bluuebunny/crossref_metadata_2025 huggingface dataset:
Packaging Cells Based on Inducible Gene Amplification for the Production of Adeno-Associated Virus Vectors
Authors: Inoue Naoki , Russell David W.
Published: December 2019

Although vectors based on adeno-associated virus (AAV) offer several unique advantages, their usage has been hampered by the difficulties encountered in vector production. In this report, we describe a new AAV packaging system based on inducible amplification of integrated helper and vector constructs containing the simian virus 40 (SV40) replication origin. The packaging and producer cell lines developed express SV40 T antigen under the control of the reverse tetracycline transactivator system, which allows inducible amplification of chromosomal loci linked to the SV40 origin. Culturing these cells in the presence of doxycycline followed by adenovirus infection resulted in helper and vector gene amplification as well as higher vector titers. Clonal producer cell lines generated vector titers that were 10 times higher than those obtained by standard methods, with approximately 10
4
vector particles produced per cell. These stocks were free of detectable replication-competent virus. The lack of a transfection step combined with the reproducibility of stable producer lines makes this packaging method ideally suited for the large-scale production of vector stocks for human gene therapy.
Pathogenicity Induced by Feline Leukemia Virus, Rickard Strain, Subgroup A Plasmid DNA (pFRA)
Authors: Chen Hang , Bechtel Marta K. , Shi Yan , Phipps Andrew , Mathes Lawrence E. , Hayes Kathleen A. , Roy-Burman Pradip
Published: December 2019

A new provirus clone of feline leukemia virus (FeLV), which we named FeLV-A (Rickard) or FRA, was characterized with respect to viral interference group, host range, complete genome sequence, and in vivo pathogenicity in specific-pathogen-free newborn cats. The in vitro studies indicated the virus to be an ecotropic subgroup A FeLV with 98% nucleotide sequence homology to another FeLV-A clone (F6A/61E), which had also been fully sequenced previously. Since subgroup B polytropic FeLVs (FeLV-B) are known to arise via recombination between ecotropic FeLV-A and endogenous FeLV (enFeLV)
env
elements, the in vivo studies were conducted by direct intradermal inoculation of the FRA plasmid DNA so as to eliminate the possibility of coinoculation of any FeLV-B which may be present in the inoculum prepared by propagating FeLV-A in feline cell cultures. The following observations were made from the in vivo experiments: (i) subgroup conversion from FeLV-A to FeLV-A and FeLV-B, as determined by the interference assay, appeared to occur in plasma between 10 and 16 weeks postinoculation (p.i.); (ii) FeLV-B-like recombinants (rFeLVs), however, could be detected in DNA isolated from buffy coats and bone marrow by PCR as early as 1 to 2 weeks p.i.; (iii) while a mixture of rFeLV species containing various amounts of N-terminal substitution of the endogenous FeLV-derived
env
sequences were detected at 8 weeks p.i., rFeLV species harboring relatively greater amounts of such substitution appeared to predominate at later infection time points; (iv) the deduced amino acid sequence of rFeLV clones manifested striking similarity to natural FeLV-B isolates, within the mid-SU region of the
env
sequenced in this work; and (v) four of the five cats, which were kept for determination of tumor incidence, developed thymic lymphosarcomas within 28 to 55 weeks p.i., with all tumor DNAs harboring both FeLV-A and rFeLV proviruses. These results provide direct evidence for how FeLV-B species evolve in vivo from FeLV-A and present a new experimental approach for efficient induction of thymic tumors in cats, which should be useful for the study of retroviral lymphomagenesis in this outbred species.
Functional Region IV of Glycoprotein D from Herpes Simplex Virus Modulates Glycoprotein Binding to the Herpesvirus Entry Mediator
Authors: Rux Ann H. , Willis Sharon H. , Nicola Anthony V. , Hou Wangfang , Peng Charline , Lou Huan , Cohen Gary H. , Eisenberg Roselyn J.
Published: December 2019

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry and has four functional regions (I to IV) important for this process. We previously showed that a truncated form of a functional region IV variant, gD1(Δ290-299t), had an enhanced ability to block virus entry and to bind to the herpesvirus entry mediator (HveAt; formerly HVEMt), a cellular receptor for HSV. To explore this phenotype further, we examined other forms of gD, especially ones with mutations in region IV. Variant proteins with deletions of amino acids between 277 and 300 (region IV), as well as truncated forms lacking C-terminal residues up to amino acid 275 of gD, were able to block HSV entry into Vero cells 1 to 2 logs better than wild-type gD1(306t). In contrast, gD truncated at residue 234 did not block virus entry into Vero cells. Using optical biosensor technology, we recently showed that gD1(Δ290-299t) had a 100-fold-higher affinity for HveAt than gD1(306t) (3.3 × 10
−8
M versus 3.2 × 10
−6
M). Here we found that the affinities of other region IV variants for HveAt were similar to that of gD1(Δ290-299t). Thus, the affinity data follow the same hierarchy as the blocking data. In each case, the higher affinity was due primarily to a faster
k
on
rather than to a slower
k
off
. Therefore, once the gDt-HveAt complex formed, its stability was unaffected by mutations in or near region IV. gD truncated at residue 234 bound to HveAt with a lower affinity (2.0 × 10
−5
M) than did gD1(306t) due to a more rapid
k
off
. These data suggest that residues between 234 and 275 are important for maintaining stability of the gDt-HveAt complex and that functional region IV is important for modulating the binding of gD to HveA. The binding properties of any gD1(234t)-receptor complex could account for the inability of this form of gDt to block HSV infection.
An Enhanced Packaging System for Helper-Dependent Herpes Simplex Virus Vectors
Authors: Stavropoulos Tom A. , Strathdee Craig A.
Published: December 2019

