bluuebunny/crossref_metadata_2025 huggingface dataset:
The NS1 polypeptide of the murine parvovirus minute virus of mice binds to DNA sequences containing the motif [ACCA]2-3
Authors: Cotmore S F , Christensen J , Nüesch J P , Tattersall P
Published: January 2020

A DNA fragment containing the minute virus of mice 3' replication origin was specifically coprecipitated in immune complexes containing the virally coded NS1, but not the NS2, polypeptide. Antibodies directed against the amino- or carboxy-terminal regions of NS1 precipitated the NS1-origin complexes, but antibodies directed against NS1 amino acids 284 to 459 blocked complex formation. Using affinity-purified histidine-tagged NS1 preparations, we have shown that the specific protein-DNA interaction is of moderate affinity, being stable in 0.1 M salt but rapidly lost at higher salt concentrations. In contrast, generalized (or nonspecific) DNA binding by NS1 could be demonstrated only in low salt. Addition of ATP or gamma S-ATP enhanced specific DNA binding by wild-type NS1 severalfold, but binding was lost under conditions which favored ATP hydrolysis. NS1 molecules with mutations in a critical lysine residue (amino acid 405) in the consensus ATP-binding site bound to the origin, but this binding could not be enhanced by ATP addition. DNase I protection assays carried out with wild-type NS1 in the presence of gamma S-ATP gave footprints which extended over 43 nucleotides on both DNA strands, from the middle of the origin bubble sequence to a position some 14 bp beyond the nick site. The DNA-binding site for NS1 was mapped to a 22-bp fragment from the middle of the 3' replication origin which contains the sequence ACCAACCA. This conforms to a reiterated motif (ACCA)2-3, which occurs, in more or less degenerate form, at many sites throughout the minute virus of mice genome (J. W. Bodner, Virus Genes 2:167-182, 1989). Insertion of a single copy of the sequence (ACCA)3 was shown to be sufficient to confer NS1 binding on an otherwise unrecognized plasmid fragment. The functions of NS1 in the viral life cycle are reevaluated in the light of this result.
Bipartite DNA-binding region of the Epstein-Barr virus BMRF1 product essential for DNA polymerase accessory function
Authors: Kiehl A , Dorsky D I
Published: January 2020

The Epstein-Barr virus (EBV) BMRF1 gene product is necessary for DNA polymerase catalytic subunit (BALF5) activity in 100 mM ammonium sulfate. To map regions of BMRF1 necessary for polymerase accessory function, linker insertion and deletion mutant BMRF1 polypeptides were expressed by in vitro transcription-translation and assayed for DNA polymerase elongation activity and binding to double-stranded DNA (dsDNA)-cellulose. Amino-terminal deletions up to residue 303 were defective for stimulation of elongation. Deletions between residues 44 and 194 and residues 238 and 303 abolished binding to dsDNA-cellulose. The region from residues 194 to 238, therefore, is necessary for stimulation of BALF5 elongation but dispensable for dsDNA-cellulose binding. Deletion analysis also localized reactive epitopes of two neutralizing monoclonal antibodies to BMRF1 to a carboxy-terminal region which is dispensable for activity. These data suggest that a bipartite DNA-binding region is an essential component of the DNA polymerase accessory function and that the two noncontiguous regions are separated by a region (residues 194 to 217) which is essential for stimulation; therefore, it may interact with the BALF5 catalytic subunit of EBV DNA polymerase. Both EBV BMRF1 and herpes simplex virus UL42 gene products are DNA polymerase accessory proteins which bind dsDNA and increase the processivity of their corresponding catalytic components. Outstanding similarities between their primary amino acid sequences are not evident. However, it appears that their structural organizations are similar.
Canine distemper virus persistence in the nervous system is associated with noncytolytic selective virus spread
Authors: Zurbriggen A , Graber H U , Wagner A , Vandevelde M
Published: January 2020

