IE62, ORF 29 encodes the major virus DNA binding protein, and ORF 21 encodes a protein associated with the developing virus nucleocapsid. We analyzed the cellular location of proteins encoded by ORF 21 (21p) and ORF 29 (29p), their phosphorylation state during productive infection, and their ability form a protein-protein complex. The locations of both 21p and 29p within infected cells mimic those of their herpes simplex virus type 1 (HSV-1) homologues (UL37 and ICP8); however, unlike these homologues, 21p is not phosphorylated and neither 21p nor 29p exhibits a protein-protein interaction. Transient transfection assays to determine the effect of 21p and 29p on transcription from VZV gene 20, 21, 28, and 29 promoters revealed no significant activation of transcription by 21p or 29p from any of the VZV gene promoters tested, and 21p did not significantly modulate the ability of IE62 to activate gene transcription. A modest increase in IE62-induced activation of gene 28 and 29 promoters was seen in the presence of 29p; however, IE62-induced activation of gene 28 and 29 promoters was reduced in the presence of 21p. A
Saccharomyces cerevisiae
two-hybrid analysis of 21p indicated that the protein can activate transcription when tethered within a responsive promoter. Together, the data reveal that while VZV gene 21 and HSV-1 UL37 share homology at the nucleic acid level, these proteins differ functionally.
Human Immunodeficiency Virus-Specific CD8+T Cells in Human Breast Milk
Authors: Sabbaj Steffanie , Edwards Bradley H. , Ghosh Mrinal K. , Semrau Katherine , Cheelo Sanford , Thea Donald M. , Kuhn Louise , Ritter G. Douglas , Mulligan Mark J. , Goepfert Paul A. , Aldrovandi Grace M. 
Published: July 2002

ABSTRACTBreast-feeding infants of human immunodeficiency virus (HIV)-infected women ingest large amounts of HIV, but most escape infection. While the factors affecting transmission risk are poorly understood, HIV-specific cytotoxic T-lymphocyte (CTL) responses play a critical role in controlling HIV levels in blood. We therefore investigated the ability of breast milk cells (BMC) from HIV-infected women from the United States and Zambia to respond to HIV-1 peptides in a gamma interferon enzyme-linked immunospot assay. All (n= 11) HIV-infected women had responses to pools of Gag peptide (range, 105 to 1,400 spot-forming cells/million; mean = 718), 8 of 11 reacted to Pol, 7 reacted to Nef, and 2 of 5 reacted to Env. Conversely, of four HIV-negative women, none responded to any of the tested HIV peptide pools. Depletion and tetramer staining studies demonstrated that CD8+T cells mediated these responses, and a chromium-release assay showed that these BMC were capable of lysing target cells in an HIV-specific manner. These data demonstrate the presence of HIV-specific major histocompatibility complex class I-restricted CD8+CTLs in breast milk. Their presence suggests a role in limiting transmission and provides a rationale for vaccine strategies to enhance these responses.
Addition of Exogenous Protease Facilitates Reovirus Infection in Many Restrictive Cells
Authors: Golden Joseph W. , Linke Jessica , Schmechel Stephen , Thoemke Kara , Schiff Leslie A. 
Published: July 2002

ABSTRACTVirion uncoating is a critical step in the life cycle of mammalian orthoreoviruses. In cell culture, and probably in extraintestinal tissues in vivo, reovirus virions undergo partial proteolysis within endosomal or/or lysosomal compartments. This process converts the virion into a form referred to as an intermediate subvirion particle (ISVP). In natural enteric reovirus infections, proteolytic uncoating takes place extracellularly within the intestinal lumen. The resultant proteolyzed particles, unlike intact virions, have the capacity to penetrate cell membranes and thereby gain access to cytoplasmic components required for viral gene expression. We hypothesized that the capacity of reovirus outer capsid proteins to be proteolyzed is a determinant of cellular host range. To investigate this hypothesis, we asked if the addition of protease to cell culture medium would expand the range of cultured mammalian cell lines that can be productively infected by reoviruses. We identified many transformed and nontransformed cell lines, as well as primary cells, that restrict viral infection. In several of these restrictive cells, virion uncoating is inefficient or blocked. Addition of proteases to the cell culture medium generates ISVP-like particles and promotes viral growth in nearly all cell lines tested. Interestingly, we found that some cell lines that restrict reovirus uncoating still express mature cathepsin L, a lysosomal protease required for virion disassembly in murine L929 cells. This finding suggests that factors in addition to cathepsin L are required for efficient intracellular proteolysis of reovirus virions. Our results demonstrate that virion uncoating is a critical determinant of reovirus cellular host range and that many cells which otherwise support productive reovirus infection cannot efficiently mediate this essential early step in the virus life cycle.
Intersubtype Human Immunodeficiency Virus Type 1 Superinfection following Seroconversion to Primary Infection in Two Injection Drug Users
Authors: Ramos Artur , Hu Dale J. , Nguyen Lily , Phan Kim-Oanh , Vanichseni Suphak , Promadej Nattawan , Choopanya Kachit , Callahan Margaret , Young Nancy L. , McNicholl Janet , Mastro Timothy D. , Folks Thomas M. , Subbarao Shambavi 
Published: July 2002

