bluuebunny/crossref_metadata_2025_split huggingface dataset:
Amino Acids of Conserved Kinase Motifs of Cytomegalovirus Protein UL97 Are Essential for Autophosphorylation
Authors: Michel Detlef , Kramer Silke , Höhn Simone , Schaarschmidt Peter , Wunderlich Kirsten , Mertens Thomas 
Published: December 2019

ABSTRACT
Thirteen point mutations targeting predicted domains conserved in homologous protein kinases were introduced into the UL97 coding region of the human cytomegalovirus. All mutagenized proteins were expressed in cells infected with recombinant vaccinia viruses (rVV). Several mutations drastically reduced ganciclovir (GCV) phosphorylation. Mutations at amino acids G340, A442, L446, and F523 resulted in a complete loss of pUL97 phosphorylation, which was strictly associated with a loss of GCV phosphorylation. Our results confirm that in rVV-infected cells pUL97 phosphorylation is due to autophosphorylation and show that several amino acids conserved within domains of protein kinases are essential for this pUL97 phosphorylation. GCV phosphorylation is dependent on pUL97 phosphorylation.
African Swine Fever Virus dUTPase Is a Highly Specific Enzyme Required for Efficient Replication in Swine Macrophages
Authors: Oliveros Mariano , García-Escudero Ramón , Alejo Alí , Viñuela Eladio , Salas María L. , Salas José 
Published: December 2019

ABSTRACT

The African swine fever virus (ASFV) gene E165R, which is homologous to dUTPases, has been characterized. A multiple alignment of dUTPases showed the conservation in ASFV dUTPase of the motifs that define this protein family. A biochemical analysis of the purified recombinant enzyme showed that the virus dUTPase is a trimeric, highly specific enzyme that requires a divalent cation for activity. The enzyme is most probably complexed with Mg
2+
, the preferred cation, and has an apparent

K
m

for dUTP of 1 μM. Northern and Western blotting, as well as immunofluorescence analyses, indicated that the enzyme is expressed at early and late times of infection and is localized in the cytoplasm of the infected cells. On the other hand, an ASFV dUTPase-deletion mutant (vΔE165R) has been obtained. Growth kinetics showed that vΔE165R replicates as efficiently as parental virus in Vero cells but only to 10% or less of parental virus in swine macrophages. Our results suggest that the dUTPase activity is dispensable for virus replication in dividing cells but is required for productive infection in nondividing swine macrophages, the natural host cell for the virus. The viral dUTPase may play a role in lowering the dUTP concentration in natural infections to minimize misincorporation of deoxyuridine into the viral DNA and ensure the fidelity of genome replication.
Nonreplicative RNA Recombination in Poliovirus
Authors: Gmyl Anatoly P. , Belousov Evgeny V. , Maslova Svetlana V. , Khitrina Elena V. , Chetverin Alexander B. , Agol Vadim I. 
Published: December 2019

ABSTRACT
Current models of recombination between viral RNAs are based on replicative template-switch mechanisms. The existence of nonreplicative RNA recombination in poliovirus is demonstrated in the present study by the rescue of viable viruses after cotransfections with different pairs of genomic RNA fragments with suppressed translatable and replicating capacities. Approximately 100 distinct recombinant genomes have been identified. The majority of crossovers occurred between nonhomologous segments of the partners and might have resulted from transesterification reactions, not necessarily involving an enzymatic activity. Some of the crossover loci are clustered. The origin of some of these “hot spots” could be explained by invoking structures similar to known ribozymes. A significant proportion of recombinant RNAs contained the entire 5′ partner, if its 3′ end was oxidized or phosphorylated prior to being mixed with the 3′ partner. All of these observations are consistent with a mechanism that involves intermediary formation of the 2′,3′-cyclic phosphate and 5′-hydroxyl termini. It is proposed that nonreplicative RNA recombination may contribute to evolutionarily significant RNA rearrangements.
A Primary Determinant of Cap-Independent Translation Is Located in the 3′-Proximal Region of the Tomato Bushy Stunt Virus Genome
Authors: Wu Baodong , White K. Andrew 
Published: December 2019

