bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Hydrophobic Patch in Ecotropic Murine Leukemia Virus Envelope Protein Is the Putative Binding Site for a Critical Tyrosine Residue on the Cellular Receptor
Authors: Zavorotinskaya Tatiana , Albritton Lorraine M. 
Published: December 2019

ABSTRACT
In the receptor for ecotropic murine leukemia viruses, tyrosine 235 contributes a critical hydrophobic side chain to the virus-receptor interaction (14). Here we report that tryptophan 142 in ecotropic Moloney murine leukemia virus envelope protein is essential to virus binding and infection. Replacement of tryptophan 142 by alanine or serine resulted in misfolding. However, replacement by methionine (W142M) allowed correct folding of the majority of glycoprotein molecules. W142M virus showed a marked reduction in virus binding and was almost noninfectious, suggesting that tryptophan 142 is involved in receptor binding. In contrast, W142Y virus containing a replacement of tryptophan 142 with an aromatic residue (tyrosine) was as efficient as wild-type virus in infection and binding of cells expressing the wild-type receptor. However, W142Y virus was 100-fold less efficient than wild-type virus in infection of cells expressing a mutant receptor containing tryptophan instead of the critical tyrosine. These results strongly support tryptophan 142 being an essential residue on the virus envelope protein that interacts directly with the critical hydrophobic residue at position 235 of the ecotropic receptor. Tryptophan 142 forms one side of a shallow hydrophobic pocket on the surface of the envelope protein, suggesting that it might comprise the complete putative binding site for tyrosine 235. We discuss the implications of our findings with respect to two models of the envelope protein trimer. Interestingly, both models place tryptophan 142 at the interface between adjacent subunits of the trimer.
A Single Rep Protein Initiates Replication of Multiple Genome Components of Faba Bean Necrotic Yellows Virus, a Single-Stranded DNA Virus of Plants
Authors: Timchenko Tatiana , de Kouchkovsky Françoise , Katul Lina , David Chantal , Vetten Heinrich Josef , Gronenborn Bruno 
Published: December 2019

ABSTRACT

Faba bean necrotic yellows virus (FBNYV) belongs to the nanoviruses, plant viruses whose genome consists of multiple circular single-stranded DNA components. Eleven distinct DNAs, 5 of which encode different replication initiator (Rep) proteins, have been identified in two FBNYV isolates. Origin-specific DNA cleavage and nucleotidyl transfer activities were shown for Rep1 and Rep2 proteins in vitro, and their essential tyrosine residues that catalyze these reactions were identified by site-directed mutagenesis. In addition, we showed that Rep1 and Rep2 proteins hydrolyze ATP, and by changing the key lysine residue in the proteins’ nucleoside triphosphate binding sites, demonstrated that this ATPase activity is essential for multiplication of virus DNA in vivo. Each of the five FBNYV Rep proteins initiated replication of the DNA molecule by which it was encoded, but only Rep2 was able to initiate replication of all the six other genome components. Furthermore, of the five
rep
components, only the Rep2-encoding DNA was always detected in 55 FBNYV samples from eight countries. These data provide experimental evidence for a master replication protein encoded by a multicomponent single-stranded DNA virus.
Interferon Regulatory Factor 3 Is Required for Viral Induction of Beta Interferon in Primary Cardiac Myocyte Cultures
Authors: Noah Diana L. , Blum Mary Ann , Sherry Barbara 
Published: December 2019

ABSTRACT
Viral myocarditis affects an estimated 5 to 20% of the human population. The antiviral cytokine beta interferon (IFN-β) is critical for protection against viral myocarditis in mice. That is, nonmyocarditic reoviruses induce myocarditis in mice that lack IFN-α/β, and nonmyocarditic reoviruses both induce more IFN-β and are more sensitive to the antiviral effects of IFN-β than myocarditic reoviruses in primary cardiac myocyte cultures. Induction of IFN-β in certain cell types involves viral activation of the transcription factor interferon regulatory factor 3 (IRF-3). To address whether IRF-3 can induce IFN-β in cardiac myocytes, primary cardiac myocyte cultures and control L929 cells were transfected with a plasmid constitutively expressing IRF-3. Overexpression of IRF-3 resulted in induction of IFN-β in the absence of viral infection in both cell types. To address whether IRF-3 is required for viral induction of IFN-β, cell cultures were transfected with a plasmid constitutively expressing a dominant negative IRF-3 protein. The dominant negative IRF-3 reduced reovirus induction of IFN-β in control L929 cells and completely eliminated induction in primary cardiac myocyte cultures. This provides the first identification of a cardiac cellular factor required for viral induction of IFN-β and the first report of any cell type requiring IRF-3 for this response.
Conformational Changes in Simian Virus 40 Rearranged Regulatory Regions: Effects of the 21-Base-Pair Promoters and Their Location
Authors: Wilderman Paula J. , Hu Bugen , Woodworth Mary E. 
Published: December 2019