ABSTRACT
In this study, we describe two cases of human immunodeficiency virus type 1 (HIV-1) intersubtype superinfection with CRF01_AE and subtype B strains, which occurred in two injection drug users participating in a prospective cohort study in Bangkok, Thailand. In both cases, the superinfecting strain was detected by molecular and serologic analyses several weeks after complete seroconversion to the primary infection with a strain belonging to a different subtype. Superinfection occurred despite specific T-cell and humoral antibody responses to the primary virus. In both cases, cross-subtype immune responses were limited or absent prior to the second infection. These data show that, in some individuals, the quality and quantity of the immune response elicited by primary HIV-1 infection may not protect against superinfection. This finding has important implications for vaccine design. HIV-1 vaccines, at a minimum, will need to include potent, broadly protective, conserved immunogens derived from several group M subtypes.
Identification of an Upstream Sequence Element Required for Vesicular Stomatitis Virus mRNA Transcription
Authors: Hinzman Edward E. , Barr John N. , Wertz Gail W. 
Published: July 2002

ABSTRACT

Vesicular stomatitis virus (VSV), the prototypic rhabdovirus, has a nonsegmented negative-sense RNA genome with five genes flanked by 3′ leader and 5′ trailer sequences. Transcription of VSV mRNAs is obligatorily sequential, starting from a single 3′ polymerase entry site, and termination of an upstream mRNA is essential for transcription of a downstream gene.
cis
-acting signals for transcription of VSV mRNAs are present within the leader region, at the leader-N junction, and at the internal gene junctions. The gene junctions of VSV consist of a conserved 23-nucleotide region that includes the gene end sequence of the upstream gene, 3′-AUACU7-5′, a nontranscribed intergenic dinucleotide, 3′-G/CA-5′, and the gene start sequence, 3′-UUGUCNNUAG-5′, at the beginning of the gene immediately downstream. Previous work has shown that the gene end sequence and intergenic region are sufficient to signal polyadenylation and termination of VSV transcripts. Mutagenesis of the gene start sequence has determined the importance of this region in the processes of initiation and 5′-end modification of mRNAs. However, because the gene end sequence is positioned directly upstream of the gene start sequence in the gene junction, and because of the requirement for termination of the upstream gene prior to transcription of the downstream gene, it has not been possible to investigate whether the gene end sequence contributes to transcription of the downstream gene. In this study, we inserted an additional gene end sequence upstream of the gene junction in a subgenomic replicon of VSV, which extended the intergenic region from 2 to 88 nucleotides. This duplication of termination signals allowed us to separate the signals required for termination from those required for initiation. We investigated the effect that the upstream gene end sequences had on downstream mRNA transcription. Our data show that the U7 tract of the upstream gene end sequence is necessary for optimal transcription of the downstream gene, independent of its role in termination of the upstream gene. Altering the sequence or changing the length of the U tract directly upstream of the gene start sequence significantly decreased transcription of the downstream gene. These results show that the U tract is a multifunctional region that is required not only for polyadenylation and termination of the upstream mRNA but also for efficient transcription of the downstream gene.
Oligomeric and Conformational Properties of a Proteolytically Mature, Disulfide-Stabilized Human Immunodeficiency Virus Type 1 gp140 Envelope Glycoprotein
Authors: Schülke Norbert , Vesanen Mika S. , Sanders Rogier W. , Zhu Ping , Lu Min , Anselma Deborah J. , Villa Anthony R. , Parren Paul W. H. I. , Binley James M. , Roux Kenneth H. , Maddon Paul J. , Moore John P. , Olson William C. 
Published: July 2002