ABSTRACT

Tomato bushy stunt virus (TBSV) is a positive-strand RNA virus and is the prototype member of the genus
Tombusvirus
. The genomes of members of this genus are not polyadenylated, and prevailing evidence supports the absence of a 5′ cap structure. Previously, a 167-nucleotide-long segment (region 3.5) located near the 3′ terminus of the TBSV genome was implicated as a determinant of translational efficiency (S.K. Oster, B. Wu and K. A. White, J. Virol. 72:5845–5851, 1998). In the present report, we provide evidence that a 3′-proximal segment of the genome, which includes region 3.5, is involved in facilitating cap-independent translation. Our results indicate that (i) a 5′ cap structure can substitute functionally for the absence of region 3.5 in viral and chimeric reporter mRNAs in vivo; (ii) deletion of region 3.5 from viral and chimeric mRNAs has no appreciable effect on message stability; (iii) region 3.5 represents part of a larger 3′ proximal element, designated as the 3′ cap-independent translational enhancer (3′CITE), that is required for proficient cap-independent translation; (iv) the 3′CITE also facilitates cap-dependent translation; (v) none of the major viral proteins are required for 3′CITE activity; and (vi) no significant 3′CITE-dependent stimulation of translation was observed when mRNAs were tested in vitro in wheat germ extract under various assay conditions. This latter property distinguishes the 3′CITE from other characterized plant viral 3′-proximal cap-independent translational enhancers. Additionally, because the 3′CITE overlaps with
cis
-acting replication signals, it could potentially participate in regulating the initiation of genome replication.
A Strong Negative Transcriptional Regulatory Region between the Human Cytomegalovirus UL127 Gene and the Major Immediate-Early Enhancer
Authors: Lundquist Christopher A. , Meier Jeffrey L. , Stinski Mark F. 
Published: December 2019

ABSTRACT

The region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region. DNase I protection analysis with human cell nuclear extracts demonstrated multiple protein binding sites in this region of the viral genome (P. Ghazal, H. Lubon, C. Reynolds-Kohler, L. Hennighausen, and J. A. Nelson, Virology 174:18–25, 1990). However, the function of this region in the context of the viral genome is not known. In wild-type human CMV-infected human fibroblasts, cells permissive for viral replication, there is little to no transcription from UL127. We determined that the unique region prevented transcription from the UL127 promoter but had no effect on the divergent MIE promoter. In transient-transfection assays, the basal level of expression from the UL127 promoter increased significantly when the wild-type unique sequences were mutated. In recombinant viruses with similar mutations in the unique region, expression from the UL127 promoter occurred only after de novo viral protein synthesis, typical of an early viral promoter. A 111-bp deletion-substitution of the unique sequence caused approximately a 20-fold increase in the steady-state level of RNA from the UL127 promoter and a 245-fold increase in the expression of a downstream indicator gene. This viral negative regulatory region was also mutated at approximately 50-bp regions proximal and distal to the UL127 promoter. Although some repressive effects were detected in the distal region, mutations of the region proximal to the UL127 promoter had the most significant effects on transcription. Within the proximal and distal regions, there are potential
cis
sites for known eucaryotic transcriptional repressor proteins. This region may also bind unknown viral proteins. We propose that the unique region upstream of the UL127 promoter and the MIE enhancer negatively regulates the expression from the UL127 promoter in permissive human fibroblast cells. This region may be a regulatory boundary preventing the effects of the very strong MIE enhancer on this promoter.
Identification and Characterization of the Functional Elements within the Tobacco Etch Virus 5′ Leader Required for Cap-Independent Translation
Authors: Niepel Mario , Gallie Daniel R. 
Published: December 2019

