bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heparin Binds to Murine Leukemia Virus and Inhibits Env-Independent Attachment and Infection
Authors: Walker Simon J. , Pizzato Massimo , Takeuchi Yasuhiro , Devereux Stephen 
Published: July 2002

ABSTRACTCertain glycosaminoglycans (GAGs), including heparin, inhibit infection by murine leukemia virus (MLV). We now show that this is due to inhibition of virus attachment independent of the interaction between viral envelope proteins (Env) and their cellular receptors. Heparin blocked the binding of both Env-deficient and amphotropic MLV (MLV-A) particles to NIH 3T3 fibroblasts, CHO cells which lack the amphotropic retroviral receptor Pit-2, and CHO cells transfected with Pit-2 (CHO-Pit-2). Heparin also inhibited the transduction of NIH 3T3 cells by MLV-A over a similar concentration range. This effect was observed within 15 min of exposure to retrovirus. Preloading target cells with heparin had no effect on transduction and both MLV-A and Env-deficient retrovirus bound efficiently to heparin-coated agarose beads, suggesting that heparin interacts with the virus rather than the target cell. This requires both a strong negative charge and a specific structure since GAGs with different charge and carbohydrate composition inhibited virus infection variably. The specificity of GAG-virus interaction also depends on the producer cells, since virus packaged by murine GP+EnvAM12 cells was 1,000-fold more sensitive to inhibition by chondroitin sulfate A than was virus packaged by human FLYA13 packaging cells. No evidence for an interaction between MLV and cell surface proteoglycans was found, however, since the attachment of MLV-A and envelope-defective virus to proteoglycan-deficient CHOpgsA-745 cells was similar to that seen with both wild-type and CHO-Pit-2 cells. Although the molecular mechanism is unclear, this study presents evidence that Env receptor-independent attachment is an important step in MLV infection.
Template Requirements for De Novo RNA Synthesis by Hepatitis C Virus Nonstructural Protein 5B Polymerase on the Viral X RNA
Authors: Kim Meehyein , Kim Hajeong , Cho Seong-Pil , Min Mi-Kyung 
Published: July 2002

ABSTRACT

The hepatitis C virus (HCV)-encoded NS5B protein is an RNA-dependent RNA polymerase which plays a substantial role in viral replication. We expressed and purified the recombinant NS5B of an HCV genotype 3a from
Esherichia coli
, and we investigated its ability to bind to the viral RNA and its enzymatic activity. The results presented here demonstrate that NS5B interacts strongly with the coding region of positive-strand RNA, although not in a sequence-specific manner. It was also determined that more than two molecules of polymerase bound sequentially to this region with the direction 3′ to 5′. Also, we attempted to determine the initiation site(s) of de novo synthesis by NS5B on X RNA, which contains the last 98 nucleotides of HCV positive-strand RNA. The initiation site(s) on X RNA was localized in the pyrimidine-rich region of stem I. However, when more than five of the nucleotides of stem I in X RNA were deleted from the 3′ end, RNA synthesis initiated at another site of the specific ribonucleotide. Our study also showed that the efficiency of RNA synthesis, which was directed by X RNA, was maximized by the GC base pair at the penultimate position from the 3′ end of the stem. These results will provide some clues to understanding the mechanism of HCV genomic RNA replication in terms of viral RNA-NS5B interaction and the initiation of de novo RNA synthesis.
Coreceptor Phenotype of Natural Human Immunodeficiency Virus with Nef Deleted Evolves In Vivo, Leading to Increased Virulence
Authors: Jekle Andreas , Schramm Birgit , Jayakumar Prerana , Trautner Verena , Schols Dominique , De Clercq Erik , Mills John , Crowe Suzanne M. , Goldsmith Mark A. 
Published: July 2002

