bluuebunny/crossref_metadata_2025_split huggingface dataset:
Concatamerization of Adeno-Associated Virus Circular Genomes Occurs through Intermolecular Recombination
Authors: Yang Jusan , Zhou Weihong , Zhang Yulong , Zidon Terese , Ritchie Terry , Engelhardt John F. 
Published: December 2019

ABSTRACT
Long-term recombinant AAV (rAAV) transgene expression in muscle has been associated with the molecular conversion of single-stranded rAAV genomes to high-molecular-weight head-to-tail circular concatamers. However, the mechanisms by which these large multimeric concatamers form remain to be defined. To this end, we tested whether concatamerization of rAAV circular intermediates occurs through intra- or intermolecular mechanisms of amplification. Coinfection of the tibialis muscle of mice with rAAV alkaline phosphatase (Alkphos)- and green fluorescent protein (GFP)-encoding vectors was used to evaluate the frequency of circular concatamer formation by intermolecular recombination of independent viral genomes. The GFP shuttle vector also encoded ampicillin resistance and contained a bacterial origin of replication to allow for bacterial rescue of circular intermediates from Hirt DNA of infected muscle samples. The results demonstrated a time-dependent increase in the abundance of rescued plasmids encoding both GFP and Alkphos, which reached 33% of the total circular intermediates by 120 days postinfection. Furthermore, these large circular concatamers were capable of expressing both GFP- and Alkphos-encoding transgenes following transient transfection in cell lines. These findings demonstrate that concatamerization of AAV genomes in vivo occurs through intermolecular recombination of independent monomer circular viral genomes and suggest new viable strategies for delivering multiple DNA segments at a single locus. Such developments will expand the utility of rAAV for splicing large gene inserts or large promoter-gene combinations carried by two or more independent rAAV vectors.
Inhibition of Cell-Free Human T-Cell Leukemia Virus Type 1 Infection at a Postbinding Step by the Synthetic Peptide Derived from an Ectodomain of the gp21 Transmembrane Glycoprotein
Authors: Jinno A. , Haraguchi Y. , Shiraki H. , Hoshino H. 
Published: December 2019

ABSTRACTTo investigate the roles of human T-cell leukemia virus type 1 (HTLV-1) envelope (Env) proteins gp46 and gp21 in the early steps of infection, the effects of the 23 synthetic peptides covering the entire Env proteins on transmission of cell-free HTLV-1 were examined by PCR and by the plaque assay using a pseudotype of vesicular stomatis virus (VSV) bearing the Env of HTLV-1 [VSV(HTLV-1)]. The synthetic peptide corresponding to amino acids 400 to 429 of the gp21 Env protein (gp21 peptide 400-429, Cys-Arg-Phe-Pro-Asn-Ile-Thr-Asn-Ser-His-Val-Pro-Ile-Leu-Gln-Glu-Arg-Pro-Pro-Leu-Glu-Asn-Arg-Val-Leu-Thr-Gly-Trp-Gly-Leu) strongly inhibited infection of cell-free HTLV-1. By using the mutant peptide, Asn407, Ser408, and Leu413, -419, -424, and -429 were confirmed to be important amino acids for neutralizing activity of the gp21 peptide 400-429. Addition of this peptide before or during adsorption of HTLV-1 at 4°C did not affect its entry. However, HTLV-1 infection was inhibited about 60% when the gp21 peptide 400-429 was added even 30 min after adsorption of HTLV-1 to cells, indicating that the amino acid sequence 400 to 429 on the gp21 Env protein plays an important role at the postbinding step of HTLV-1 infection. In contrast, a monoclonal antibody reported to recognize the gp46 191-196 peptide inhibited the infection of HTLV-1 at the binding step.
Translating Ribosomes Inhibit Poliovirus Negative-Strand RNA Synthesis
Authors: Barton David J. , Morasco B. Joan , Flanegan James B. 
Published: December 2019