Helper-dependent herpes simplex virus (HSV) vectors (amplicons) show considerable promise to provide for long-term transduced-gene expression in most cell types. The current packaging system of choice for these vectors involves cotransfection with a set of five overlapping cosmids that encode the full HSV type 1 (HSV-1) helper virus genome from which the packaging (
pac
) elements have been deleted. Although both the helper virus and the HSV amplicon can replicate, only the latter is packaged into infectious viral particles. Since the titers obtained are too low for practical application, an enhanced second-generation packaging system was developed by modifying both the helper virus and the HSV amplicon vector. The helper virus was reverse engineered by using the original five cosmids to generate a single HSV-bacterial artificial chromosome (BAC) clone in
Escherichia coli
from which the
pac
elements were deleted to generate a replication-proficient but packaging-defective HSV-1 genome. The HSV amplicon was modified to contain the simian virus 40 origin of replication, which acts as an HSV-independent replicon to provide for the replicative expansion of the vector. The HSV amplicon is packaged into infectious particles by cotransfection with the HSV-BAC helper virus into the 293T cell line, and the resulting cell lysate is free of detectable helper virus contamination. The combination of both modifications to the original packaging system affords an eightfold increase in the packaged-vector yield.
Control of Murine Cytomegalovirus in the Lungs: Relative but Not Absolute Immunodominance of the Immediate-Early 1 Nonapeptide during the Antiviral Cytolytic T-Lymphocyte Response in Pulmonary Infiltrates
Authors: Holtappels Rafaela , Podlech Jürgen , Geginat Gernot , Steffens Hans-Peter , Thomas Doris , Reddehase Matthias J.
Published: December 2019

The lungs are a major organ site of cytomegalovirus (CMV) infection, pathogenesis, and latency. Interstitial CMV pneumonia represents a critical manifestation of CMV disease, in particular in recipients of bone marrow transplantation (BMT). We have employed a murine model for studying the immune response to CMV in the lungs in the specific scenario of immune reconstitution after syngeneic BMT. Control of pulmonary infection was associated with a vigorous infiltration of the lungs, which was characterized by a preferential recruitment and massive expansion of the CD8 subset of α/β T cells. The infiltrate provided a microenvironment in which the CD8 T cells differentiated into mature effector cells, that is, into functionally active cytolytic T lymphocytes (CTL). This gave us the opportunity for an ex vivo testing of the antigen specificities of CTL present at a relevant organ site of viral pathogenesis. The contribution of the previously identified immediate-early 1 (IE1) nonapeptide of murine CMV was evaluated by comparison with the CD3ɛ-redirected cytolytic activity used as a measure of the overall CTL response in the lungs. The IE1 peptide was detected by pulmonary CTL, but it accounted for a minor part of the response. Interestingly, no additional viral or virus-induced antigenic peptides were detectable among naturally processed peptides derived from infected lungs, even though infected fibroblasts were recognized in a major histocompatibility complex-restricted manner. We conclude that the antiviral pulmonary immune response is a collaborative function that involves many antigenic peptides, among which the IE1 peptide is immunodominant in a relative sense.
Rotavirus Infection Reduces Sucrase-Isomaltase Expression in Human Intestinal Epithelial Cells by Perturbing Protein Targeting and Organization of Microvillar Cytoskeleton
Authors: Jourdan Nathalie , Brunet Jean Philippe , Sapin Catherine , Blais Anne , Cotte-Laffitte Jacqueline , Forestier Françoise , Quero Anne-Marie , Trugnan Germain , Servin Alain L.
Published: December 2019

Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. These viruses infect mature enterocytes of the small intestine and cause structural and functional damage, including a reduction in disaccharidase activity. It was previously hypothesized that reduced disaccharidase activity resulted from the destruction of rotavirus-infected enterocytes at the villus tips. However, this pathophysiological model cannot explain situations in which low disaccharidase activity is observed when rotavirus-infected intestine exhibits few, if any, histopathologic changes. In a previous study, we demonstrated that the simian rotavirus strain RRV replicated in and was released from human enterocyte-like Caco-2 cells without cell destruction (N. Jourdan, M. Maurice, D. Delautier, A. M. Quero, A. L. Servin, and G. Trugnan, J. Virol. 71:8268–8278, 1997). In the present study, to reinvestigate disaccharidase expression during rotavirus infection, we studied sucrase-isomaltase (SI) in RRV-infected Caco-2 cells. We showed that SI activity and apical expression were specifically and selectively decreased by RRV infection without apparent cell destruction. Using pulse-chase experiments and cell surface biotinylation, we demonstrated that RRV infection did not affect SI biosynthesis, maturation, or stability but induced the blockade of SI transport to the brush border. Using confocal laser scanning microscopy, we showed that RRV infection induces important alterations of the cytoskeleton that correlate with decreased SI apical surface expression. These results lead us to propose an alternate model to explain the pathophysiology associated with rotavirus infection.
Functional Interaction between the Bovine Papillomavirus Virus Type 1 Replicative Helicase E1 and Cyclin E-Cdk2
Authors: Cueille Nathalie , Nougarede Romain , Mechali Francisca , Philippe Michel , Bonne-Andrea Catherine
Published: December 2019