Canine distemper virus (CDV), a negative-strand RNA morbillivirus, causes a progressive demyelinating disease in which virus persistence plays an essential role. The antiviral immune response leads to virus clearance in the inflammatory lesions. However, CDV can replicate and persist outside these inflammatory lesions within the brain. How CDV is capable of persisting in the presence of an effective antiviral immune response is poorly understood. In the present investigation, we studied several aspects of virus replication in primary dog brain cell cultures (DBCC), comparing an attenuated CDV strain and a virulent CDV strain. Confluent DBCC were infected with either virulent A75/17-CDV or attenuated Onderstepoort-CDV and monitored for 60 days. Persistence was not associated with defective virus production, because all mRNAs and corresponding proteins were continuously expressed in the noncytolytic infection. Quantitative measurements did not detect a difference between the two types of infection in the rate of virus transcription and protein synthesis at the level of the single cell. However, electron microscopy and virus titration experiments showed that in the persistent CDV infection virus budding is strongly limited compared with that of the attenuated virus. Morphometry and immunocytochemistry showed profound differences in the way the two viruses spread in the culture. The attenuated CDV spread randomly to immediately adjacent cells, whereas persistent CDV spread selectively to more-distant cells by way of cell processes. In conclusion, the present study supports a mechanism of CDV persistence through selective spread by way of cell processes, enabling virulent CDV to invade the central nervous system without the need of releasing much virus into the extracellular space.
Human cytomegalovirus UL102 gene
Authors: Smith J A , Pari G S
Published: January 2020

We have identified and characterized the transcript for the human cytomegalovirus (HCMV) UL102 gene. The UL102 gene product is proposed to encode the primase-associated factor. The primase-associated factor is one of the three components of the helicase-primase complex, along with UL105 (helicase) and UL70 (primase). In order to characterize the UL102 transcription unit we used single-stranded antisense RNA probes to identify an abundant 2.7-kb transcript originating from the UL102 region. This transcript can be initially detected at 24 h postinfection and in the presence of phosphonoformic acid but not in the presence of cycloheximide. A 2.7-kb cDNA clone containing this transcript was isolated from a 72-h HCMV (strain Towne) cDNA library. Sequence analysis of this clone revealed a continuous unspliced transcript between the region of UL101X and UL102; the only in-frame translational stop codon is 2,619 bp downstream from the first ATG in the message. Genome sequencing of the UL102 region from strains AD169 and Towne revealed that the UL101X stop codon TAA was actually TAC and that the cDNA and genomic sequences were in agreement. The cDNA clone starts 5 nucleotides (nt) upstream of the UL101X ATG, continues through the putative ATG of UL102, and ends 97 nt downstream of the putative termination codon of the UL102 open reading frame. Primer extension analysis indicated a transcriptional start site 23 nt upstream of the UL101X open reading frame.
An amino-terminal domain of the hepatitis C virus NS3 protease is essential for interaction with NS4A
Authors: Failla C , Tomei L , De Francesco R
Published: January 2020

Hepatitis C virus (HCV) genomic RNA is translated into a large polyprotein that is processed into structural and nonstructural proteins. Processing at the N termini of several nonstructural proteins requires sequences contained in both NS3 and NS4A. NS3 contains a serine protease, whereas the function of NS4A in proteolysis is yet to be determined. By using the vaccinia virus-T7 hybrid expression system to transiently express HCV polypeptides in HeLa cells, we studied the effect of several N-terminal and C-terminal deletions of HCV NS3 on the processing activity at all the downstream cleavage sites. In this way, we have delineated the minimal domain of NS3 required for the serine protease activity associated with this protein. In addition, we demonstrate the formation of a stable complex between NS3 and NS4A: analysis of the deletion mutants reveals a region at the N terminus of NS3 that is necessary for both complex formation and modulation of the proteolytic activity by NS4A but not for the NS4A-independent serine protease activity of NS3.
The central globular domain of the nucleocapsid protein of human immunodeficiency virus type 1 is critical for virion structure and infectivity
Authors: Ottmann M , Gabus C , Darlix J L
Published: January 2020

