bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Analysis of the Human Immunodeficiency Virus Type 1 Rev Protein Oligomerization Interface
Authors: Thomas Sarah L. , Oft Martin , Jaksche Herbert , Casari Georg , Heger Peter , Dobrovnik Marika , Bevec Dorian , Hauber Joachim 
Published: December 2019

ABSTRACT

The expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the action of the viral
trans
-regulatory protein Rev. Rev is a nuclear shuttle protein that directly binds to its
cis
-acting Rev response element (RRE) RNA target sequence. Subsequent oligomerization of Rev monomers on the RRE and interaction of Rev with a cellular cofactor(s) result in the cytoplasmic accumulation of RRE-containing viral mRNAs. Moreover, Rev by itself is exported from the nucleus to the cytoplasm. Although it has been demonstrated that Rev multimerization is critically required for Rev activity and hence for HIV-1 replication, the number of Rev monomers required to form a
trans
-activation-competent complex on the RRE is unknown. Here we report a systematic analysis of the putative multimerization domains within the Rev
trans
-activator protein. We identify the amino acid residues which are part of the proposed single hydrophobic surface patch in the Rev amino terminus that mediates intermolecular interactions. Furthermore, we show that the expression of a multimerization-deficient Rev mutant blocks HIV-1 replication in a
trans
-dominant (dominant-negative) fashion.
A Leucine Zipper-Like Domain Is Essential for Dimerization and Encapsidation of Bluetongue Virus Nucleocapsid Protein VP4
Authors: Ramadevi N. , Rodriguez Javier , Roy Polly 
Published: December 2019

ABSTRACT

The bluetongue virus (BTV) minor protein VP4, with molecular mass of 76 kDa, is one of the seven structural proteins and is located within the inner capsid of the virion. The protein has a putative leucine zipper near the carboxy terminus of the protein. In this study, we have investigated the functional activity of this putative leucine zipper by a number of approaches. The putative leucine zipper region (amino acids [aa] 523 to 551) was expressed initially as a fusion protein by using the pMAL vector of
Escherichia coli
, which expresses a maltose binding monomeric protein. The expressed fusion protein was purified by affinity chromatography, and its size was determined by gel filtration chromatography. Proteins of two sizes, 51 and 110 kDa, were recovered, one equivalent to the monomeric form and the other equivalent to the dimeric form of the fusion protein. To prove that the VP4-derived sequence was responsible for dimerization of this protein, a mutated fusion protein was created in which a VP4 leucine residue (at aa 537) within the zipper was replaced by a proline residue. Analyses of the mutated protein demonstrated that the single mutation indeed prevented dimerisation of the protein. The dimeric nature of VP4 was further confirmed by using purified full-length BTV-10 VP4 recovered from recombinant baculovirus-expressing BTV-10 VP4-infected insect cells. Using chemical cross-linking and gel filtration chromatography, we documented that the native VP4 indeed exists as a dimer in solution. Subsequently, Leu537 was replaced by either a proline or an alanine residue and the full-length mutated VP4 was expressed in the baculovirus system. By sucrose density gradient centrifugation and gel filtration chromatography, these mutant forms of VP4 were shown to lack the ability to form dimers. The biological significance of the dimeric forms of VP4 was examined by using a functional assay system, in which the encapsidation activity of VP4 into core-like particles (CLPs) was studied (H. LeBlois, T. French, P. P. C. Mertens, J. N. Burroughs, and P. Roy, Virology 189:757–761, 1992). We demonstrated conclusively that dimerization of VP4 was essential for encapsidation by CLPs.
Expression of Enzymatically Active Rabbit Hemorrhagic Disease Virus RNA-Dependent RNA Polymerase in
Escherichia coli
Authors: Vázquez Ana López , Martín Alonso José M. , Casais Rosa , Boga José A. , Parra Francisco 
Published: December 2019