We have found that the replicative helicase E1 of bovine papillomavirus type 1 (BPV-1) interacts with a key cell cycle regulator of S phase, the cyclin E-Cdk2 kinase. The E1 helicase, which interacts with cyclin E and not with Cdk2, presents the highest affinity for catalytically active kinase complexes. In addition, E1, cyclin E, and Cdk2 expressed in
Xenopus
egg extracts are quantitatively coimmunoprecipitated from crude extracts by either anti-Cdk2 or anti-E1 antibodies. E1 protein is also a substrate of the cyclin E-Cdk2 kinase in vitro. Using the viral components required for in vitro BPV-1 replication and free-membrane cytosol from
Xenopus
eggs, we show that efficient replication of BPV plasmids is dependent on the addition of E1-cyclin E-Cdk2 complexes. Thus, the BPV initiator of replication and cyclin E-Cdk2 are likely to function together as a protein complex which may be the key to the cell cycle regulation of papillomavirus replication.
Equine Infectious Anemia Virus Is Found in Tissue Macrophages during Subclinical Infection
Authors: Oaks J. Lindsay , McGuire Travis C. , Ulibarri Catherine , Crawford Timothy B.
Published: December 2019

The equine infectious anemia virus (EIAV) often results in lifelong subclinical infection following early episodes of clinical disease. To identify the cellular reservoirs of EIAV during subclinical infection, horses were infected with EIAV and allowed to develop subclinical infections. Horses with acute disease served as a basis for comparison. The tissue distribution, replication status, location of infected cells, and viral load were characterized by PCR for proviral DNA and reverse transcriptase PCR for viral RNA, in situ hybridization, and in situ PCR. Proviral DNA was widespread in tissues regardless of disease status. Viral
gag
and
env
RNAs were also detected in tissues of all horses regardless of disease status. Plasma viral RNA (viremia) could be detected in some, but not all, horses with subclinical infections. In situ assays determined that a primary cellular reservoir and site of viral replication during subclinical infection is the macrophage. During subclinical infection, viral load was decreased 4- to 733-fold and there was decreased viral RNA expression within infected cells. These data indicate that viral replication continues at all times, even in horses that are clinically quiescent. Moreover, restricted viral replication at the cellular level is associated with clinical remission.
DNA Vaccination Affords Significant Protection against Feline Immunodeficiency Virus Infection without Inducing Detectable Antiviral Antibodies
Authors: Hosie Margaret J. , Flynn J. Norman , Rigby Mark A. , Cannon Celia , Dunsford Thomas , Mackay Nancy A. , Argyle David , Willett Brian J. , Miyazawa Takayuki , Onions David E. , Jarrett Oswald , Neil James C.
Published: December 2019

To test the potential of a multigene DNA vaccine against lentivirus infection, we generated a defective mutant provirus of feline immunodeficiency virus (FIV) with an in-frame deletion inpol(FIVΔRT). In a first experiment, FIVΔRT DNA was administered intramuscularly to 10 animals, half of which also received feline gamma interferon (IFN-γ) DNA. The DNA was administered in four 100-μg doses at 0, 10, and 23 weeks. Immunization with FIVΔRT elicited cytotoxic T-cell (CTL) responses to FIV Gag and Env in the absence of a serological response. After challenge with homologous virus at week 26, all 10 of the control animals became seropositive and viremic but 4 of the 10 vaccinates remained seronegative and virus free. Furthermore, quantitative virus isolation and quantitative PCR analysis of viral DNA in peripheral blood mononuclear cells revealed significantly lower virus loads in the FIVΔRT vaccinates than in the controls. Immunization with FIVΔRT in conjunction with IFN-γ gave the highest proportion of protected cats, with only two of five vaccinates showing evidence of infection following challenge. In a second experiment involving two groups (FIVΔRT plus IFN-γ and IFN-γ alone), the immunization schedule was reduced to 0, 4, and 8 weeks. Once again, CTL responses were seen prior to challenge in the absence of detectable antibodies. Two of five cats receiving the proviral DNA vaccine were protected against infection, with an overall reduction in virus load compared to the five infected controls. These findings demonstrate that DNA vaccination can elicit protection against lentivirus infection in the absence of a serological response and suggest the need to reconsider efficacy criteria for lentivirus vaccines.
Rotavirus RNA Replication Requires a Single-Stranded 3′ End for Efficient Minus-Strand Synthesis
Authors: Chen Dayue , Patton John T.
Published: December 2019

The segmented double-stranded (ds) RNA genome of the rotaviruses is replicated asymmetrically, with viral mRNA serving as the template for the synthesis of minus-strand RNA. Previous studies with cell-free replication systems have shown that the highly conserved termini of rotavirus gene 8 and 9 mRNAs contain
cis
-acting signals that promote the synthesis of dsRNA. Based on the location of the
cis
-acting signals and computer modeling of their secondary structure, the ends of the gene 8 or 9 mRNAs are proposed to interact in
cis
to form a modified panhandle structure that promotes the synthesis of dsRNA. In this structure, the last 11 to 12 nucleotides of the RNA, including the
cis
-acting signal that is essential for RNA replication, extend as a single-stranded tail from the panhandled region, and the 5′ untranslated region folds to form a stem-loop motif. To understand the importance of the predicted secondary structure in minus-strand synthesis, mutations were introduced into viral RNAs which affected the 3′ tail and the 5′ stem-loop. Analysis of the RNAs with a cell-free replication system showed that, in contrast to mutations which altered the structure of the 5′ stem-loop, mutations which caused complete or near-complete complementarity between the 5′ end and the 3′ tail significantly inhibited (≥10-fold) minus-strand synthesis. Likewise, incubation of wild-type RNAs with oligonucleotides which were complementary to the 3′ tail inhibited replication. Despite their replication-defective phenotype, mutant RNAs with complementary 5′ and 3′ termini were shown to competitively interfere with the replication of wild-type mRNA and to bind the viral RNA polymerase VP1 as efficiently as wild-type RNA. These results indicate that the single-strand nature of the 3′ end of rotavirus mRNA is essential for efficient dsRNA synthesis and that the specific binding of the RNA polymerase to the mRNA template is required but not sufficient for the synthesis of minus-strand RNA.
Temporal Analyses of Virus Replication, Immune Responses, and Efficacy in Rhesus Macaques Immunized with a Live, Attenuated Simian Immunodeficiency Virus Vaccine
Authors: Connor Ruth I. , Montefiori David C. , Binley James M. , Moore John P. , Bonhoeffer Sebastian , Gettie Agegnehu , Fenamore Elizabeth A. , Sheridan Kristine E. , Ho David D. , Dailey Peter J. , Marx Preston A.
Published: December 2019