ABSTRACT
Poliovirus has a single-stranded RNA genome of positive polarity that serves two essential functions at the start of the viral replication cycle in infected cells. First, it is translated to synthesize viral proteins and, second, it is copied by the viral polymerase to synthesize negative-strand RNA. We investigated these two reactions by using HeLa S10 in vitro translation-RNA replication reactions. Preinitiation RNA replication complexes were isolated from these reactions and then used to measure the sequential synthesis of negative- and positive-strand RNAs in the presence of different protein synthesis inhibitors. Puromycin was found to stimulate RNA replication overall. In contrast, RNA replication was inhibited by diphtheria toxin, cycloheximide, anisomycin, and ricin A chain. Dose-response experiments showed that precisely the same concentration of a specific drug was required to inhibit protein synthesis and to either stimulate or inhibit RNA replication. This suggested that the ability of these drugs to affect RNA replication was linked to their ability to alter the normal clearance of translating ribosomes from the input viral RNA. Consistent with this idea was the finding that the protein synthesis inhibitors had no measurable effect on positive-strand synthesis in normal RNA replication complexes. In marked contrast, negative-strand synthesis was stimulated by puromycin and was inhibited by cycloheximide. Puromycin causes polypeptide chain termination and induces the dissociation of polyribosomes from mRNA. Cycloheximide and other inhibitors of polypeptide chain elongation “freeze” ribosomes on mRNA and prevent the normal clearance of ribosomes from viral RNA templates. Therefore, it appears that the poliovirus polymerase was not able to dislodge translating ribosomes from viral RNA templates and mediate the switch from translation to negative-strand synthesis. Instead, the initiation of negative-strand synthesis appears to be coordinately regulated with the natural clearance of translating ribosomes to avoid the dilemma of ribosome-polymerase collisions.
Efficient Processing of the Immunodominant, HLA-A*0201-Restricted Human Immunodeficiency Virus Type 1 Cytotoxic T-Lymphocyte Epitope despite Multiple Variations in the Epitope Flanking Sequences
Authors: Brander Christian , Yang Otto O. , Jones Norman G. , Lee Yun , Goulder Philip , Johnson R. Paul , Trocha Alicja , Colbert David , Hay Christine , Buchbinder Susan , Bergmann Cornelia C. , Zweerink Hans J. , Wolinsky Steven , Blattner William A. , Kalams Spyros A. , Walker Bruce D. 
Published: December 2019

ABSTRACTImmune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.
Evolution of the Human Immunodeficiency Virus Type 1 Long Terminal Repeat Promoter by Conversion of an NF-κB Enhancer Element into a GABP Binding Site
Authors: Verhoef Koen , Sanders Rogier W. , Fontaine Veronique , Kitajima Shigetaka , Berkhout Ben 
Published: December 2019

ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) transcription is regulated by the viral Tat protein and cellular factors, of which the concentration and activity may depend on the cell type. Viral long terminal repeat (LTR) promoter sequences are therefore optimized to suit the specific nuclear environment of the target host cell. In long-term cultures of a Tat-defective, poorly replicating HIV-1 mutant, we selected for a faster-replicating virus with a 1-nucleotide deletion in the upstream copy of two highly conserved NF-κB binding sites. The variant enhancer sequence demonstrated a severe loss of NF-κB binding in protein binding assays. Interestingly, we observed a new binding activity that is specific for the variant NF-κB sequence and is present in the nuclear extract of unstimulated cells that lack NF-κB. These results suggest that inactivation of the NF-κB site coincides with binding of another transcription factor. Fine mapping of the sequence requirements for binding of this factor revealed a core sequence similar to that of Ets binding sites, and supershift assays with antibodies demonstrated the involvement of the GABP transcription factor. Transient transfection experiments with LTR-chloramphenicol acetyltransferase constructs indicated that the variant LTR promoter is specifically inhibited by GABP in the absence of Tat, but this promoter was dramatically more responsive to Tat than the wild-type LTR. Introduction of this GABP site into the LAI virus yielded a specific gain of fitness in SupT1 cells, which contain little NF-κB protein. These results suggest that GABP potentiates Tat-mediated activation of LTR transcription and viral replication in some cell types. Conversion of an NF-κB into a GABP binding site is likely to have occurred also during the worldwide spread of HIV-1, as we noticed the same LTR modification in subtype E isolates from Thailand. This typical LTR promoter configuration may provide these viruses with unique biological properties.
Gamma Interferon Is a Major Suppressive Factor Produced by Activated Human Peripheral Blood Lymphocytes That Is Able To Inhibit Foamy Virus-Induced Cytopathic Effects
Authors: Falcone Valeria , Schweizer Matthias , Toniolo Antonio , Neumann-Haefelin Dieter , Meyerhans Andreas 
Published: December 2019