ABSTRACT

Simian virus 40 (SV40) is an excellent model system for investigating the
cis
- and
trans
-acting factors involved in eukaryotic DNA replication because it uses host enzymes, with the exception of the virus-encoded T-antigen (T-ag), to replicate its genome. Although its origin of replication (
ori
) is essential for DNA replication, there are transcriptional promoters and enhancers that affect DNA replication efficiency. T-ag binds to sites I to III within and around
ori
with different affinities and induces structural changes. We were interested in determining if the position of the promoters relative to
ori
influences the binding of T-ag to these regions. Furthermore, we characterized the DNA structural changes that occur as a result of protein binding when the promoters are absent and also when the promoters are moved from their wild-type position upstream of
ori
to a position downstream of
ori
. Using sequence- and conformation-specific chemical probes, our data indicate that (i) the conformation of site I is influenced by T-ag binding and by flanking sequences, (ii) the conformation of the promoters after T-ag binding is dependent on their location, and (iii) unwinding of
ori
is influenced by the location of the promoters and their presence or absence. These differences in DNA conformation may help explain decreases in relative DNA replication efficiency that occur when the promoters are absent or located downstream of
ori.
Transfer of Human CD4+T Lymphocytes Producing Beta Interferon in Hu-PBL-SCID Mice Controls Human Immunodeficiency Virus Infection
Authors: Vieillard Vincent , Jouveshomme Stephane , Leflour Nicole , Jean-Pierre Eric , Debre Patrice , De Maeyer Edward , Autran Brigitte 
Published: December 2019

ABSTRACTBeta interferon (IFN-β) exerts pleiotropic antiretroviral activities and affects many different stages of the human immunodeficiency virus (HIV) infectious cycle in IFN-treated cells. To explore whether transfer of genetically engineered human CD4+T cells producing constitutively low amounts of IFN-β can eradicate HIV in vivo, we developed a new Hu-PBL-SCID mouse model supporting a persistent, replicative HIV infection maintained by periodic reinoculations of activated human CD4+T cells. Transferring human CD4+T cells containing the IFN-β retroviral vector drastically reduced the preexisting HIV infection and enhanced CD4+T-cell survival and Th1 cytokine expression. Furthermore, in 40% of the Hu-PBL-SCID mice engrafted with IFN-β-transduced CD4+T cells, HIV-1 was undetectable in vivo as well as after cocultivation of mouse tissues with human phytohemagglutinin-stimulated lymphoblasts. These results indicate that a therapeutic strategy based upon IFN-β transduction of CD4+T cells may be an approach to controlling a preexisting HIV infection and allowing immune restoration.
Brome Mosaic Virus RNA Replication Proteins 1a and 2a Colocalize and 1a Independently Localizes on the Yeast Endoplasmic Reticulum
Authors: Restrepo-Hartwig María , Ahlquist Paul 
Published: December 2019

ABSTRACTThe universal membrane association of positive-strand RNA virus RNA replication complexes is implicated in their function, but the intracellular membranes used vary among viruses. Brome mosaic virus (BMV) encodes two mutually interacting RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and the polymerase-like 2a protein. In cells from the natural plant hosts of BMV, 1a and 2a colocalize on the endoplasmic reticulum (ER). 1a and 2a also direct BMV RNA replication and subgenomic mRNA synthesis in the yeastSaccharomyces cerevisiae, but whether the distribution of 1a, 2a, and active replication complexes in yeast duplicates that in plant cells has not been determined. For yeast expressing 1a and 2a and replicating BMV genomic RNA3, we used double-label confocal immunofluorescence to define the localization of 1a, 2a, and viral RNA and to explore the determinants of replication complex targeting. As in plant cells, 1a and 2a colocalized on and were retained on the yeast ER, with no detectable accumulation in the Golgi apparatus. 1a and 2a were distributed over most of the ER surface, with strongest accumulation on the perinuclear ER. In vivo labeling with bromo-UTP showed that the sites of 1a and 2a accumulation were the sites of nascent viral RNA synthesis. In situ hybridization showed that completed viral RNA products accumulated predominantly in the immediate vicinity of replication complexes but that some, possibly more mature cells also accumulated substantial viral RNA in the surrounding cytoplasm distal to replication complexes. Additionally, we find that 1a localizes to the ER when expressed in the absence of other viral factors. These results show that BMV RNA replication in yeast duplicates the normal localization of replication complexes, reveal the intracellular distribution of RNA replication products, and show that 1a is at least partly responsible for the ER localization and retention of the RNA replication complex.
Determinants of CD4 Independence for a Human Immunodeficiency Virus Type 1 Variant Map outside Regions Required for Coreceptor Specificity
Authors: LaBranche Celia C. , Hoffman Trevor L. , Romano Josephine , Haggarty Beth S. , Edwards Terri G. , Matthews Thomas J. , Doms Robert W. , Hoxie James A. 
Published: December 2019