Despite evidence that live, attenuated simian immunodeficiency virus (SIV) vaccines can elicit potent protection against pathogenic SIV infection, detailed information on the replication kinetics of attenuated SIV in vivo is lacking. In this study, we measured SIV RNA in the plasma of 16 adult rhesus macaques immunized with a live, attenuated strain of SIV (SIVmac239Δnef). To evaluate the relationship between replication of the vaccine virus and the onset of protection, four animals per group were challenged with pathogenic SIVmac251 at either 5, 10, 15, or 25 weeks after immunization. SIVmac239Δnef replicated efficiently in the immunized macaques in the first few weeks after inoculation. SIV RNA was detected in the plasma of all animals by day 7 after inoculation, and peak levels of viremia (105to 107RNA copies/ml) occurred by 7 to 12 days. Following challenge, SIVmac251 was detected in all of the four animals challenged at 5 weeks, in two of four challenged at 10 weeks, in none of four challenged at 15 weeks, and one of four challenged at 25 weeks. One animal immunized with SIVmac239Δnef and challenged at 10 weeks had evidence of disease progression in the absence of detectable SIVmac251. Although complete protection was not achieved at 5 weeks, a transient reduction in viremia (approximately 100-fold) occurred in the immunized macaques early after challenge compared to the nonimmunized controls. Two weeks after challenge, SIV RNA was also reduced in the lymph nodes of all immunized macaques compared with control animals. Taken together, these results indicate that host responses capable of reducing the viral load in plasma and lymph nodes were induced as early as 5 weeks after immunization with SIVmac239Δnef, while more potent protection developed between 10 and 15 weeks. In further experiments, we found that resistance to SIVmac251 infection did not correlate with the presence of antibodies to SIV gp130 and p27 antigens and was achieved in the absence of significant neutralizing activity against the primary SIVmac251 challenge stock.
Identification of Specific Nucleotide Sequences within the Conserved 3′-SL in the Dengue Type 2 Virus Genome Required for Replication
Authors: Zeng Lingling , Falgout Barry , Markoff Lewis
Published: December 2019

The flavivirus genome is a positive-stranded ∼11-kb RNA including 5′ and 3′ noncoding regions (NCR) of approximately 100 and 400 to 600 nucleotides (nt), respectively. The 3′ NCR contains adjacent, thermodynamically stable, conserved short and long stem-and-loop structures (the 3′-SL), formed by the 3′-terminal ∼100 nt. The nucleotide sequences within the 3′-SL are not well conserved among species. We examined the requirement for the 3′-SL in the context of dengue virus type 2 (DEN2) replication by mutagenesis of an infectious cDNA copy of a DEN2 genome. Genomic full-length RNA was transcribed in vitro and used to transfect monkey kidney cells. A substitution mutation, in which the 3′-terminal 93 nt constituting the wild-type (wt) DEN2 3′-SL sequence were replaced by the 96-nt sequence of the West Nile virus (WN) 3′-SL, was sublethal for virus replication. An analysis of the growth phenotypes of additional mutant viruses derived from RNAs containing DEN2-WN chimeric 3′-SL structures suggested that the wt DEN2 nucleotide sequence forming the bottom half of the long stem and loop in the 3′-SL was required for viability. One 7-bp substitution mutation in this domain resulted in a mutant virus that grew well in monkey kidney cells but was severely restricted in cultured mosquito cells. In contrast, transpositions of and/or substitutions in the wt DEN2 nucleotide sequence in the top half of the long stem and in the short stem and loop were relatively well tolerated, provided the stem-loop secondary structure was conserved.
Transient Inhibition of CD28 and CD40 Ligand Interactions Prolongs Adenovirus-Mediated Transgene Expression in the Lung and Facilitates Expression after Secondary Vector Administration
Authors: Wilson Christopher B. , Embree Lisa J. , Schowalter David , Albert Richard , Aruffo Alejandro , Hollenbaugh Diane , Linsley Peter , Kay Mark A.
Published: December 2019