ABSTRACT

The rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) RNA-dependent RNA-polymerase (3D
pol
) coding region was expressed in
Escherichia coli
by using a glutathione
S
-transferase-based vector, which allowed milligram purification of a homogeneous enzyme with an expected molecular mass of about 58 kDa. The recombinant polypeptide exhibited rifampin- and actinomycin D-resistant, poly(A)-dependent poly(U) polymerase. The enzyme also showed RNA polymerase activity in in vitro reactions with synthetic RHDV subgenomic RNA in the presence or absence of an oligo(U) primer. Template-size products were synthesized in the oligo(U)-primed reactions, whereas in the absence of added primer, RNA products up to twice the length of the template were made. The double-length RNA products were double stranded and hybridized to both positive- and negative-sense probes.
Hepatitis C Virus Core from Two Different Genotypes Has an Oncogenic Potential but Is Not Sufficient for Transforming Primary Rat Embryo Fibroblasts in Cooperation with the H-rasOncogene
Authors: Chang Jun , Yang Se-Hwan , Cho Young-Gyu , Hwang Soon Bong , Hahn Young Shin , Sung Young Chul 
Published: December 2019

ABSTRACTPersistent infection with hepatitis C virus (HCV) is associated with the development of liver cirrhosis and hepatocellular carcinoma. To examine the oncogenic potential of the HCV core gene product, primary rat embryo fibroblasts (REFs) were transfected with the core gene in the presence or absence of the H-rasoncogene. In contrast to a previous report (R. B. Ray, L. M. Lagging, K. Meyer, and R. Ray, J. Virol. 70:4438–4443, 1996), HCV core proteins from two different genotypes (type 1a and type 1b) were not found to transform REFs to tumorigenic phenotype in cooperation with the H-rasoncogene, although the core protein was successfully expressed 20 days after transfection. In addition, REFs transfected with E1A- but not core-expressing plasmid showed the phenotype of immortalized cells when selected with G418. The biological activity was confirmed by observing the transcription activation from two viral promoters, Rous sarcoma virus long terminal repeat and simian virus 40 promoter, which are known to be activated by the core protein from HCV-1 isolate. In contrast to the result with primary cells, the Rat-1 cell line, stably expressing HCV core protein, exhibited focus formation, anchorage-independent growth, and tumor formation in nude mice. HCV core protein was able to induce the transformation of Rat-1 cells with various efficiencies depending on the expression level of the core protein. These results indicate that HCV core protein has an oncogenic potential to transform the Rat-1 cell line but is not sufficient to either immortalize primary REFs by itself or transform primary cells in conjunction with the H-rasoncogene.
Herpesvirus Saimiri Transforms Human T-Cell Clones to Stable Growth without Inducing Resistance to Apoptosis
Authors: Kraft Michael S. , Henning Golo , Fickenscher Helmut , Lengenfelder Doris , Tschopp Jürg , Fleckenstein Bernhard , Meinl Edgar 
Published: December 2019

ABSTRACTHerpesvirus saimiri (HVS) transforms human T cells to stable growth in vitro. Since HVS codes for two different antiapoptotic proteins, growth transformation by HVS might be expected to confer resistance to apoptosis. We found that the expression of both viral antiapoptotic genes was restricted to cultures with viral replication and absent in growth-transformed human T cells. A comparative examination of HVS-transformed T-cell clones and their native parental clones revealed that the expression of Bcl-2, Bcl-XL, Bax, and members of the tumor necrosis factor receptor (TNF-R) superfamily with a death domain, namely, TNF-RI, CD95, and TRAMP, were not modulated by HVS. Expression of CD30 was induced in HVS-transformed T cells, and these cells also expressed the CD30 ligand. Uninfected and transformed T cells were sensitive to CD95 ligation but resistant to apoptosis mediated by TRAIL or soluble TNF-α. CD95 ligand was constitutively expressed on transformed but not uninfected parental T cells. Both cell types showed similar sensitivity to cell death induction or inhibition of T-cell activation mediated by irradiation, oxygen radicals, dexamethasone, cyclosporine, and prostaglandin E2. Altogether, this study strongly suggests that growth transformation by HVS is based not on resistance to apoptosis but, rather, on utilization of normal cellular activation pathways.
Inducible Gene Expression from African Swine Fever Virus Recombinants: Analysis of the Major Capsid Protein p72
Authors: García-Escudero Ramón , Andrés Germán , Almazán Fernando , Viñuela Eladio 
Published: December 2019