ABSTRACTAlthough infection by human immunodeficiency virus (HIV) typically requires an interaction between the viral envelope glycoprotein (Env), CD4, and a chemokine receptor, CD4-independent isolates of HIV and simian immunodeficiency virus have been described. The structural basis and underlying mechanisms for this phenotype are unknown. We have derived a variant of HIV-1/IIIB, termed IIIBx, that acquired the ability to utilize CXCR4 without CD4. This virus infected CD4-negative T and B cells and fused with murine 3T3 cells that expressed human CXCR4 alone. A functional IIIBxenvclone exhibited several mutations compared to the CD4-dependent HXBc2env, including the striking loss of five glycosylation sites. By constructingenvchimeras with HXBc2, the determinants for CD4 independence were shown to map outside the V1/V2 and V3 hypervariable loops, which determine chemokine receptor specificity, and at least partly within an area on the gp120 core that has been implicated in forming a conserved chemokine receptor binding site. We also identified a point mutation in the C4 domain that could render the IIIBxenvclone completely CD4 dependent. Mutations in the transmembrane protein (TM) were also required for CD4 independence. Remarkably, when the V3 loop of a CCR5-tropic Env was substituted for the IIIBx Env, the resulting chimera was found to utilize CCR5 but remained CD4 independent. These findings show that Env determinants for chemokine receptor specificity are distinct from those that mediate CD4-independent use of that receptor for cell fusion and provide functional evidence for multiple steps in the interaction of Env with chemokine receptors. Combined with our observation that the conserved chemokine receptor binding site on gp120 is more exposed on the IIIBx gp120 (T. L. Hoffman, C. C. LaBranche, W. Zhang, G. Canziani, J. Robinson, I. Chaiken, J. A. Hoxie, and R. W. Doms, Proc. Natl. Acad. Sci. USA 96:6359–6364, 1999), the findings from this study suggest novel approaches to derive and design Envs with exposed chemokine receptor binding sites for vaccine purposes.
The Activation Domain of Herpesvirus Saimiri R Protein Interacts with the TATA-Binding Protein
Authors: Hall Kersten T. , Stevenson Alex J. , Goodwin Delyth J. , Gibson Paul C. , Markham Alex F. , Whitehouse Adrian 
Published: December 2019

ABSTRACT
The herpesvirus saimiri open reading frame (ORF) 50 produces two transcripts. The first is spliced, contains a single intron, and is detected at early times during the productive cycle, whereas the second is expressed later and is produced from a promoter within the second exon. Analysis of their gene products has shown that they function as sequence specific transactivators. In this report, we demonstrate that the carboxy terminus of ORF 50b contains an activation domain which is essential for transactivation. This domain contains positionally conserved hydrophobic residues found in a number of activation domains, including the herpes simplex virus VP16 and the Epstein-Barr virus R proteins. Mutational analysis of this domain demonstrates that these conserved hydrophobic residues are essential for ORF 50 transactivation capability. Furthermore, this domain is required for the interaction between the ORF 50 proteins and the basal transcription factor TATA-binding protein.
Expression of the Pseudorabies Virus Latency-Associated Transcript Gene during Productive Infection of Cultured Cells
Authors: Jin Ling , Scherba Gail 
Published: December 2019