Recombinant adenovirus vectors have been used to transfer genes to the lungs in animal models, but the extent and duration of primary transgene expression and the ability to achieve expression after repeated vector administration have been limited by the development of antigen-specific immunity to the vector and, in some cases, to vector-transduced foreign proteins. To determine if focused modulation of the immune response could overcome some of these limitations, costimulatory interactions between T cells and B cells/antigen-presenting cells were transiently blocked around the time of vector administration. Systemic treatment at the time of primary-vector administration with a monoclonal antibody (MR1) against murine CD40 ligand, combined with recombinant murine CTLA4Ig and intratracheal coadministration of an adenovirus vector transducing the expression of murine CTLA4Ig, prolonged adenovirus-transduced β-galactosidase expression in the airways for up to 28 days and resulted in persistent alveolar expression for >90 days (the duration of the experiment). Consistent with these results, this treatment regimen reduced local inflammation and markedly reduced the T-cell and T-cell-dependent antibody response to the vector. A secondary adenovirus vector, administered >90 days after the last systemic dose of MR1 and muCTLA4Ig, resulted in alkaline phosphatase expression at levels comparable to those seen with primary-vector administration. Expression of the secondary transgene persisted in the alveoli (but not in the airways) for up to 24 days (the longest period of observation) at levels similar to those observed on days 3 to 4. These results indicate that transient inhibition of costimulatory molecule interactions substantially enhanced gene transfer to the alveoli but was much less effective in the airways. This suggests that there are differences in the efficiency or nature of mechanisms limiting transgene expression in the airways and in the alveoli.
The Poliovirus Empty Capsid Specifically Recognizes the Poliovirus Receptor and Undergoes Some, but Not All, of the Transitions Associated with Cell Entry
Authors: Basavappa Ravi , Gómez-Yafal Alicia , Hogle James M.
Published: December 2019

Experimental results presented here demonstrate that the poliovirus empty capsid binds with saturable character to poliovirus-susceptible cells, binds preferentially to susceptible cells, and competes with mature virus for binding sites on cells. Hence, induced changes in the structure and/or stability of the particle by RNA encapsidation and virus maturation are not necessary for recognition by receptor. In mature virus, heat-induced rearrangements mimic those induced by receptor at physiological temperatures in several important respects, namely, expulsion of VP4 and externalization of the VP1 N-terminal arm. It is shown here that in the empty capsid the VP1 N-terminal arm is externalized but the VP4 portion of VP0 is not. Thus, these two hallmark rearrangements associated with cell entry can be uncoupled.
Molecular Characterization of Proteolytic Processing of the Pol Proteins of Human Foamy Virus Reveals Novel Features of the Viral Protease
Authors: Pfrepper Klaus-Ingmar , Rackwitz Hans-Richard , Schnölzer Martina , Heid Hans , Löchelt Martin , Flügel Rolf M.
Published: December 2019

Spumaviruses, or foamy viruses, express a
pol
-specific transcript that codes for a Pol polyprotein that consists of the protease, reverse transcriptase, ribonuclease H, and the integrase domains. To delineate the proteolytic cleavage sites between the Pol subdomains, recombinant human foamy virus (HFV) Pol proteins were expressed, purified by affinity chromatography, and subjected to either HFV protease assays or autocatalytic processing. In control experiments, HFV protease-deficient mutant proteins in which the active site Asp was replaced by an Ala residue were used to rule out unspecific processing by nonviral proteases. Specific proteolytic cleavage products were isolated, and the cleavage sites were analyzed by amino acid sequencing. Peptides spanning the resulting cleavage sites were chemically synthesized and assayed with HFV protease, and the cleaved peptides were subjected to mass spectrometry. The cleavage site sequences obtained were in complete agreement with the amino-terminal sequences from amino acid sequencing of authentic cleavage products of the HFV Pol proteins. Analysis by fast-protein liquid chromatography of a short version of the active HFV protease revealed that the enzyme predominantly formed dimeric molecules.
Lipofection of Purified Adeno-Associated Virus Rep68 Protein: toward a Chromosome-Targeting Nonviral Particle
Authors: Lamartina Stefania , Roscilli Giuseppe , Rinaudo Daniela , Delmastro Paola , Toniatti Carlo
Published: December 2019

Adeno-associated virus (AAV) integrates very efficiently into a specific site (AAVS1) of human chromosome 19. Two elements of the AAV genome are sufficient: the inverted terminal repeats (ITRs) and the Rep78 or Rep68 protein. The incorporation of the AAV integration machinery in nonviral delivery systems is of great interest for gene therapy. We demonstrate that purified recombinant Rep68 protein is functionally active when directly delivered into human cells by using the polycationic liposome Lipofectamine, promoting the rescue-replication of a codelivered ITR-flanked cassette in adenovirus-infected cells and its site-specific integration in noninfected cells. The sequencing of cloned virus-host DNA junctions confirmed that lipofected Rep68 protein triggers site-specific integration at the same sites in chromosome 19 already characterized in cells latently infected with AAV.
Classical Swine Fever Virus Leader Proteinase N
pro
Is Not Required for Viral Replication in Cell Culture
Authors: Tratschin Jon-Duri , Moser Christian , Ruggli Nicolas , Hofmann Martin A.
Published: December 2019

The sequence encoding the viral leader proteinase N
pro
was replaced by the murine ubiquitin gene in a full-length cDNA clone of the classical swine fever virus (CSFV) strain Alfort/187. The recombinant virus vA187-Ubi showed growth characteristics similar to those of the parent vA187-1 virus. At two occasions cells infected with vA187-Ubi exhibited a cytopathic effect and were found to contain a subgenomic viral RNA. This RNA lacked the same viral genes as the subgenomic RNA which has been found in all cytopathogenic CSFV strains analyzed so far, but it maintained the ubiquitin sequence.
CrFK Feline Kidney Cells Produce an RD114-Like Endogenous Virus That Can Package Murine Leukemia Virus-Based Vectors
Authors: Baumann Jörg G. , Günzburg Walter H. , Salmons Brian
Published: December 2019