ABSTRACT

Translation in plants is highly cap dependent, and the only plant mRNAs known to naturally lack a cap structure (m
7
GpppN) are viral in origin. The genomic RNA of tobacco etch virus (TEV), a potyvirus that belongs to the picornavirus superfamily, is a polyadenylated mRNA that is naturally uncapped and yet is a highly competitive mRNA during translation. The 143-nucleotide 5′ leader is responsible for conferring cap-independent translation even on reporter mRNAs. We have carried out a deletion analysis of the TEV 5′ leader to identify the elements responsible for its regulatory function and have identified two centrally located cap-independent regulatory elements (CIREs) that promote cap-independent translation. The introduction of a stable stem-loop structure upstream of each element demonstrated that CIRE-1 is less 5′ end dependent in function than CIRE-2. In a dicistronic mRNA, the presence of the TEV 5′ leader sequence in the intercistronic region increased expression of the second cistron, suggesting that the viral sequence can function in a 5′-distal position. Interestingly, the introduction of a stable stem-loop upstream of the TEV leader sequence or upstream of either CIRE in dicistronic constructs markedly increased their regulatory function. These data suggest that the TEV 5′ leader contains two elements that together promote internal initiation but that the function of one element, in particular, is facilitated by proximity to the 5′ end.
Human Immunodeficiency Virus Type 1-Induced Hematopoietic Inhibition Is Independent of Productive Infection of Progenitor Cells In Vivo
Authors: Koka Prasad S. , Jamieson Beth D. , Brooks David G. , Zack Jerome A. 
Published: December 2019

ABSTRACTHuman immunodeficiency virus (HIV)-infected individuals exhibit a variety of hematopoietic dysfunctions. The SCID-hu mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues) can be used to model the loss of human hematopoietic precursor cell function following HIV infection and has a distinct advantage in that data can be obtained in the absence of confounding factors often seen in infected humans. In this study, we establish that HIV type 1 (HIV-1) bearing a reporter gene inserted into the viralvprgene is highly aggressive in depleting human myeloid and erythroid colony-forming precursor activity in vivo. Human CD34+progenitor cells can be efficiently recovered from infected implants yet do not express the viral reporter gene, despite severe functional defects. Our results indicate that HIV-1 infection alone leads to hematopoietic inhibition in vivo; however, this effect is due to indirect mechanisms rather than to direct infection of CD34+cells in vivo.
Evaluation of Novel Human Immunodeficiency Virus Type 1 Gag DNA Vaccines for Protein Expression in Mammalian Cells and Induction of Immune Responses
Authors: Qiu Jian-Tai , Song Ruijiang , Dettenhofer Markus , Tian Chunjuan , August Thomas , Felber Barbara K. , Pavlakis George N. , Yu Xiao-Fang 
Published: December 2019

ABSTRACTHuman immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) are an important parameter of host defenses that limit viral replication after infection. Induction of effective CTL against conserved viral proteins such as Gag may be essential to the development of a safe and effective HIV type 1 (HIV-1) vaccine. DNA vaccination represents a novel strategy for inducing potent CD8+CTL responses in vivo. However, expression of HIV-1 structural proteins by DNA vectors has been hampered by a stringent requirement for coexpression with other viral components, such as Rev and RRE. Furthermore, even with Rev and RRE present, the level of expression of HIV-1 Gag, Pol, or Env is very low in murine cells. These problems have limited our ability to address the key issue of how to generate effective CTL responses to Gag in a mouse model. To overcome this problem, we compared several novel DNA expression vectors for HIV-1 Gag protein expression in primate and mouse cells and for generating immune responses in mice after DNA vaccination. A DNA vector containing wild type HIV-1gagcoding sequences did not induce detectable Gag expression in any of the cells tested. Attempts to increase nuclear export of Gag expression RNA by adding the constitutive transport element yielded only a moderate increase in Gag expression in monkey-derived COS cells and an even lower increase in Gag expression in HeLa cells or several mouse cell lines. In contrast, silent-site mutations in the HIV-1gagcoding sequences significantly increased Gag expression levels in all cells tested. Furthermore, this construct induced both Gag-specific antibody and CTL responses in mice after DNA vaccination. Using this construct, we achieved stable expression of HIV-1 Gag in the mouse cell line p815, which can now be used as a target cell for measuring HIV-1 Gag-specific CTL responses in immunized mice. The DNA vectors described in this study should make it possible to systematically evaluate the approaches for maximizing the induction of CTL responses against HIV-1 Gag in mouse and other animal systems.
Longitudinal Phenotypic Analysis of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes: Correlation with Disease Progression
Authors: Ogg Graham S. , Kostense Stefan , Klein Michel R. , Jurriaans Suzanne , Hamann Dörte , McMichael Andrew J. , Miedema Frank 
Published: December 2019