ABSTRACT

The Sydney Blood Bank Cohort is a group of patients with slowly progressive infection by a human immunodeficiency virus strain containing spontaneous deletions within the
nef
long terminal repeat region. In 1999, 18 years after the initial infection, one of the members (D36) developed AIDS. In this work, we used an ex vivo human lymphoid cell culture system to analyze two viral isolates obtained from this patient, one prior to the onset of AIDS in 1995 and one after disease progression in 1999. Both D36 isolates were less potent in depleting CD4
+
T cells than a reference dualtropic,
nef
-bearing viral isolate. However, the 1999 isolate was measurably more cytotoxic to CD4
+
T cells than the 1995 isolate. Interestingly, although both isolates were nearly equally potent in depleting CCR5
+
CD4
+
T cells, the cytotoxic effect of the 1999 isolate toward CCR5
−
CD4
+
T cells was significantly higher. Furthermore, GHOST cell infection assays and blocking experiments with the CXCR4 inhibitor AMD3100 showed that the later D36 1999 isolate could infect both CCR5
+
and CCR5
−
CXCR4
+
cells efficiently, while infection by the 1995 isolate was nearly completely restricted to CCR5
+
cells. Sequence analysis of the V1/V2 and V3 regions of the viral envelope protein gp120 revealed that the more efficient CXCR4 usage of the later isolate might be caused by an additional potential N-glycosylation site in the V1/V2 loop. In conclusion, these data show that an in vivo evolution of the tropism of this
nef
-deleted strain toward an X4 phenotype was associated with a higher cytopathic potential and progression to AIDS.
Origin of Human Immunodeficiency Virus Type 1 Quasispecies Emerging after Antiretroviral Treatment Interruption in Patients with Therapeutic Failure
Authors: Kijak Gustavo H. , Simon Viviana , Balfe Peter , Vanderhoeven Jeroen , Pampuro Sandra E. , Zala Carlos , Ochoa Claudia , Cahn Pedro , Markowitz Martin , Salomon Horacio 
Published: July 2002

ABSTRACT

The emergence of antiretroviral (ARV) drug-resistant human immunodeficiency virus type 1 (HIV-1) quasispecies is a major cause of treatment failure. These variants are usually replaced by drug-sensitive ones when the selective pressure of the drugs is removed, as the former have reduced fitness in a drug-free environment. This was the rationale for the design of structured ARV treatment interruption (STI) studies for the management of HIV-1 patients with treatment failure. We have studied the origin of drug-sensitive HIV-1 quasispecies emerging after STI in patients with treatment failure due to ARV drug resistance. Plasma and peripheral blood mononuclear cell samples were obtained the day of treatment interruption (day 0) and 30 and 60 days afterwards. HIV-1
pol
and
env
were partially amplified, cloned, and sequenced. At day 60 drug-resistant variants were replaced by completely or partially sensitive quasispecies. Phylogenetic analyses of
pol
revealed that drug-sensitive variants emerging after STI were not related to their immediate temporal ancestors but formed a separate cluster, demonstrating that STI leads to the recrudescence and reemergence of a sequestrated viral population rather than leading to the back mutation of drug-resistant forms. No evidence for concomitant changes in viral tropism was seen, as deduced from
env
sequences. This study demonstrates the important role that the reemergence of quasispecies plays in HIV-1 population dynamics and points out the difficulties that may be found when recycling ARV therapies with patients with treatment failure.
Activity of Polymerase Proteins of Vaccine and Wild-Type Measles Virus Strains in a Minigenome Replication Assay
Authors: Bankamp Bettina , Kearney Sean P. , Liu Xin , Bellini William J. , Rota Paul A. 
Published: July 2002

ABSTRACTThe relative activities of five measles virus (MV) polymerase (L) proteins were compared in an intracellular, plasmid-based replication assay. When coexpressed with N and P proteins from an attenuated strain, L proteins from two attenuated viruses directed the production of up to eight times more reporter protein from an MV minigenome than the three wild-type L proteins. Northern blot analysis demonstrated that the differences in reporter protein production correlated with mRNA transcription levels. Increased activity of polymerases from attenuated viruses equally affected mRNA transcription and minigenome replication. The higher level of transcription may be a consequence of increased template availability or may be an independent effect of the elevated activity of the attenuated polymerases. Coexpression of wild-type L proteins with homologous N and P proteins did not affect the activity of the wild-type polymerases, indicating that the differential activity was a function of the L proteins alone. Use of a minigenome that incorporated two nucleotide changes found in the genomic leader of the three wild-type viruses did not raise the activity of the wild-type L proteins. These data demonstrate that increased polymerase activity differentiates attenuated from wild-type viruses and suggest that functions involved in RNA synthesis contribute to the attenuated phenotype of MV vaccine strains.
The RNA Polymerase of Influenza A Virus Is Stabilized by Interaction with Its Viral RNA Promoter
Authors: Brownlee George G. , Sharps Jane L. 
Published: July 2002