The feline kidney cell line CrFK is used extensively for viral infectivity assays and for study of the biology of various retroviruses and derived vectors. We demonstrate the production of an endogenous, RD114-like, infectious retrovirus from CrFK cells. This virus also is shown to efficiently package Moloney murine leukemia virus vectors.
Spatial Perturbations within an RNA Promoter Specifically Recognized by a Viral RNA-Dependent RNA Polymerase (RdRp) Reveal That RdRp Can Adjust Its Promoter Binding Sites
Authors: Stawicki Scott Stevenson , Kao C. Cheng
Published: December 2019

RNA synthesis during viral replication requires specific recognition of RNA promoters by the viral RNA-dependent RNA polymerase (RdRp). Four nucleotides (−17, −14, −13, and −11) within the brome mosaic virus (BMV) subgenomic core promoter are required for RNA synthesis by the BMV RdRp (R. W. Siegel et al., Proc. Natl. Acad. Sci. USA 94:11238–11243, 1997). The spatial requirements for these four nucleotides and the initiation (+1) cytidylate were examined in RNAs containing nucleotide insertions and deletions within the BMV subgenomic core promoter. Spatial perturbations between nucleotides −17 and −11 resulted in decreased RNA synthesis in vitro. However, synthesis was still dependent on the key nucleotides identified in the wild-type core promoter and the initiation cytidylate. In contrast, changes between nucleotides −11 and +1 had a less severe effect on RNA synthesis but resulted in RNA products initiated at alternative locations in addition to the +1 cytidylate. The results suggest a degree of flexibility in the recognition of the subgenomic promoter by the BMV RdRp and are compared with functional regions in other DNA and RNA promoters.
The 5′ Terminal Trailer Region of Vesicular Stomatitis Virus Contains a Position-Dependent
cis
-Acting Signal for Assembly of RNA into Infectious Particles
Authors: Whelan Sean P. J. , Wertz Gail W.
Published: December 2019

The
cis
-acting genomic RNA requirements for the assembly of vesicular stomatitis virus (VSV) ribonucleocapsids into infectious particles were investigated. Using a biological assay based on particle infectivity, we demonstrated that subgenomic replicons that contained all four possible combinations of the natural genomic termini, the 3′ leader (Le) and 5′ trailer (Tr) regions, were replication competent; however, a 3′ copyback replicon (3′CB), containing the natural 3′ terminus but having the 5′ Tr replaced by a sequence complementary to the 3′ Le for 46 nucleotides, was unable to assemble infectious particles, despite efficient replication. When a copy of Tr was inserted 51 nucleotides from the 5′ end of 3′CB, infectious particles were produced. However, analysis of the replication products of these particles showed that the 51 nucleotides which corresponded to the Le complement sequences at the 5′ terminus were removed during RNA replication, thus restoring the wild-type 5′ Tr to the exact 5′ terminus. These data showed that a
cis
-acting signal was necessary for assembly of VSV RNAs into infectious particles and that this signal was supplied by Tr when located at the 5′ end. The regions within Tr required for assembly were analyzed by a series of deletions and exchanges for Le complement sequences, which demonstrated that the 5′ terminal 29 nucleotides of Tr allowed assembly of infectious particles but that the 5′ terminal 22 nucleotides functioned poorly. Deletions in Tr also altered the balance between negative- and positive-strand genomic RNA and affected levels of replication. RNAs that retained fewer than 45 but at least 22 nucleotides of the 5′ terminus could replicate but were impaired in RNA replication, and RNAs that retained only 14 nucleotides of the 5′ terminus were severely reduced in ability to replicate. These data define the VSV Tr as a position-dependent,
cis
-acting element for the assembly of RNAs into infectious particles, and they delineate RNA sequences that are essential for negative-strand RNA synthesis. These observations are consistent with, and offer an explanation for, the absence of 3′ copyback defective interfering particles in nature.
Accessing Epstein-Barr Virus-Specific T-Cell Memory with Peptide-Loaded Dendritic Cells
Authors: Redchenko I. V. , Rickinson A. B.
Published: December 2019

The conventional means of studying Epstein-Barr virus (EBV)-induced cytotoxic T-lymphocyte (CTL) memory, by in vitro stimulation with the latently infected autologous lymphoblastoid cell line (LCL), has important limitations. First, it gives no information on memory to lytic cycle antigens; second, it preferentially amplifies the dominant components of latent antigen-specific memory at the expense of key subdominant reactivities. Here we describe an alternative approach, based on in vitro stimulation with epitope peptide-loaded dendritic cells (DCs), which allows one to probe the CTL repertoire for any individual reactivity of choice; this method proved significantly more efficient than stimulation with peptide alone. Using this approach we first show that reactivities to the immunodominant and subdominant lytic cycle epitopes identified by T cells during primary EBV infection are regularly detectable in the CTL memory of virus carriers; this implies that in such carriers chronic virus replication remains under direct T-cell control. We further show that subdominant latent cycle reactivities to epitopes in the latent membrane protein LMP2, though rarely undetectable in LCL-stimulated populations, can be reactivated by DC stimulation and selectively expanded as polyclonal CTL lines; the adoptive transfer of such preparations may be of value in targeting certain EBV-positive malignancies.
Mammary Gland Expression of Mouse Mammary Tumor Virus Is Regulated by a Novel Element in the Long Terminal Repeat
Authors: Qin Wei , Golovkina Tatyana V. , Peng Tao , Nepomnaschy Irene , Buggiano Valeria , Piazzon Isabel , Ross Susan R.
Published: December 2019