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is a 72-amino-acid peptide containing two CCHC-type zinc fingers linked by a short basic sequence, 29RAPRKKG35, which is conserved in HIV-1 and simian immunodeficiency virus. The complete three-dimensional structure of NCp7 has been determined by 1H-nuclear magnetic resonance spectroscopy (N. Morellet, H. de Rocquigny, Y. Mely, N. Jullian, H. Demene, M. Ottmann, D. Gerard, J. L. Darlix, M. C. Fournié-Zaluski, and B. P. Roques, J. Mol. Biol. 235:287-301, 1994) and revealed a central globular domain where the two zinc fingers are brought in close proximity by the RAPRKKG linker. To examine the role of this globular structure and more precisely of the RAPRKKG linker in virion structure and infectivity, we generated HIV-1 DNA mutants in the RAPRKK sequence of NCp7 and analyzed the mutant virions produced by transfected cells. Mutations that probably alter the structure of NCp7 structure led to the formation of very poorly infectious virus (A30P) or noninfectious virus (P31L and R32G). In addition, the P31L mutant did not contain detectable amounts of reverse transcriptase and had an immature core morphology, as determined by electron microscopy. On the other hand, mutations changing the basic nature of NCp7 had poor effect. R29S had a wild-type phenotype, and the replacement of 32RKK34 by SSS (S3 mutant) resulted in a decrease by no more than 100-fold of the virus titer. These results clearly show that the RAPRKKG linker contains residues that are critical for virion structure and infectivity.
Vaccinia virus gene H5R encodes a protein that is phosphorylated by the multisubstrate vaccinia virus B1R protein kinase
Authors: Beaud G , Beaud R , Leader D P
Published: January 2020

Vaccinia virus gene B1R encodes a protein kinase, the previously identified substrates of which include the proteins S2 and Sa of 40S ribosomal subunits. This work characterizes another substrate of the B1R kinase: a 36-kDa protein induced at the early stage of infection. Partially purified 36-kDa protein, eluted from a single-stranded DNA-cellulose column by 0.5 M NaCl, was separated by two-dimensional gel electrophoresis. Phosphorylation in vitro yielded multiple forms of the 36-kDa protein with approximate isoelectric points (pI) of 5.5, 5.7, 5.9, and 6.3, in addition to the apparently unphosphorylated form with a pI of approximately 6.8. The tryptic peptides derived from 36-kDa proteins with pI values of 5.7, 5.9, and 6.3 yielded almost identical high-pressure liquid chromatography profiles, strongly suggesting that the 36-kDa protein was modified by the phosphorylation of at least four sites, which were characterized as threonine residues. The amino acid sequence of several tryptic peptides derived from the 36-kDa protein showed that the 36-kDa protein was encoded by gene H5R of vaccinia virus. Consistent with this, the B1R kinase--either expressed in Escherichia coli or highly purified from HeLa cells--phosphorylated a recombinant trpE-H5R fusion protein in vitro. Fingerprints of the trpE-H5R and 36-kDa proteins phosphorylated by recombinant B1R kinase revealed common sites of phosphorylation, although some tryptic peptides were specific to either protein. Comparison was made of fingerprints of tryptic phosphopeptides derived from 36-kDa single-stranded DNA-binding protein labelled in vivo or in vitro. A common subset of peptides was observed, suggesting that some sites on H5R protein are phosphorylated by the B1R kinase in infected cells. These results suggest that some of the multiple threonine sites in the H5R protein are phosphorylated in vivo by the B1R protein kinase.
Direct interaction of the hepatitis B virus HBx protein with p53 leads to inhibition by HBx of p53 response element-directed transactivation
Authors: Truant R , Antunovic J , Greenblatt J , Prives C , Cromlish J A
Published: January 2020