ABSTRACTThe activation of human peripheral blood lymphocytes by mitogens or by triggering the T-cell receptor with anti-CD3 antibodies leads to the production of a potent soluble inhibitory activity against foamy virus-induced cytopathic effects in vitro. The inhibitory activity acts in a species-specific manner. As a consequence, the isolation of foamy viruses from blood lymphocytes of infected humans is accelerated in a heterologous coculture system. Antibodies against gamma interferon (IFN-γ) are able to suppress most of the inhibitory activity, suggesting that IFN-γ is the dominant component.
A Herpes Simplex Virus Type 1 Latency-Associated Transcript Mutant with Increased Virulence and Reduced Spontaneous Reactivation
Authors: Perng Guey-Chuen , Slanina Susan M. , Yukht Ada , Drolet Barbara S. , Keleher William , Ghiasi Homayon , Nesburn Anthony B. , Wechsler Steven L. 
Published: December 2019

ABSTRACT

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation of HSV-1 from latency. We previously reported that insertion of the LAT promoter and just the first 1.5 kb of the 8.3-kb LAT gene into an ectopic location in the virus restored wild-type spontaneous reactivation to a LAT null mutant. This mutant, LAT3.3A (previously designated LAT1.5a), thus showed that the expression of just the first 1.5 kb of LAT is sufficient for wild-type spontaneous reactivation. We also showed that in the context of the entire LAT gene, deletion of LAT nucleotides 76 to 447 (LAT mutant
d
LAT371) had no effect on spontaneous reactivation or virulence. We report here on a LAT mutant designated LAT2.9A. This mutant is similar to LAT3.3A, except that the ectopic LAT insert contains the same 371-nucleotide deletion found in
d
LAT371. We found that LAT2.9A had a significantly reduced rate of spontaneous reactivation compared to marker-rescued and wild-type viruses. This was unexpected, since the combined results of
d
LAT371 and LAT3.3A predicted that spontaneous reactivation of LAT2.9A would be wild type. We also found that LAT2.9A was more virulent than wild-type or marker-rescued viruses after ocular infection of rabbits. This was unexpected, since LAT null mutants and LAT3.3A have wild-type virulence. These results suggest for the first time (i) that regions past the first 1.5 kb of LAT can compensate for deletions in the first 1.5kb of LAT and may therefore play a role in LAT dependent spontaneous reactivation and (ii) that regions of LAT affect viral virulence.
Intracellular Redistribution of Truncated La Protein Produced by Poliovirus 3C
pro
-Mediated Cleavage
Authors: Shiroki Kazuko , Isoyama Takeshi , Kuge Shusuke , Ishii Toshihiko , Ohmi Shinobu , Hata Syoji , Suzuki Koichi , Takasaki Yoshinari , Nomoto Akio 
Published: December 2019

ABSTRACT

The La autoantigen (also known as SS-B), a cellular RNA binding protein, may shuttle between the nucleus and cytoplasm, but it is mainly located in the nucleus. La protein is redistributed to the cytoplasm after poliovirus infection. An in vitro translation study demonstrated that La protein stimulated the internal initiation of poliovirus translation. In the present study, a part of the La protein was shown to be cleaved in poliovirus-infected HeLa cells, and this cleavage appeared to be mediated by poliovirus-specific protease 3C (3C
pro
). Truncated La protein (dl-La) was produced in vitro from recombinant La protein by cleavage with purified 3C
pro
at only one Gln
358
-Gly
359
peptide bond in the 408-amino-acid (aa) sequence of La protein. The dl-La expressed in L cells was detected in the cytoplasm. However, green fluorescence protein linked to the C-terminal 50-aa sequence of La protein was localized in the nucleus, suggesting that this C-terminal region contributes to the steady-state nuclear localization of the intact La protein in uninfected cells. The dl-La retained the enhancing activity of translation initiation driven by poliovirus RNA in rabbit reticulocyte lysates. These results suggest that La protein is cleaved by 3C
pro
in the course of poliovirus infection and that the dl-La is redistributed to the cytoplasm. dl-La, as well as La protein, may play a role in stimulating the internal initiation of poliovirus translation in the cytoplasm.
V3 Recombinants Indicate a Central Role for CCR5 as a Coreceptor in Tissue Infection by Human Immunodeficiency Virus Type 1
Authors: Chan Stephen Y. , Speck Roberto F. , Power Christopher , Gaffen Sarah L. , Chesebro Bruce , Goldsmith Mark A. 
Published: December 2019