ABSTRACTFew studies have examined longitudinal changes in human immunodeficiency virus type 1 (HIV)-specific cytotoxic T lymphocytes (CTL). To more closely define the natural history of HIV-specific CTL, we used HLA-peptide tetrameric complexes to study the longitudinal CD8+T-cell response evolution in 16 A*0201-positive untreated individuals followed clinically for up to 14 years. As early as 1 to 2 years after seroconversion, we found a significant association between high frequencies of A*0201-restricted p17Gag/Poltetramer-binding cells and slower disease progression (P< 0.01). We observed that responses could remain stable over many months, but any longitudinal changes that occurred were typically accompanied by reciprocal changes in RNA viral load. Phenotypic analysis with markers CD45RO, CD45RA, and CD27 identified distinct subsets of antigen-specific cells and the preferential loss of CD27+CD45RO+cells during periods of rapid decline in the frequency of tetramer-binding cells. In addition we were unable to confirm previous studies showing a consistent selective loss of HIV-specific cells in the context of sustained Epstein-Barr virus-specific cell frequencies. Overall, these data support a role of HIV-specific CTL in the control of disease progression and suggest that the ultimate loss of such CTL may be preferentially from the CD27+CD45RO+subset.
Genetic Regulation of Long-Term Nonprogression in E-55+ Murine Leukemia Virus Infection in Mice
Authors: Panoutsakopoulou Vily , Hunter Kathryn , Sieck Thomas G. , Blankenhorn Elizabeth P. , Blank Kenneth J. 
Published: December 2019

ABSTRACTCertain inbred mouse strains display progression to lymphoma development after infection with E-55+ murine leukemia virus (E-55+ MuLV), while others demonstrate long-term nonprogression. This difference in disease progression occurs despite the fact that E-55+ MuLV causes persistent infection in both immunocompetent BALB/c–H-2k(BALB.K) progressor (P) and C57BL/10–H-2k(B10.BR) long-term nonprogressor (LTNP) mice. In contrast to immunocompetent mice, immunosuppressed mice from both P and LTNP strains develop lymphomas about 2 months after infection, indicating that the LTNP phenotype is determined by the immune response of the infected mouse. In this study, we used bone marrow chimeras to demonstrate that the LTNP phenotype is associated with the genotype of donor bone marrow and not the recipient microenvironment. In addition, we have mapped a genetic locus that may be responsible for the LTNP trait. Microsatellite-based linkage analysis demonstrated that a non-major histocompatibility complex gene on chromosome 15 regulates long-term survival and is located in the same region as theRfv3gene.Rfv3is involved in recovery from Friend virus-induced leukemia and has been demonstrated to regulate neutralizing virus antibody titers. In our studies, however, both P and LTNP strains produce similar titers of neutralizing and cytotoxic anti-E-55+ MuLV. Therefore, while it is possible thatRfv3influences the course of E-55+ MuLV infection, it is more likely that the LTNP phenotype in E-55+ MuLV-infected mice is regulated by a different, closely linked gene.
trans
-Complementation Analysis of the Flavivirus Kunjin ns5 Gene Reveals an Essential Role for Translation of Its N-Terminal Half in RNA Replication
Authors: Khromykh Alexander A. , Sedlak Petra L. , Westaway Edwin G. 
Published: December 2019

ABSTRACT

Recently we described rescue of defective Kunjin virus (KUN) RNAs with small deletions in the methyltransferase and RNA polymerase motifs of the ns5 gene, using BHK cells stably expressing KUN replicon RNA (repBHK cells) as helper (A. A. Khromykh et al., J. Virol. 72:7270–7279, 1998). We have now extended our previous observations and report successful
trans
-complementation of defective KUN RNAs with most of the ns5 gene deleted or substituted with a heterologous (dengue virus) ns5 sequence. Replication of full-length KUN RNAs with 3′-terminal deletions of 136 (5%), 933 (34%), and 1526 (56%) nucleotides in the ns5 gene was complemented efficiently in transfected repBHK cells. RNA with a larger deletion of 2,042 nucleotides (75%) was complemented less efficiently, and RNA with an even larger deletion of 2,279 nucleotides (84%) was not complemented at all. Chimeric KUN genomic RNA containing 87% of the KUN ns5 gene replaced by the corresponding sequence of the dengue virus type 2 ns5 gene was unable to replicate in normal BHK cells but was complemented in repBHK cells. These results demonstrate for the first time complementation of flavivirus RNAs with large deletions (as much as 75%) in the RNA polymerase gene and establish that translation of most of the N-terminal half of NS5 is essential for complementation in
trans
. A model of formation of the flavivirus replication complex implicating a possible role in RNA replication of conserved coding sequences in the N-terminal half of NS5 is proposed based on the complementation and earlier results with KUN and on reported data with other flaviviruses.
Generation of Adenovirus Vectors Devoid of All Viral Genes by Recombination between Inverted Repeats
Authors: Steinwaerder Dirk S. , Carlson Cheryl A. , Lieber André 
Published: December 2019