ABSTRACT
The RNA polymerase of the influenza virus is responsible for the transcription and replication of the segmented RNA viral genome during infection of host cells. Polymerase function is known to be strictly dependent on interaction with its RNA promoter, but no attempts to investigate whether the virion RNA (vRNA) promoter stabilizes the polymerase have been reported previously. Here we tested whether the vRNA promoter protects the polymerase against heat inactivation. We prepared partially purified recombinant influenza A virus RNA polymerase, in the absence of influenza virus vRNA promoter sequences, by transient transfection of expression plasmids into human kidney 293T cells. The polymerase was found to be heat labile at 40°C in the absence of added vRNA. However, it was protected from heat inactivation if both the 5′ and 3′ strands of the vRNA promoter were present. By using the ability of vRNA to protect the enzyme against heat inactivation, we established a novel assay, in conjunction with a mutagenic approach, that was used to test the secondary structure requirement of the vRNA promoter for polymerase binding. Binding required a panhandle structure and the presence of local hairpin loop structures in both the 5′ and 3′ ends of vRNA, as suggested by the corkscrew model. The interaction of the vRNA promoter with the influenza virus RNA polymerase heterotrimeric complex is likely to favor a particular closed conformation of the complex, thereby ensuring the stability of the RNA polymerase within both the infected cell and the isolated virus.
Evidence for Segment-Nonspecific Packaging of the Influenza A Virus Genome
Authors: Bancroft Christa T. , Parslow Tristram G. 
Published: July 2002

ABSTRACT
The influenza A virus genome is composed of eight negative-sense RNA segments (called vRNAs), all of which must be packaged to produce an infectious virion. It is not clear whether individual vRNAs are packaged specifically or at random, however, and the total vRNA capacity of the virion is unknown. We have created modified forms of the viral nucleoprotein (NP), neuraminidase (NA), and nonstructural (NS) vRNAs that encode green or yellow fluorescent proteins and studied the efficiency with which these are packaged by using a plasmid-based influenza A virus assembly system. Packaging was assessed precisely and quantitatively by scoring transduction of the fluorescent markers in a single-round infectivity assay with a flow cytometer. We found that, under conditions in which virions are limiting, pairs of alternatively tagged vRNAs compete for packaging but do so in a nonspecific manner. Reporters representing different vRNAs were not packaged additively, as would be expected under specific packaging, but instead appeared to compete for a common niche in the virion. Moreover, 3 to 5% of transduction-competent viruses were found to incorporate two alternative reporters, regardless of whether those reporters represented the same or different vRNAs — a finding compatible with random, but not with specific, packaging. Probabilistic estimates suggest that in order to achieve this level of dual transduction by chance alone, each influenza A virus virion must package an average of 9 to 11 vRNAs.
Herpes Simplex Virus Type 1/Adeno-Associated Virus Hybrid Vectors Mediate Site-Specific Integration at the Adeno-Associated Virus Preintegration Site, AAVS1, on Human Chromosome 19
Authors: Heister Thomas , Heid Irma , Ackermann Mathias , Fraefel Cornel 
Published: July 2002

ABSTRACT

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have a large transgene capacity and can efficiently infect many different cell types. One disadvantage of HSV-1 vectors is their instability of transgene expression. By contrast, vectors based on adeno-associated virus (AAV) can either persist in an episomal form or integrate into the host cell genome, thereby supporting long-term gene expression. AAV expresses four
rep
genes,
rep68
, -
78
, -
40
, and -
52
. Of those,
rep68
or
rep78
are sufficient to mediate site-specific integration of the AAV DNA into the host cell genome. The major disadvantage of AAV vectors is the small transgene capacity (∼4.6 kb). In this study, we constructed HSV/AAV hybrid vectors that contained, in addition to the standard HSV-1 amplicon elements, AAV
rep68
,
rep78
, both
rep68
and -
78
, or all four
rep
genes and a reporter gene that was flanked by the AAV inverted terminal repeats (ITRs). Southern blots of Hirt DNA from cells transfected with the hybrid vectors and HSV-1 helper DNA demonstrated that both the AAV elements and the HSV-1 elements were functional in the context of the hybrid vector. All hybrid vectors could be packaged into HSV-1 virions, although those containing
rep
sequences had lower titers than vectors that did not. Site-specific integration at AAVS1 on human chromosome 19 was directly demonstrated by PCR and sequence analysis of ITR-AAVS1 junctions in hybrid vector-transduced 293 cells. Cell clones that stably expressed the transgene for at least 12 months could easily be isolated without chemical selection. In the majority of these clones, the transgene cassette was integrated at AAVS1, and no sequences outside the ITR cassette,
rep
in particular, were present as determined by PCR, ITR rescue/replication assays, and Southern analysis. Some of the clones contained random integrations of the transgene cassette alone or together with sequences outside the ITR cassette. These data indicate that the long-term transgene expression observed following transduction with HSV/AAV hybrid vectors is, at least in part, supported by chromosomal integration of the transgene cassette, both randomly and site specifically.
Disruption of Adherens Junctions Liberates Nectin-1 To Serve as Receptor for Herpes Simplex Virus and Pseudorabies Virus Entry
Authors: Yoon Miri , Spear Patricia G. 
Published: July 2002