ABSTRACTBinding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 to both CD4 and one of several chemokine receptors (coreceptors) permits entry of virus into target cells. Infection of tissues may establish latent viral reservoirs as well as cause direct pathologic effects that manifest as clinical disease such as HIV-associated dementia. We sought to identify the critical coreceptors recognized by HIV-1 tissue-derived strains as well as to correlate these coreceptor preferences with site of infection and dementia diagnosis. To reconstitute coreceptor use, we cloned HIV-1 envelope V3 sequences encoding the primary determinants of coreceptor specificity from 13 brain-derived and 6 colon-derived viruses into an isogenic (NL4-3) viral background. All V3 recombinants utilized the chemokine receptor CCR5 uniformly and efficiently as a coreceptor but not CXCR4, BOB/GPR15, or Bonzo/STRL33. Other receptors such as CCR3, CCR8, and US28 were inefficiently and variably used as coreceptors by various envelopes. CCR5 without CD4 present did not allow for detectable infection by any of the tested recombinants. In contrast to the pathogenic switch in coreceptor specificity frequently observed in comparisons of blood-derived viruses early after HIV-1 seroconversion and after onset of AIDS, the characteristics of these V3 recombinants suggest that CCR5 is a primary coreceptor for brain- and colon-derived viruses regardless of tissue source or diagnosis of dementia. Therefore, tissue infection may not depend significantly on viral envelope quasispeciation to broaden coreceptor range but rather selects for CCR5 use throughout disease progression.
CCR2-64I Polymorphism Is Not Associated with Altered CCR5 Expression or Coreceptor Function
Authors: Mariani Roberto , Wong Sally , Mulder Lubbertus C. F. , Wilkinson David A. , Reinhart Amy L. , LaRosa Gregory , Nibbs Robert , O’Brien Thomas R. , Michael Nelson L. , Connor Ruth I. , Macdonald Marcy , Busch Michael , Koup Richard A. , Landau Nathaniel R. 
Published: December 2019

ABSTRACTA polymorphism in the gene encoding CCR2 is associated with a delay in progression to AIDS in human immunodeficiency virus (HIV)-infected individuals. The polymorphism,CCR2-64I, changes valine 64 of CCR2 to isoleucine. However, it is not clear whether the effect on AIDS progression results from the amino acid change or whether the polymorphism marks a genetically linked, yet unidentified mutation that mediates the effect. Because the gene encoding CCR5, the major coreceptor for HIV type 1 primary isolates, lies 15 kb 3′ toCCR2, linked mutations in theCCR5promoter or other regulatory sequences could explain the association ofCCR2-64I with slowed AIDS pathogenesis. Here, we show thatCCR2-64I is efficiently expressed on the cell surface but does not have dominant negative activity on CCR5 coreceptor function. A panel of peripheral blood mononuclear cells (PBMC) from uninfected donors representing the variousCCR5/CCR2genotypes was assembled. Activated primary CD4+T cells ofCCR264I/64I donors expressed cell surface CCR5 at levels comparable to those ofCCR2+/+ donors. A slight reduction in CCR5 expression was noted, although this was not statistically significant.CCR5andCCR2mRNA levels were nearly identical for each of the donor PBMC, regardless of genotype. Cell surface CCR5 and CCR2 levels were more variable than mRNA transcript levels, suggesting that an alternative mechanism may influence CCR5 cell surface levels.CCR2-64I is linked to theCCR5promoter polymorphisms 208G, 303A, 627C, and 676A; however, in transfected promoter reporter constructs, these did not affect transcriptional activity. Taken together, these findings suggest thatCCR2-64I does not act by influencingCCR5transcription or mRNA levels.
In Vivo and In Vitro Analysis of Baculovirus
ie-2
Mutants
Authors: Prikhod’ko Elena A. , Lu Albert , Wilson Joyce A. , Miller Lois K. 
Published: December 2019