ABSTRACT

Direct or inverse repeated sequences are important functional features of prokaryotic and eukaryotic genomes. Considering the unique mechanism, involving single-stranded genomic intermediates, by which adenovirus (Ad) replicates its genome, we investigated whether repetitive homologous sequences inserted into E1-deleted adenoviral vectors would affect replication of viral DNA. In these studies we found that inverted repeats (IRs) inserted into the E1 region could mediate predictable genomic rearrangements, resulting in vector genomes devoid of all viral genes. These genomes (termed ΔAd.IR) contained only the transgene cassette flanked on both sides by precisely duplicated IRs, Ad packaging signals, and Ad inverted terminal repeat sequences. Generation of ΔAd.IR genomes could also be achieved by coinfecting two viruses, each providing one inverse homology element. The formation of ΔAd.IR genomes required Ad DNA replication and appeared to involve recombination between the homologous inverted sequences. The formation of ΔAd.IR genomes did not depend on the sequence within or adjacent to the inverted repeat elements. The small ΔAd.IR vector genomes were efficiently packaged into functional Ad particles. All functions for ΔAd.IR replication and packaging were provided by the full-length genome amplified in the same cell. ΔAd.IR vectors were produced at a yield of ∼10
4
particles per cell, which could be separated from virions with full-length genomes based on their lighter buoyant density. ΔAd.IR vectors infected cultured cells with the same efficiency as first-generation vectors; however, transgene expression was only transient due to the instability of deleted genomes within transduced cells. The finding that IRs present within Ad vector genomes can mediate precise genetic rearrangements has important implications for the development of new vectors for gene therapy approaches.
The Mode and Duration of Anti-CD28 Costimulation Determine Resistance to Infection by Macrophage-Tropic Strains of Human Immunodeficiency Virus Type 1 In Vitro
Authors: Creson Jennifer R. , Lin Andy A. , Li Qun , Broad David F. , Roberts Margo R. , Anderson Stephen J. 
Published: December 2019

ABSTRACTWe have investigated the ability of anti-CD28 antibody costimulation to induce resistance to macrophage (M)-tropic strains of human immunodeficiency virus type 1 (HIV-1) in vitro. Our results confirm the observations of Levine et al. (15) that stimulation of CD4 T cells with anti-CD3/anti-CD28 antibodies coimmobilized on magnetic beads renders the cells resistant to infection by M-tropic strains of HIV-1. The resistance was strongest when the beads were left in the cultures throughout the experiment. In contrast, stimulation of CD4 T cells with the same antibodies immobilized on the surface of plastic culture dishes failed to induce resistance and resulted in high levels of p24 production. This was true even if the cells were passaged continuously on freshly coated plates. If the beads were removed after initial stimulation, p24 production increased over time and produced a result intermediate to the other forms of stimulation. For beads-in, beads-out, and one-time plate stimulated cultures, resistance to infection correlated with down-regulation of CCR5 expression at the cell surface and with increased production of β-chemokines. However, cultures of CD4 T cells continuously passaged on anti-CD3/anti-CD28-coated plates produced large amounts of p24 despite decreased levels of CCR5 expression and increasing production of β-chemokines. Expression of the T-cell activation markers CD25 and CD69 and production of gamma interferon further supported the differences in plate versus bead stimulation. Our results explain the apparent contradiction between the ability of anti-CD28 antibody costimulation to induce resistance to HIV infection when presented on magnetic beads and the increased ability to recover virus from the cells of HIV-positive donors who are on highly active antiretroviral therapy when cells are stimulated by anti-CD3/anti-CD28 immobilized on plastic dishes.