ABSTRACT

We describe the further properties of a protein, designated SOS gp140, wherein the association of the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is stabilized by an intersubunit disulfide bond. HIV-1
JR-FL
SOS gp140, proteolytically uncleaved gp140 (gp140
UNC
), and gp120 were expressed in stably transfected Chinese hamster ovary cells and analyzed for antigenic and structural properties before and after purification. Compared with gp140
UNC
, SOS gp140 reacted more strongly in surface plasmon resonance and radioimmunoprecipitation assays with the neutralizing monoclonal antibodies (MAbs) 2G12 (anti-gp120), 2F5 (anti-gp41), and 17b (to a CD4-induced epitope that overlaps the CCR5-binding site). In contrast, gp140
UNC
displayed the greater reactivity with nonneutralizing anti-gp120 and anti-gp41 MAbs. Immunoelectron microscopy studies suggested a model for SOS gp140 wherein the gp41 ectodomain (gp41
ECTO
) occludes the “nonneutralizing” face of gp120, consistent with the antigenic properties of this protein. We also report the application of Blue Native polyacrylamide gel electrophoresis (BN-PAGE), a high-resolution molecular sizing method, to the study of viral envelope proteins. BN-PAGE and other biophysical studies demonstrated that SOS gp140 was monomeric, whereas gp140
UNC
comprised a mixture of noncovalently associated and disulfide-linked dimers, trimers, and tetramers. The oligomeric and conformational properties of SOS gp140 and gp140
UNC
were largely unaffected by purification. An uncleaved gp140 protein containing the SOS cysteine mutations (SOS gp140
UNC
) was also oligomeric. Surprisingly, variable-loop-deleted SOS gp140 proteins were expressed (although not yet purified) as cleaved, noncovalently associated oligomers that were significantly more stable than the full-length protein. Overall, our findings have relevance for rational vaccine design.
Similarities in the Induction of Post-Golgi Vesicles by the Vaccinia Virus F13L Protein and Phospholipase D
Authors: Husain Matloob , Moss Bernard 
Published: July 2002

ABSTRACT
Intracellular mature vaccinia virions are wrapped by cisternae, derived from virus-modified trans-Golgi or endosomal membranes, and then transported via microtubules to the cell periphery. Two viral proteins, encoded by the F13L and B5R open reading frames, are essential for the membrane-wrapping step. Previous transfection studies indicated that F13L induces the formation of post-Golgi vesicles that incorporate the B5R protein and that this activity depends on an intact F13L phospholipase motif. Here we show that the F13L protein has a general effect on the trafficking of integral membrane proteins from the Golgi apparatus, as both the vaccinia virus A36R protein and the vesicular stomatitis virus G protein also colocalized with the F13L protein in vesicles. In addition, increased expression of cellular phospholipase D, which has a similar phospholipase motif as, but little amino acid sequence identity with, F13L, induced post-Golgi vesicles that contained B5R and A36R proteins. Butanol-1, which prevents the formation of phosphatidic acid by phospholipase D and specifically inhibits phospholipase D-mediated vesicle formation, also inhibited F13L-induced vesicle formation, whereas secondary and tertiary alcohols had no effect. Moreover, inhibition of phospholipase activity by butanol-1 also reduced plaque size and decreased the formation of extracellular vaccinia virus without affecting the yield of intracellular mature virus. Phospholipase D, however, could not complement a vaccinia virus F13L deletion mutant, indicating that F13L has additional virus-specific properties. Taken together, these data support an important role for F13L in inducing the formation of vesicle precursors of the vaccinia virus membrane via phospholipase activity or activation.
A Chimeric Human T-Cell Lymphotropic Virus Type 1 with the Envelope Glycoprotein of Moloney Murine Leukemia Virus Is Infectious for Murine Cells
Authors: Delebecque Frédéric , Pramberger Karin , Prévost Marie-Christine , Brahic Michel , Tangy Frédéric 
Published: July 2002