ABSTRACT

Upon transient expression in cell culture, the
ie-2
gene of
Autographa californica
nuclear polyhedrosis virus (Ac
M
NPV) displays three functions:
trans
activation of viral promoters, direct or indirect stimulation of virus origin-specific DNA replication, and arrest of the cell cycle. The ability of IE2 to
trans
stimulate DNA replication and coupled late gene expression is observed in a cell line derived from
Spodoptera frugiperda
but not in a cell line derived from
Trichoplusia ni
. This finding suggested that IE-2 may exert cell line-specific or host-specific effects. To examine the role of
ie-2
in the context of infection and its possible influence on the host range, we constructed recombinants of Ac
M
NPV containing deletions of different functional regions within
ie-2
and characterized them in cell lines and larvae of
S. frugiperda
and
T. ni
. The
ie-2
mutant viruses exhibited delays in viral DNA synthesis, late gene expression, budded virus production, and occlusion body formation in SF-21 cells but not in TN-5B1-4 cells. In TN-5B1-4 cells, the
ie-2
mutants produced more budded virus and fewer occlusion bodies but the infection proceeded without delay. Examination of the effects of
ie-2
and the respective mutants on immediate-early viral promoters in transient expression assays revealed striking differences in the relative levels of expression and differences in responses to
ie-2
and its mutant forms in different cell lines. In
T. ni
and
S. frugiperda
larvae, the infectivities of the occluded form of
ie-2
mutant viruses by the normal oral route of infection was 100- and 1,000-fold lower, respectively, than that of wild-type Ac
M
NPV. The reduction in oral infectivity was traced to the absence of virions within the occlusion bodies. The infectivity of the budded form of
ie-2
mutants by hemocoelic injection was similar to that of wild-type virus in both species. Thus,
ie-2
mutants are viable but exhibit cell line-specific effects on temporal regulation of the infection process. Due to its effect on virion occlusion, mutants of IE-2 were essentially noninfectious by the normal route of infection in both species tested. However, since budded viruses exhibited normal infectivity upon hemocoelic injection, we conclude that
ie-2
does not affect host range per se. The possibility that IE-2 exerts tissue-specific effects has not been ruled out.
Development of Animal Models for Adeno-Associated Virus Site-Specific Integration
Authors: Rizzuto Gabriella , Gorgoni Barbara , Cappelletti Manuela , Lazzaro Domenico , Gloaguen Isabelle , Poli Valeria , Sgura Antonella , Cimini Daniela , Ciliberto Gennaro , Cortese Riccardo , Fattori Elena , La Monica Nicola 
Published: December 2019

ABSTRACT

The adeno-associated virus (AAV) is unique in its ability to target viral DNA integration to a defined region of human chromosome 19 (AAVS1). Since AAVS1 sequences are not conserved in a rodent’s genome, no animal model is currently available to study AAV-mediated site-specific integration. We describe here the generation of transgenic rats and mice that carry the AAVS1 3.5-kb DNA fragment. To test the response of the transgenic animals to Rep-mediated targeting, primary cultures of mouse fibroblasts, rat hepatocytes, and fibroblasts were infected with wild-type wt AAV. PCR amplification of the inverted terminal repeat (ITR)-AAVS1 junction revealed that the AAV genome integrated into the AAVS1 site in fibroblasts and hepatocytes. Integration in rat fibroblasts was also observed upon transfection of a plasmid containing the
rep
gene under the control of the p5 and p19 promoters and a dicistronic cassette carrying the green fluorescent protein (GFP) and neomycin (
neo
) resistance gene between the ITRs of AAV. The localization of the GFP-Neo sequence in the AAVS1 region was determined by Southern blot and FISH analysis. Lastly, AAV genomic DNA integration into the AAVS1 site in vivo was assessed by virus injection into the quadriceps muscle of transgenic rats and mice. Rep-mediated targeting to the AAVS1 site was detected in several injected animals. These results indicate that the transgenic lines are proficient for Rep-mediated targeting. These animals should allow further characterization of the molecular aspects of site-specific integration and testing of the efficacy of targeted integration of AAV recombinant vectors designed for human gene therapy.