Mouse mammary tumor virus (MMTV) infects both lymphoid tissue and lactating mammary gland during its infectious cycle, but some endogenous MMTVs are transcribed only in lymphoid cells. We found a lymphoid cell-specific endogenous MMTV that was converted to a milk-borne, infectious virus through recombination with an exogenously transmitted MMTV. The changed expression pattern correlated with the alteration of a single base pair in the long terminal repeat of the lymphoid cell-specific virus. Transgenic mice with the element from either the milk-borne or lymphoid cell-specific virus upstream of the chloramphenicol acetyltransferase reporter gene showed the same pattern of expression as the virus from which the regulatory sequences were derived. Electrophoretic mobility shift assays with mammary cell extracts showed that the site from the milk-borne virus was preferentially bound by a prolactin-inducible factor that poorly bound the altered site from the lymphoid cell-specific virus. The complex that formed on the milk-borne virus-specific oligonucleotide supershifted with anti-Stat5b antibody. Mice lacking either Stat5a or Stat5b had dramatically reduced levels of MMTV transcripts in mammary gland but not in lymphoid tissue. Thus, a member of the STAT family of transcription factors is involved in the tissue-specific expression of mouse mammary tumor virus in vivo. This is the first example of the involvement of a member of the STAT family of transcription factors in the control of tissue-specific expression.
Analysis of Murine CD8+T-Cell Clones Specific for the Dengue Virus NS3 Protein: Flavivirus Cross-Reactivity and Influence of Infecting Serotype
Authors: Spaulding Anne C. , Kurane Ichiro , Ennis Francis A. , Rothman Alan L.
Published: December 2019

Serotype-cross-reactive dengue virus-specific cytotoxic T lymphocytes (CTL) induced during a primary dengue virus infection are thought to play a role in the immunopathogenesis of dengue hemorrhagic fever (DHF) during a secondary dengue virus infection. Although there is no animal model of DHF, we previously reported that murine dengue virus-specific CTL responses are qualitatively similar to human dengue virus-specific CTL responses. We used BALB/c mice to study the specificity of the CTL response to an immunodominant epitope on the dengue virus NS3 protein. We mapped the minimalH-2Kd-restricted CTL epitope to residues 298 to 306 of the dengue type 2 virus NS3 protein. In short-term T-cell lines and clones, the predominant CD8+CTL to this epitope in mice immunized with dengue type 2 virus or vaccinia virus expressing the dengue type 4 virus NS3 protein were cross-reactive with dengue type 2 or type 4 virus, while broadly serotype-cross-reactive CTL were a minority population. In dengue type 3 virus-immunized mice, the predominant CTL response to this epitope was broadly serotype cross-reactive. All of the dengue virus-specific CTL clones studied also recognized the homologous NS3 sequences of one or more closely related flaviviruses, such as Kunjin virus. The critical contact residues for the CTL clones with different specificities were mapped with peptides having single amino acid substitutions. These data demonstrate that primary dengue virus infection induces a complex population of flavivirus-cross-reactive NS3-specific CTL clones in mice and suggest that CTL responses are influenced by the viral serotype. These findings suggest an additional mechanism by which the order of sequential flavivirus infections may influence disease manifestations.
In Vivo Modulation of Vaccine-Induced Immune Responses toward a Th1 Phenotype Increases Potency and Vaccine Effectiveness in a Herpes Simplex Virus Type 2 Mouse Model
Authors: Sin Jeong-Im , Kim Jong J. , Boyer Jean D. , Ciccarelli Richard B. , Higgins Terry J. , Weiner David B.
Published: December 2019

Several vaccines have been investigated experimentally in the herpes simplex virus type 2 (HSV-2) model system. While it is believed that CD4+-T-cell responses are important for protection in general, the correlates of protection from HSV-2 infection are still under investigation. Recently, the use of molecular adjuvants to drive vaccine responses induced by DNA vaccines has been reported in a number of experimental systems. We sought to take advantage of this immunization model to gain insight into the correlates of immune protection in the HSV-2 mouse model system and to further explore DNA vaccine technology. To investigate whether the Th1- or Th2-type immune responses are more important for protection from HSV-2 infection, we codelivered the DNA expression construct encoding the HSV-2 gD protein with the gene plasmids encoding the Th1-type (interleukin-2 [IL-2], IL-12, IL-15, and IL-18) and Th2-type (IL-4 and IL-10) cytokines in an effort to drive immunity induced by vaccination. We then analyzed the modulatory effects of the vaccine on the resulting immune phenotype and on the mortality and the morbidity of the immunized animals following a lethal challenge with HSV-2. We observed that Th1 cytokine gene coadministration not only enhanced the survival rate but also reduced the frequency and severity of herpetic lesions following intravaginal HSV challenge. On the other hand, coinjection with Th2 cytokine genes increased the rate of mortality and morbidity of the challenged mice. Moreover, of the Th1-type cytokine genes tested, IL-12 was a particularly potent adjuvant for the gD DNA vaccination.
Pathogenicity of Different Rabies Virus Variants Inversely Correlates with Apoptosis and Rabies Virus Glycoprotein Expression in Infected Primary Neuron Cultures
Authors: Morimoto Kinjiro , Hooper D. Craig , Spitsin Sergei , Koprowski Hilary , Dietzschold Bernhard
Published: December 2019