ABSTRACT

We constructed a chimeric human T-cell lymphotropic virus type 1 (HTLV-1) provirus in which the original envelope precursor sequence was replaced by that of ecotropic Moloney murine leukemia virus (Mo-MuLV). Chimeric particles produced by transient transfection of this chimeric provirus were infectious for murine cells, such as NIH 3T3 fibroblasts, lymphoid EL4 cells, and primary CD4
+
T lymphocytes, whereas HTLV-1 particles were not. The infectivity of chimeric particles increased 10 times when the R peptide located at the carboxy terminus of the MuLV envelope glycoprotein was deleted. Primary murine CD4
+
T lymphocytes, infected by the ΔR chimeric virus, released particles that could spread the infection to other naive murine lymphoid cells. This chimeric virus, with the Mo-MuLV envelope glycoprotein and the replication characteristics of HTLV-1, should be useful in studying the pathogenesis of HTLV-1 in a mouse model.
Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Contains Two Functional Lytic Origins of DNA Replication
Authors: AuCoin David P. , Colletti Kelly S. , Xu Yiyang , Cei Sylvia A. , Pari Gregory S. 
Published: July 2002

ABSTRACT
We used a transient-transfection replication assay to identify two functional copies of the human herpesvirus 8 (HHV8) lytic origin of DNA replication (oriLyt). BCLB-1 cells were transfected with HHV8 subgenomic fragments containing the putative lytic origin along with a plasmid expressing viral transactivator open reading frame (ORF) 50. The HHV8 left-end oriLyt (oriLyt-L) lies between ORFs K4.2 and K5 and is composed of a region encoding various transcription factor binding sites and an A+T-rich region and a G+C repeat region. The right-end oriLyt (oriLyt-R) maps between ORF 69 and vFLIP, a region similar to the RRV oriLyt, and is an inverted duplication of oriLyt-L.
Comparative Sequence Analysis of the Largest E1A Proteins of Human and Simian Adenoviruses
Authors: Avvakumov Nikita , Wheeler Russ , D'Halluin Jean Claude , Mymryk Joe S. 
Published: July 2002

ABSTRACT

The early region 1A (
E1A
) gene is the first gene expressed after infection with adenovirus and has been most extensively characterized in human adenovirus type 5 (hAd5). The E1A proteins interact with numerous cellular regulatory proteins, influencing a variety of transcriptional and cell cycle events. For this reason, these multifunctional proteins have been useful as tools for dissecting pathways regulating cell growth and gene expression. Despite the large number of studies using hAd5 E1A, relatively little is known about the function of the E1A proteins of other adenoviruses. In 1985, a comparison of E1A sequences from three human and one simian adenovirus identified three regions with higher overall levels of sequence conservation designated conserved regions (CR) 1, 2, and 3. As expected, these regions are critical for a variety of E1A functions. Since that time, the sequences of several other human and simian adenovirus E1A proteins have been determined. Using these, and two additional sequences that we determined, we report here a detailed comparison of the sequences of 15 E1A proteins representing each of the six hAd subgroups and several simian adenoviruses. These analyses refine the positioning of CR1, 2, and 3; define a fourth CR located near the carboxyl terminus of E1A; and suggest several new functions for E1A.
Survey of Transcript Expression in Rainbow Trout Leukocytes Reveals a Major Contribution of Interferon-Responsive Genes in the Early Response to a Rhabdovirus Infection
Authors: O'Farrell Caroline , Vaghefi Nikta , Cantonnet Monique , Buteau Bénédicte , Boudinot Pierre , Benmansour Abdenour 
Published: July 2002

ABSTRACT
Virus infections induce changes in the expression of host cell genes. A global knowledge of these modifications should help to better understand the virus/host cell interactions. To obtain a more comprehensive view of the rainbow trout response to a viral infection, we used the subtractive suppressive hybridization methodology in the viral hemorrhagic septicemia model of infection. We infected rainbow trout leukocytes with viral hemorrhagic septicemia virus (VHSV), and total RNA from infected and mock-infected cells was compared at 40 h postinfection. Twenty-four virus-induced genes were ultimately retrieved from the subtracted cDNA library, and their differential expression was further confirmed by semiquantitative reverse transcription-PCR and Northern blot analysis. Among these sequences, three were already described as VHSV-induced genes. Eight sequences with known homologs were extended to full-length cDNA using 5′ and 3′ rapid amplification of cDNA ends, and they were subsequently divided into three functional subsets. Four genes were homologous to mammalian interferon responsive genes, three were similar to chemo-attractant molecules (CXC chemokine, galectin), and two had nucleic acid binding domains. All of the virus-induced genes were also induced by rainbow trout interferon, indicating that the interferon pathway is the predominant component of the anti-VHSV response. They were also expressed in vivo in experimentally infected fish, indicating their biological relevance in natural infection.