ABSTRACT
Nectin-1, a cell adhesion molecule belonging to the immunoglobulin superfamily, can bind to virion glycoprotein D (gD) to mediate entry of herpes simplex viruses (HSV) and pseudorabies virus (PRV). Nectin-1 colocalizes with E-cadherin at adherens junctions in epithelial cells. The disruption of cell junctions can result in the redistribution of nectin-1. To determine whether disruption of junctions by calcium depletion influenced the susceptibility of epithelial cells to viral entry, Madin-Darby canine kidney cells expressing endogenous nectin-1 or transfected human nectin-1 were tested for the ability to bind soluble forms of viral gD and to be infected by HSV and PRV, before and after calcium depletion. Confocal microscopy revealed that binding of HSV and PRV gD was localized to adherens junctions in cells maintained in normal medium but was distributed, along with nectin-1, over the entire cell surface after calcium depletion. Both the binding of gD and the fraction of cells that could be infected by HSV-1 and PRV were enhanced by calcium depletion. Taken together, these results provide evidence that nectin-1 confined to adherens junctions in epithelial cells is not very accessible to virus, whereas dissociation of cell junctions releases nectin-1 to serve more efficiently as an entry receptor.
The EBNA2 Polyproline Region Is Dispensable for Epstein-Barr Virus-Mediated Immortalization Maintenance
Authors: Gordadze Alexey V. , Poston David , Ling Paul D. 
Published: July 2002

ABSTRACT
Epstein-Barr virus nuclear antigen 2 (EBNA2) is required for EBV-mediated immortalization of primary human B cells and is a direct transcriptional activator of viral and cellular genes. The prototype EBNA2 protein contains a unique motif in which 43 out of 45 amino acids are prolines (polyproline region [PPR]). Previous genetic analysis has shown that deletion of the PPR resulted in viruses unable to immortalize B cells, although the protein did appear transcriptionally functional (R. Yalamanchili, S. Harada, and E. Kieff, J. Virol. 70:2468-2473, 1996). The PPR's uniqueness and requirement for immortalization make it an attractive therapeutic target. However, the role of this highly unusual motif for immortalization remains enigmatic. We have recently developed a transcomplementation assay that allows both genetic and functional analyses of EBNA2 in EBV-mediated immortalization maintenance (A. V. Gordadze, R. Peng, J. Tan, G. Liu, R. Sutton, B. Kempkes, G. W. Bornkamm, and P. D. Ling, J. Virol. 75:5899-5912, 2001). Surprisingly, we found that ΔPPR-EBNA2 was able to support B-cell proliferation similar to that of wild-type EBNA2 in this assay, indicating that deletion of the PPR from EBNA2 does not result in a loss of function required for immortalization maintenance. Further analysis of this mutant EBNA2 revealed that it consistently activated the viral LMP1 and LMP2A promoters severalfold better than wild-type EBNA2 in transient cotransfection assays. In addition, one striking difference between lymphoblastoid cell lines expressing wild-type EBNA2 from those expressing ΔPPR-EBNA2 is that the latter cells have significantly reduced EBV genomic levels. The data are consistent with a model in which lower EBNA2 target gene dosage may be selected for in ΔPPR-EBNA2-dependent cell lines to compensate for hyperactive stimulation of viral genes, such as LMP-1, which is cytostatic for B cells when overexpressed. It is conceivable that the hyperactivity rather than the loss of function, as hypothesized previously, could be responsible for the inability of recombinant ΔPPR-EBNA2 EBVs to immortalize B cells.
Changes in Human Immunodeficiency Virus Type 1 Gag at Positions L449 and P453 Are Linked to I50V Protease Mutants In Vivo and Cause Reduction of Sensitivity to Amprenavir and Improved Viral Fitness In Vitro
Authors: Maguire Michael F. , Guinea Rosario , Griffin Philip , Macmanus Sarah , Elston Robert C. , Wolfram Josie , Richards Naomi , Hanlon Mary H. , Porter David J. T. , Wrin Terri , Parkin Neil , Tisdale Margaret , Furfine Eric , Petropoulos Chris , Snowden B. Wendy , Kleim Jörg-Peter 
Published: July 2002