The mouse-adapted rabies virus strain CVS-24 has stable variants, CVS-B2c and CVS-N2c, which differ greatly in their pathogenicity for normal adult mice and in their ability to infect nonneuronal cells. The glycoprotein (G protein), which has previously been implicated in rabies virus pathogenicity, shows substantial structural differences between these variants. Although prior studies have identified antigenic site III of the G protein as the major pathogenicity determinant, CVS-B2c and CVS-N2c do not vary at this site. The possibility that pathogenicity is inversely related to G protein expression levels is suggested by the finding that CVS-B2c, the less pathogenic variant, expresses at least fourfold-higher levels of G protein than CVS-N2c in infected neurons. Although there is some difference between CVS-B2c- and CVS-N2c-infected neurons in G protein mRNA expression levels, the differential expression of G protein appears to be largely determined by posttranslational mechanisms that affect G protein stability. Pulse-chase experiments indicated that the G protein of CVS-B2c is degraded more slowly than that of CVS-N2c. The accumulation of G protein correlated with the induction of programmed cell death in CVS-B2c-infected neurons. The extent of apoptosis was considerably lower in CVS-N2c-infected neurons, where G protein expression was minimal. While nucleoprotein (N protein) expression levels were similar in neurons infected with either variant, the transport of N protein into neuronal processes was strongly inhibited in CVS-B2c-infected cells. Thus, downregulation of G protein expression in neuronal cells evidently contributes to rabies virus pathogenesis by preventing apoptosis and the apparently associated failure of the axonal transport of N protein.
A Packaging Cell Line for Lentivirus Vectors
Authors: Kafri Tal , van Praag Henriette , Ouyang Ling , Gage Fred H. , Verma Inder M.
Published: December 2019

Lentivirus vectors can transduce dividing and nondividing cells. Using three-plasmid transient transfections, high-titer (>10
9
IU/ml) recombinant lentivirus vectors pseudotyped with vesicular stomatitis virus G (VSV-G) protein can be generated (T. Kafri et al., Nat. Genet. 17:314–317, 1997; H. Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319–10323, 1997; L. Naldini et al., Science 272:263–267, 1996). The recombinant lentiviruses can efficiently infect brain, liver, muscle, and retinal tissue in vivo. Furthermore, the transduced tissues demonstrated long-term expression of reporter genes in immunocompetent rodents. We now report the generation of a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line which can generate virus particles at titers greater than 10
6
IU/ml for at least 3 to 4 days. The vector produced by the inducible cell line can be concentrated to titers of 10
9
IU/ml and can efficiently transduce nondividing cells in vitro and in vivo. The availability of a lentivirus packaging cell line will significantly facilitate the production of high-titer lentivirus vectors for gene therapy and study of human immunodeficiency virus biology.
Human Cytomegalovirus UL69 Protein Induces Cells To Accumulate in G
1
Phase of the Cell Cycle
Authors: Lu Mansuo , Shenk Thomas
Published: December 2019

Earlier studies have revealed that human cytomegalovirus rapidly inhibits the growth of fibroblasts, blocking cell cycle progression at multiple points, including the G
1
-to-S-phase transition. The present study demonstrates that the UL69 protein, a virus-encoded constituent of the virion, is able to arrest cell cycle progression when introduced into uninfected cells. Expression of the UL69 protein causes U2 OS cells and primary human fibroblasts to accumulate within the G
1
compartment of the cell cycle, and serum fails to induce the progression of quiescent human fibroblasts into the S phase when the protein is present. Therefore, the UL69 protein is at least partially responsible for the cell cycle block that is instituted after infection of permissive cells with human cytomegalovirus.
Phosphorylation of the Human T-Cell Leukemia Virus Type 1 Transactivator Tax on Adjacent Serine Residues Is Critical for Tax Activation
Authors: Bex Françoise , Murphy Kathy , Wattiez Ruddy , Burny Arsène , Gaynor Richard B.
Published: December 2019

The Tax transactivator protein of human T-cell leukemia virus type 1 (HTLV-1) plays a central role in the activation of viral gene expression. In addition, Tax is capable of activating the expression of specific cellular genes and is involved in the transformation of T-lymphocytes resulting in the development of adult T-cell leukemia. Tax is a phosphoprotein that colocalizes in nuclear bodies with RNA polymerase II, splicing complexes, and specific transcription factors including members of the ATF/CREB and NF-κB families. In this study, we identified adjacent serine residues at positions 300 and 301 in the carboxy terminus of Tax as the major sites for phosphorylation. Phosphorylation of at least one of these serine residues is required for Tax localization in nuclear bodies and for Tax-mediated activation of gene expression via both the ATF/CREB and NF-κB pathways. Introduction of amino acid substitutions which are phosphoserine mimetics at positions 300 and 301 restored the ability of a phosphorylation-defective Tax mutant to form nuclear bodies and to activate gene expression. These studies define sites for regulatory phosphorylation events in Tax which are critical for its ability to activate gene transcription.
Fine Structure and Morphogenesis of Borna Disease Virus
Authors: Kohno Takehiro , Goto Toshiyuki , Takasaki Tomohiko , Morita Chizuko , Nakaya Takaaki , Ikuta Kazuyoshi , Kurane Ichiro , Sano Kouichi , Nakai Masuyo
Published: December 2019

Borna disease virus (BDV), a negative nonsegmented single-stranded RNA virus, has not been fully characterized morphologically. Here we present what is to our knowledge the first data on the fine ultrastructure and morphogenesis of BDV. The supernatant of MDCK cells persistently infected with BDV treated with
n
-butyrate contained many virus-like particles and more BDV-specific RNA than that of untreated samples. The particles were spherical, enveloped, and approximately 130 nm in diameter; had spikes 7 nm in length; and reacted with BDV p40 antibody. A thin nucleocapsid, 4 nm in width, was present peripherally in contrast to the thick nucleocapsid of hemagglutinating virus of Japan. The BDV particles reproduced by budding on the cell surface.