ABSTRACT

Like other alphaherpesviruses, pseudorabies virus (PrV) exhibits restricted gene expression during latency. These latency-associated transcripts (LATs) are derived from the region located within 0.69 to 0.77 map units of the viral genome. However, the presence of such viral RNAs during a productive infection has not been described. Although several transcripts originating between 0.706 to 0.737 map units have been detected in PrV-infected cultured cells, their relationship to the LATs has not been examined. Therefore, to determine if any correlation exists between PrV LAT gene expression in the natural and laboratory systems, transcription from the LAT gene region during lytic infection of cultured neuronal and nonneuronal cells was evaluated. A Northern blot assay using single-stranded RNA probes complementary to the spliced in vivo 8.4-kb largest latency transcript (LLT) detected 1.0-, 2.0-, and 8.0-kb poly(A) RNAs in all PrV-infected cells lines. The 1.0- and 8.0-kb transcripts partially overlapped the first and second exons of the LLT, respectively. In contrast, portions of both LLT exons comprised the 2.0-kb RNA sequence, which lacked the same intron as the LLT. Generation of this transcript began about 243 bp downstream of the LLT initiation site and terminated near the junction of BamHI fragments 8′ and 8. Its synthesis was inhibited by cycloheximide but not by cytosine β-
d
-arabinofuranoside, which suggests that the 2.0-kb RNA is not an immediate-early gene product. Thus, although the PrV LAT gene is transcriptionally active during a productive infection of cultured cells, the resulting RNAs are distinctive from the LLT.
Latent Nuclear Antigen of Kaposi’s Sarcoma-Associated Herpesvirus Interacts with RING3, a Homolog of the
Drosophila
Female Sterile Homeotic (
fsh
) Gene
Authors: Platt Georgina M. , Simpson Guy R. , Mittnacht Sibylle , Schulz Thomas F. 
Published: December 2019

ABSTRACT

Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) is the likely infectious cause of Kaposi’s sarcoma, primary effusion lymphoma, and some cases of multicentric Castleman’s disease. Its latent nuclear antigen (LANA) is expressed in the nuclei of latently infected cells and may play a role in the persistence of episomal viral DNA in dividing cells. Here we report that LANA interacts with RING3, a nuclear protein and member of the
Drosophila
fsh (female sterile homeotic) family of proteins, some of which have previously been implicated in controlling gene expression. Binding of RING3 to LANA involves the ET domain, characteristic of fsh-related proteins, suggesting that this highly conserved region is involved in protein-protein interactions. The interaction between RING3 and LANA results in phosphorylation of serine and threonine residues located between amino acids 951 and 1107 in the carboxy-terminal region of LANA. However, RING3 is not itself a kinase but appears to recruit an as yet unidentified serine/threonine protein kinase into the complex which it forms with LANA.
Replication of
Mus dunni
Endogenous Retrovirus Depends on Promoter Activation Followed by Enhancer Multimerization
Authors: Wolgamot Greg , Miller A. Dusty 
Published: December 2019

ABSTRACT

Mus dunni
endogenous virus (MDEV) is an apparently intact retrovirus that normally lies transcriptionally silent in cultured
M. dunni
cells, but the provirus can be activated by treatment of the cells with hydrocortisone or 5-iodo-2′-deoxyuridine. Sequence analysis of a molecular clone of the replicating virus revealed a simple retrovirus with a chimeric VL30/GALV-like structure. Interestingly, in the region of the long terminal repeat (LTR) that typically contains the retroviral transcription enhancers, we found over six 80-bp repeats with only a single mismatch, indicating that acquisition of the repeats was a recent event. Here we provide evidence for the following model of MDEV activation and replication. The MDEV provirus in
M. dunni
cells has a chimeric structure similar to that of the molecular clone but has only 1.15 copies of the 80-bp repeat sequence found in the molecular clone. Activating chemicals directly stimulate transcription from the LTR, allowing a low level of virus replication. Copying errors made during reverse transcription allow multimerization of the 80-bp enhancer region, resulting in viruses with higher transcriptional rates and improved fitness, but increased enhancer copy number is likely balanced by the natural instability of retroviral repeats and constraints imposed by virion packaging limits. The resultant population of replicating MDEV is widely heterogeneous, having from 2.15 to 13.15 enhancer repeats in the LTR. These results reveal a novel mechanism for regulation of transcription and replication of an endogenous retrovirus, in terms of both activation of the virus by the steroid hydrocortisone and the large number and variation in enhancer repeats observed.
Mouse Mammary Tumor Virus Carrying a Bacterial
supF
Gene Has Wild-Type Pathogenicity and Enables Rapid Isolation of Proviral Integration Sites
Authors: Jiang Zhaorong , Shackleford Gregory M. 
Published: December 2019