ABSTRACT

A method to study the function of individual African swine fever virus (ASFV) gene products utilizing the
Escherichia coli lac
repressor-operator system has been developed. Recombinant viruses containing both the
lacI
gene encoding the
lac
repressor and a strong virus late promoter modified by the insertion of one or two copies of the
lac
operator sequence at various positions were constructed. The ability of each modified promoter to regulate expression of the firefly luciferase gene was assayed in the presence and in the absence of the inducer isopropyl β-
d
-thiogalactoside (IPTG). Induction and repression of gene activity were dependent on the position(s) of the operator(s) with respect to the promoter and on the number of operators inserted. The ability of this system to regulate the expression of ASFV genes was analyzed by constructing a recombinant virus inducibly expressing the major capsid protein p72. Electron microscopy analysis revealed that under nonpermissive conditions, electron-dense membrane-like structures accumulated in the viral factories and capsid formation was inhibited. Induction of p72 expression allowed the progressive building of the capsid on these structures, leading to assembly of ASFV particles. The results of this report demonstrate that the transferred inducible expression system is a powerful tool for analyzing the function of ASFV genes.
The Bovine Leukemia Virus Encapsidation Signal Is Composed of RNA Secondary Structures
Authors: Mansky Louis M. , Wisniewski Rebecca M. 
Published: December 2019

ABSTRACT

The encapsidation signal of bovine leukemia virus (BLV) was previously shown by deletion analysis to be discontinuous and to extend into the 5′ end of the
gag
gene (L. Mansky et al., J. Virol. 69:3282–3289, 1995). The global minimum-energy optimal folding for the entire BLV RNA, including the previously mapped primary and secondary encapsidation signal regions, was analyzed. Two stable stem-loop structures (located just downstream of the
gag
start codon) were predicted within the primary signal region, and one stable stem-loop structure (in the
gag
gene) was predicted in the secondary signal region. Based on these predicted structures, we introduced a series of mutations into the primary and secondary encapsidation signals in order to explore the sequence and structural information contained within these regions. The replication efficiency and levels of cytoplasmic and virion RNA were analyzed for these mutants. Mutations that disrupted either or both of the predicted stem-loop structures of the primary signal reduced the replication efficiency by factors of 7 and 40, respectively; similar reductions in RNA encapsidation efficiency were observed. The mutant with both stem-loop structures disrupted had a phenotype similar to that of a mutant containing a deletion of the entire primary signal region. Mutations that disrupted the predicted stem-loop structure of the secondary signal led to similar reductions (factors of 4 to 6) in both the replication and RNA encapsidation efficiencies. The introduction of compensatory mutations into mutants from both the primary and secondary signal regions, which restored the predicted stem-loop structures, led to levels of replication and RNA encapsidation comparable to those of virus containing the wild-type encapsidation signal. Replacement of the BLV RNA region containing the primary and secondary encapsidation signals with a similar region from human T-cell leukemia virus (HTLV) type 1 or type 2 led to virus replication at three-quarters or one-fifth of the level of the parental virus, respectively. The results from both the compensatory mutants and BLV-HTLV chimeras indicate that the encapsidation sequences are recognized largely by their secondary or tertiary structures.
The Receptor Binding Site of Feline Leukemia Virus Surface Glycoprotein Is Distinct from the Site Involved in Virus Neutralization
Authors: Ramsey I. K. , Spibey N. , Jarrett O. 
Published: December 2019

ABSTRACT
The external surface glycoprotein (SU) of feline leukemia virus (FeLV) contains sites which define the viral subgroup and induce virus-neutralizing antibodies. The subgroup phenotypic determinants have been located to a small variable region, VR1, towards the amino terminus of SU. The sites which function as neutralizing epitopes in vivo are unknown. Recombinant SU proteins were produced by using baculoviruses that contained sequences encoding the SUs of FeLV subgroup A (FeLV-A), FeLV-C, and two chimeric FeLVs (FeLV-215 and FeLV-VC) in which the VR1 domain of FeLV-A had been replaced by the corresponding regions of FeLV-C isolates. The recombinant glycoproteins, designated Bgp70-A, -C, -215, and -VC, respectively, were similar to their wild-type counterparts in several immunoblots and inhibited infection of susceptible cell lines in a subgroup-specific manner. Thus, Bgp70-A interfered with infection by FeLV-A, whereas Bgp70-C, -VC, and -215 did not. Conversely, Bgp70-C, -VC, and -215 blocked infection with FeLV-C, while Bgp70-A had no effect. These results indicate that the site on SU which binds to the FeLV cell surface receptor was preserved in the recombinant glycoproteins. It was also found that the recombinant proteins were able to bind naturally occurring neutralizing antibodies. Bgp70-A, -VC, and -215 interfered with the action of anti-FeLV-A neutralizing antibodies, whereas Bgp70-C did not. Furthermore, Bgp70-C interfered with the action of anti-FeLV-C neutralizing antibodies, while the other proteins did not. These results indicate that the neutralizing epitope(s) of FeLV SU lies outside the subgroup-determining VR1 domain.
Studies of the Nucleopolyhedrovirus Infection Process in Insects by Using the Green Fluorescence Protein as a Reporter
Authors: Barrett John W. , Brownwright Andy J. , Primavera Mark J. , Palli Subba Reddy 
Published: December 2019