Hepatitis B virus is a major risk factor in human hepatocellular carcinomas. We have used protein affinity chromatography to show that the 17-kDa hepatitis B virus gene product, HBx, binds directly to the human tumor suppressor gene product, p53. Interaction of HBx with p53 did not prevent p53 from specifically binding DNA. Instead, HBx enhanced p53's oligomerization state on a DNA oligonucleotide containing a p53 response element. Optimal binding of HBx to p53 required intact p53, but weaker binding to both the N-terminal activation domain of p53 and a protein fragment containing the C-terminal DNA-binding and oligomerization domains of p53 was observed. In transient transfection experiments with human Calu-6 cells, HBx inhibited transactivation by p53 of a reporter gene containing a p53 response element. Also, HBx inhibited p53-stimulated transcription in vitro even when added to the reaction mixture after the formation of the preinitiation complex. Interaction of HBx with p53 did not prevent the activation domain of p53 from binding two general initiation factors, the TATA-box binding protein subunit of TFIID and the p62 subunit of TFIIH. To explain these results, we propose that localization of HBx to a promoter by interaction with DNA-bound p53 enables a repression domain in HBx to directly contact the basal transcription machinery and thereby repress transcription.
Cellular proteins bind to the downstream component of the lytic origin of DNA replication of Epstein-Barr virus
Authors: Gruffat H , Renner O , Pich D , Hammerschmidt W
Published: January 2020

The lytic origin of DNA replication of Epstein-Barr virus, oriLyt, is a complex eukaryotic origin which is activated during the lytic phase of the viral life cycle. It consists of at least two independent cis-acting components, one of which plays a dual role in transcription and DNA replication. The binding of the viral factor BZLF1, a member of the AP1 family of transcription factors, to this upstream component is crucial for oriLyt function (A. Schepers, D. Pich, and W. Hammerschmidt, EMBO J. 12:3921-3929, 1993). The second cis-acting element, the downstream component of oriLyt, is equally indispensable; however, its function is unknown. In this study, the downstream component was found to be the binding target of several cellular proteins. One could be identified as Sp1 or as a related protein which binds twice to the downstream component of oriLyt. Mutational analysis indicated that Sp1 alone is not directly involved in mediating DNA replication; however, other factors which share the same binding sequence or bind closely to one of the Sp1 binding sites are likely candidates to contribute to a replication protein complex at the downstream component of oriLyt. The sequence requirements for the downstream component are remarkably stringent, indicating that at least one of the putative factors is a sequence-specific DNA-binding protein which is required for the activation of oriLyt.
Structural analysis of the principal immunodominant domain of the feline immunodeficiency virus transmembrane glycoprotein
Authors: Pancino G , Camoin L , Sonigo P
Published: January 2020

In the transmembrane envelope glycoprotein (TM) of lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV), two cysteine residues, conserved in most retroviruses, are thought to form a loop containing five to seven amino acids. These elements make up a B-cell epitope recognized by nearly 100% of sera from infected patients or animals, designated the principal immunodominant domain (PID). The PID amino acid sequences are highly conserved between isolates of the same lentivirus but are unrelated, except for the two cysteines, when divergent lentiviruses are compared. The aim of this study was to analyze the relationship between amino acid sequence in the PID and envelope function. We introduced two kinds of mutations in the PID of FIV: mutations which impeded the formation of a loop and mutations which substituted the sequence of FIV with the corresponding sequences from other lentiviruses, HIV-1, visna virus, and equine infectious anemia virus. We analyzed antibody recognition, processing, and fusogenic properties of the modified envelopes, using two methods of Env expression: a cell-free expression system and transfection of a feline fibroblast cell line with gag-pol-deleted FIV proviruses. Most mutations in the PID of FIV severely affected envelope processing and abolished syncytium formation. Only the chimeric envelope containing the HIV-1 PID sequence was correctly processed and maintained the capacity to induce syncytium formation, although less efficiently than the wild-type envelope. We computed three-dimensional structural models of the PID, which were consistent with mutagenesis data and confirmed the similarity of FIV and HIV-1 PID structures, despite their divergence in amino acid sequence. Considering these results, we discussed the respective importance of selection exerted by functional requirements or host antibodies to explain the observed variations of the PIDs in lentiviruses.
Interactions between human immunodeficiency virus type 1 and human cytomegalovirus in human term syncytiotrophoblast cells coinfected with both viruses
Authors: Tóth F D , Mosborg-Petersen P , Kiss J , Aboagye-Mathiesen G , Hager H , Juhl C B , Gergely L , Zdravkovic M , Aranyosi J , Lampé L
Published: January 2020