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) Gag protease cleavage sites (CS) undergo sequence changes during the development of resistance to several protease inhibitors (PIs). We have analyzed the association of sequence variation at the p7/p1 and p1/p6 CS in conjunction with amprenavir (APV)-specific protease mutations following PI combination therapy with APV. Querying a central resistance data repository resulted in the detection of significant associations (
P
< 0.001) between the presence of APV protease signature mutations and Gag L449F (p1/p6 LP1′F) and P453L (p1/p6 PP5′L) CS changes. In population-based sequence analyses the I50V mutant was invariably linked to either L449F or P453L. Clonal analysis revealed that both CS mutations were never present in the same genome. Sequential plasma samples from one patient revealed a transition from I50V M46L P453L viruses at early time points to I50V M46I L449F viruses in later samples. Various combinations of the protease and Gag mutations were introduced into the HXB2 laboratory strain of HIV-1. In both single- and multiple-cycle assay systems and in the context of I50V, the L449F and P453L changes consistently increased the 50% inhibitory concentration of APV, while the CS changes alone had no measurable effect on inhibitor sensitivity. The decreased in vitro fitness of the I50V mutant was only partially improved by addition of either CS change (I50V M46I L449F mutant replicative capacity ≈ 16% of that of wild-type virus). Western blot analysis of Pr55 Gag precursor cleavage products from infected-cell cultures indicated accumulation of uncleaved Gag p1-p6 in all I50V viruses without coexisting CS changes. Purified I50V protease catalyzed cleavage of decapeptides incorporating the L449F or P453L change 10-fold and 22-fold more efficiently than cleavage of the wild-type substrate, respectively. HIV-1 protease CS changes are selected during PI therapy and can have effects on both viral fitness and phenotypic resistance to PIs.
Analysis of CD4
+
T-Cell Responses to Human Papillomavirus (HPV) Type 11 L1 in Healthy Adults Reveals a High Degree of Responsiveness and Cross-Reactivity with Other HPV Types
Authors: Williams O. Martin , Hart Keith W. , Wang Eddie C. Y. , Gelder Colin M. 
Published: July 2002

ABSTRACT

Human papillomavirus type 11 (HPV-11) infection causes genital warts and recurrent respiratory papillomatosis. While there is compelling evidence that CD4
+
T cells play an important role in immune surveillance of HPV-associated diseases, little is known about human CD4
+
T-cell recognition of HPV-11. We have investigated the CD4
+
T-cell responses of 25 unrelated healthy donors to HPV-11 L1 virus-like particles (VLP). CD4
+
T-cell lines from 21 of 25 donors were established. Cell sorting experiments carried out on cells from six donors demonstrated that the response was located in the CD45RA
low
CD45RO
high
memory T-cell population. To determine the peptide specificity of these responses, epitope selection was analyzed by using 95 15-mer peptides spanning the entire HPV-11 L1 protein. No single region of L1 was immunodominant; responders recognized between 1 and 10 peptides, located throughout the protein, and peptide responses fell into clear HLA class II restricted patterns. Panels of L1 peptides specific for skin and genital HPV were used to show that the L1 CD4
+
T-cell responses were cross-reactive. The degree of cross-reactivity was inversely related to the degree of L1 sequence diversity between these viruses. Finally, responses to HPV-11 L1 peptides were elicited from ex vivo CD45RO
+
peripheral blood mononuclear cells, demonstrating that recognition of HPV-11 was a specific memory response and not due to in vitro selection during tissue culture. This is the first study of CD4
+
T-cell responses to HPV-11 in healthy subjects and demonstrates marked cross-reactivity with other skin and genital HPV types. This cross-reactivity may be of significance for vaccine strategies against HPV-associated clinical diseases.