ABSTRACT

A recombinant
Autographa californica
multicapsid nucleopolyhedrovirus (Ac
M
NPV) expressing the green fluorescence protein (GFP) under the control of the Ac
M
NPV polyhedrin promoter was constructed to study the spatial and temporal regulation of baculovirus infection in a permissive host. Larvae that ingested Ac
M
NPV-GFP showed localized expression of GFP in the midgut epithelial cells, as well as hemocytes, at 24 h postinfection. The presence of fluorescence in these tissues indicated not only that the virus was replicating but also that the very late viral proteins were being synthesized. Secondary infection occurred within the tracheal cells throughout the body cavity, confirming earlier reports, and these foci of infection allowed entry of the virus into other tissues, such as the epidermis and the fat body.
Mutational Analysis of the Rous Sarcoma Virus DR Posttranscriptional Control Element
Authors: Ogert Robert A. , Beemon Karen L. 
Published: December 2019

ABSTRACT

The direct repeat (DR) sequences flanking the
src
gene in Rous sarcoma virus are essential posttranscriptional control elements; at least one copy of this sequence is necessary for cytoplasmic accumulation of unspliced viral RNA. These sequences promote Rev-independent human immunodeficiency virus type 1 expression, suggesting they act as constitutive transport elements (CTEs). To determine which regions of this sequence are critical for CTE function, mutations in the downstream DR were generated and tested in a viral deletion construct lacking
src
and the upstream DR. Two single-point mutations and three different clustered mutations caused substantial reductions in reverse transcriptase activity, Gag protein levels, and unspliced viral RNA in the cytoplasm. Three conserved regions of the CTE, including nucleotides 8844 to 8847, 8862 to 8864, and 8868 to 8870, were most sensitive to inactivation by mutagenesis.
Evidence for Cooperation between Murine Leukemia Virus Env Molecules in Mixed Oligomers
Authors: Rein Alan , Yang Chinglai , Haynes Jacqueline A. , Mirro Jane , Compans Richard W. 
Published: December 2019

ABSTRACT
A retroviral Env molecule consists of a surface glycoprotein (SU) complexed with a transmembrane protein (TM). In turn, these complexes are grouped into oligomers on the surfaces of the cell and of the virion. In the case of murine leukemia viruses (MuLVs), the SU moieties are polymorphic, with SU proteins of different viral isolates directed towards different cell surface receptors. During maturation of the released virus particle, the 16 C-terminal residues of TM (the R peptide or p2E) are removed from the protein by the viral protease; this cleavage is believed to activate the membrane-fusing potential of MuLV Env. We have tested the possibility that different MuLV Env proteins in the same cell can interact with each other, both physically and functionally, in mixed oligomers. We found that coexpressed Env molecules can be precipitated out of cell lysates by antiserum which reacts with only one of them. Furthermore, they can evidently cooperate with each other: if one Env species lacks the R peptide, then it can apparently induce fusion if the SU protein of the other Env species encounters its cognate receptor on the surface of another cell. This functional interaction between different Env molecules has a number of implications with respect to the mechanism of induction of membrane fusion, for the genetic analysis of Env function, and for the design of targeted retroviral vectors for gene therapy.
Persistent Antibody Responses but Declining Cytotoxic T-Lymphocyte Responses to Multiple Human Immunodeficiency Virus Type 1 Antigens in a Long-Term Nonprogressing Individual with a Defective p17 Proviral Sequence and No Detectable Viral RNA Expression
Authors: Binley James M. , Jin Xia , Huang Yaoxing , Zhang Linqi , Cao Yunzhen , Ho David D. , Moore John P. 
Published: December 2019