Human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1) may interact in the pathogenesis of AIDS. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. We analyzed the patterns of replication of HIV-1 and HCMV in singly an dually infected human term syncytiotrophoblast cells cultured in vitro. Syncytiotrophoblast cells exhibited restricted permissiveness for HIV-1, while HCMV replication was restricted at the level of immediate-early and early gene products in the singly infected cells. We found that the syncytiotrophoblasts as an overlapping cell population could be coinfected with HIV-1 and HCMV. HIV-1 replication was markedly upregulated by previous or simultaneous infection of the cells with HCMV, whereas prior HIV-1 infection of the cells converted HCMV infection from a nonpermissive to a permissive one. No simultaneous enhancement of HCMV and HIV-1 expression was observed in the dually infected cell cultures. Major immediate-early proteins of HCMV were necessary for enhancement of HIV-1 replication, and interleukin-6 production induced by HCMV and further increased by replicating HIV-1 synergized with these proteins to produce this effect. Permissive replication cycle of HCMV was induced by the HIV-1 tat gene product. We were unable to detect HIV-1 (HCMV) or HCMV (HIV-1) pseudotypes in supernatant fluids from dually infected cell cultures. Our results suggest that interactions between HIV-1 and HCMV in coinfected syncytiotrophoblast cells may contribute to the transplacental transmission of both viruses.
Physical association of moesin and CD46 as a receptor complex for measles virus
Authors: Schneider-Schaulies J , Dunster L M , Schwartz-Albiez R , Krohne G , ter Meulen V
Published: January 2020

Recently, two cellular membrane proteins, the membrane cofactor protein CD46 and the membrane-organizing external spike protein, moesin, have been identified to be functionally associated with measles virus (MV) infectivity of cells. We investigated the functional consequences of binding of monoclonal antibodies to both molecules individually and combined on MV attachment, fusion, and plaque formation and the putative direct physical interaction of moesin and CD46. We found that antibodies to moesin or CD46 separately inhibited MV-cell interactions to a high percentage in the plaque test, by approximately 85 and 75%, respectively. The inhibition by combinations of antibodies was additive at low concentrations and complete at high concentrations. This indicates that similar sites of interaction were blocked by steric hindrance. Furthermore, antimoesin antibodies blocked the infection of CD46-negative mouse cell lines with MV. Chemical cross-linking of cell surface proteins indicated the close proximity of CD46 and moesin in the membrane of human cells, and coimmunoprecipitation of moesin with CD46 suggested their physical interaction. Immunohistochemically by electron microscopy, CD46 and moesin were found to be localized at sites of the cellular membrane where MV particles adsorbed. These data support a model of direct interaction of CD46 and moesin in the cellular membrane and suggest that this complex is functionally involved in the uptake of MV into cells.
Characterization of neutralization epitopes in the V2 region of human immunodeficiency virus type 1 gp120: role of glycosylation in the correct folding of the V1/V2 domain
Authors: Wu Z , Kayman S C , Honnen W , Revesz K , Chen H , Vijh-Warrier S , Tilley S A , McKeating J , Shotton C , Pinter A
Published: January 2020