ABSTRACT

Mouse mammary tumor virus (MMTV) has frequently been used as an insertional mutagen to identify provirally activated mammary proto-oncogenes. To expedite and facilitate the process of cloning MMTV insertion sites, we have introduced a bacterial
supF
suppressor tRNA gene into the long terminal repeat (LTR) of MMTV, thus allowing selection of clones containing it in lambda vectors bearing amber mutations. The presence of
supF
in the LTR should circumvent the screening process for proviral insertion sites, since only those lambda clones with
supF
-containing proviral-cellular junction fragments should be able to form plaques on a lawn of wild-type
Escherichia coli
(i.e., lacking
supF
). The resulting virus (MMTV
supF
) induced mammary tumors at the expected rate in infected mice, deleted the appropriate T-cell population by virtue of its superantigen gene, and stably retained the
supF
gene after passage via the milk to female offspring. To test the selective function of the system, size-selected DNA containing two proviral-cellular junction fragments from an MMTV
supF
-induced mammary tumor was ligated into λgtWES.λB, packaged, and plated on a
supF
-deficient bacterial host for selection of
supF
-containing clones. All plaques tested contained the desired cloned fragments, thus demonstrating the utility of this modified provirus for the rapid cloning of MMTV insertion sites.
Oncogenic Role of Epstein-Barr Virus-Encoded RNAs in Burkitt’s Lymphoma Cell Line Akata
Authors: Komano Jun , Maruo Seiji , Kurozumi Koichi , Oda Takanori , Takada Kenzo 
Published: December 2019

ABSTRACT
Our previous reports indicated that Epstein-Barr virus (EBV) contributes to the malignant phenotype and resistance to apoptosis in Burkitt’s lymphoma (BL) cell line Akata (N. Shimizu, A. Tanabe-Tochikura, Y. Kuroiwa, and K. Takada, J. Virol. 68:6069–6073, 1994; J. Komano, M. Sugiura, and K. Takada, J. Virol. 72:9150–9156, 1998). Here we report that the EBV-encoded small RNAs (EBERs) are responsible for these phenotypes. Transfection of the EBER genes into EBV-negative Akata clones restored the capacity for growth in soft agar, tumorigenicity in SCID mice, resistance to apoptotic inducers, and upregulated expression of bcl-2 oncoprotein that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. This is the first report which provides evidence that virus-encoded RNAs (EBERs) have oncogenic functions in BL cells.
Role of the Epstein-Barr Virus Rta Protein in Activation of Distinct Classes of Viral Lytic Cycle Genes
Authors: Ragoczy Tobias , Miller George 
Published: December 2019

ABSTRACTInitiation of the Epstein-Barr virus (EBV) lytic cycle is controlled by two immediate-early genes, BZLF1 and BRLF1. In certain epithelial and B-cell lines, their protein products, ZEBRA and Rta, stimulate their own expression, reciprocally stimulate each other’s expression, and activate downstream viral targets. It has been difficult to examine the individual roles of these two transactivators in EBV-infected lymphocytes, as they are expressed simultaneously upon induction of the lytic cycle. Here we show that the Burkitt lymphoma cell line Raji represents an experimental system that allows the study of Rta’s role in the lytic cycle of EBV in the absence and presence of ZEBRA. When expressed in Raji cells, exogenous Rta does not activate endogenous BZLF1 expression, yet Rta remains competent to transactivate certain downstream viral targets. Some genes, such as BaRF1, BMLF1, and a late gene, BLRF2, are maximally activated by Rta itself in the absence of detectable ZEBRA. The use of the Z(S186A) mutant form of ZEBRA, whose transactivation function is manifest only by coexpression of Rta, allows identification of a second class of lytic cycle genes, such as BMRF1 and BHRF1, that are activated in synergy by Rta and ZEBRA. It has already been documented that of the two activators, only ZEBRA stimulates the BRLF1 gene in Raji cells. Thus, there is a third class of viral genes activated by ZEBRA but not Rta. Moreover, ZEBRA exhibits an inhibitory effect on Rta’s capacity to stimulate the late gene, BLRF2. Consequently ZEBRA may function to repress Rta’s potential to activate some late genes. Raji cells thus allow delineation of the combinatorial roles of Rta and ZEBRA in control of several distinct classes of lytic cycle genes.