ABSTRACTLong-term nonprogressor AD-18 has been infected with human immunodeficiency virus type 1 (HIV-1) for at least 16 years. During the past 5 years, he has had undetectable levels of plasma viremia, and HIV-1 cannot be isolated from him. Sequencing of proviral DNA indicates that the only HIV-1 sequences that can be identified in AD-18 have gross defects in the p17-encoding regions of thegaggene (Y. Huang, L. Zhang, and D. D. Ho, Virology 240:36–49, 1998). However, AD-18 has strong, sustained antibody responses to several HIV-1 antigens, including p17. Cytotoxic T-lymphocyte responses to Env and Gag antigens have gradually diminished over the past 4 years, at a time when the titers of antibodies to the same proteins have remained stable. We discuss what these observations might mean for the generation and maintenance of immunological memory.
CD4-Immunoglobulin G2 Protects Hu-PBL-SCID Mice against Challenge by Primary Human Immunodeficiency Virus Type 1 Isolates
Authors: Gauduin Marie-Claire , Allaway Graham P. , Olson William C. , Weir Raymond , Maddon Paul J. , Koup Richard A. 
Published: December 2019

ABSTRACT

CD4-immunoglobulin G2 (IgG2) is a fusion protein comprising human IgG2 in which the Fv portions of both heavy and light chains have been replaced by the V1 and V2 domains of human CD4. Previous studies found that CD4-IgG2 potently neutralizes a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates in vitro and ex vivo. The current report demonstrates that CD4-IgG2 protects against infection by primary isolates of HIV-1 in vivo, using the hu-PBL-SCID mouse model. Passive administration of 10 mg of CD4-IgG2 per kg of body weight protected all animals against subsequent challenge with 10 mouse infectious doses of the laboratory-adapted T-cell-tropic isolate HIV-1
LAI
, while 50 mg of CD4-IgG2 per kg protected four of five mice against the primary isolates HIV-1
JR-CSF
and HIV-1
AD6
. In contrast, a polyclonal HIV-1 Ig fraction exhibited partial protection against HIV-1
LAI
at 150 mg/kg but no significant protection against the primary HIV-1 isolates. The results demonstrate that CD4-IgG2 effectively neutralizes primary HIV-1 isolates in vivo and can prevent the initiation of infection by these viruses.
Foot-and-Mouth Disease Virus Virulent for Cattle Utilizes the Integrin α
v
β
3
as Its Receptor
Authors: Neff Sherry , Sá-Carvalho Daniel , Rieder Elizabeth , Mason Peter W. , Blystone Scott D. , Brown Eric J. , Baxt Barry 
Published: December 2019

ABSTRACT

Adsorption and plaque formation of foot-and-mouth disease virus (FMDV) serotype A
12
are inhibited by antibodies to the integrin α
v
β
3
(A. Berinstein et al., J. Virol. 69:2664–2666, 1995). A human cell line, K562, which does not normally express α
v
β
3
cannot replicate this serotype unless cells are transfected with cDNAs encoding this integrin (K562-α
v
β
3
cells). In contrast, we found that a tissue culture-propagated FMDV, type O
1
BFS, was able to replicate in nontransfected K562 cells, and replication was not inhibited by antibodies to the endogenously expressed integrin α
5
β
1
. A recent report indicating that cell surface heparan sulfate (HS) was required for efficient infection of type O
1
(T. Jackson et al., J. Virol. 70:5282–5287, 1996) led us to examine the role of HS and α
v
β
3
in FMDV infection. We transfected normal CHO cells, which express HS but not α
v
β
3
, and two HS-deficient CHO cell lines with cDNAs encoding human α
v
β
3
, producing a panel of cells that expressed one or both receptors. In these cells, type A
12
replication was dependent on expression of α
v
β
3
, whereas type O
1
BFS replicated to high titer in normal CHO cells but could not replicate in HS-deficient cells even when they expressed α
v
β
3
. We have also analyzed two genetically engineered variants of type O
1
Campos, vCRM4, which has greatly reduced virulence in cattle and can bind to heparin-Sepharose columns, and vCRM8, which is highly virulent in cattle and cannot bind to heparin-Sepharose. vCRM4 replicated in wild-type K562 cells and normal, nontransfected CHO (HS
+
α
v
β

3
−

) cells, whereas vCRM8 replicated only in K562 and CHO cells transfected with α
v
β
3
cDNAs. A similar result was also obtained in assays using a vCRM4 virus with an engineered RGD→KGE mutation. These results indicate that virulent FMDV utilizes the α
v
β
3
integrin as a primary receptor for infection and that adaptation of type O
1
virus to cell culture results in the ability of the virus to utilize HS as a receptor and a concomitant loss of virulence.