A number of monoclonal antibodies (MAbs) with various levels of neutralizing activity that recognize epitopes in the V1/V2 domain of LAI-related gp120s have been described. These include rodent antibodies directed against linear and conformational epitopes and a chimpanzee MAb, C108G, with extremely potent neutralizing activity directed against a glycan-dependent epitope. A fusion glycoprotein expression system that expressed the isolated V1/V2 domain of gp120 in native form was used to analyze the structural characteristics of these epitopes. A number of MAbs (C108G, G3-4, 684-238, SC258, 11/68b, 38/66a, 38/66c, 38/62c, and CRA3) that did not bind with high affinity to peptides immunoprecipitated a fusion glycoprotein expressing the V1/V2 domain of HXB2 gp120 in the absence of other human immunodeficiency virus sequences, establishing that their epitopes were fully specified within this region. Biochemical analyses indicated that in the majority of V1/V2 fusion molecules only five of the six glycosylation signals in the V1/V2 domain were utilized, and the glycoforms were found to be differentially recognized by particular MAbs. Both C108G and MAbs directed against conformational epitopes reacted with large fractions of the fully glycosylated molecules but with only small fractions of the incompletely glycosylated molecules. Mutational analysis of the V1 and V2 glycosylation signals indicated that in most cases the unutilized site was located either at position 156 or at position 160, suggesting the occurrence of competition for glycan addition at these neighboring positions. Mutation of glycosylation site 160 destroyed the C108G epitope but increased the fraction of the molecules that presented the conformational epitopes, while mutation of the highly conserved glycosylation site at position 156 greatly diminished the expression of the conformational epitopes and increased expression of the C108G epitope. Similar heterogeneity in glycosylation was also observed when the HXB2 V1/V2 fusion glycoprotein was expressed without most of the gp70 carrier protein, and thus, this appeared to be an intrinsic property of the V1/V2 domain. Heterogeneity in expression of conformational and glycan-dependent epitopes was also observed for the natural viral env precursor, gPr160, but not for gp120. These results suggested that the closely spaced glycosylation sites 156 and 160 are often alternatively utilized and that the pattern of glycosylation at these positions affects the formation of the conformational structures needed for both expression of native epitopes in this region and processing of gPr160 to mature env products.
Intraspecific phylogenetics: support for dental transmission of human immunodeficiency virus
Authors: Crandall K A
Published: January 2020

A new method to estimate within-species gene genealogies was used to establish linkages among individuals associated with the Florida dental human immunodeficiency virus transmission case. Phylogenetic relationships were estimated from 103 nucleotide sequences from the V3 region of the env gene representing the Florida dentist, eight of his seropositive patients, and many local controls. The cladogram estimation procedure supports linkages among individuals within the previously described dental clade, whereas local controls and other patients form independent networks or are outliers in the main network, indicating more distant evolutionary relationships. A nested statistical analysis also indicates significant cohesion of the dental clade group.
Immunogenic targeting of recombinant peptide vaccines to human antigen-presenting cells by chimeric anti-HLA-DR and anti-surface immunoglobulin D antibody Fab fragments in vitro
Authors: Baier G , Baier-Bitterlich G , Looney D J , Altman A
Published: January 2020

To increase the inherently weak immunogenicity of synthetic peptide vaccines, we used recombinant DNA techniques to generate chimeras between immunogenic determinants of human immunodeficiency virus type 1 (HIV-1) gp120 and antibody Fab fragments reactive with surface structures displayed specifically on human antigen-presenting cells (APCs), including surface immunoglobulin D (sIgD) and class II major histocompatibility complex (MHC) molecules. Hybridomas producing anti-human MHC class II (HLA-DR) or surface immunoglobulin D monoclonal antibodies (MAbs) that recognize nonpolymorphic determinants were used to clone chimeric Fab gene fragments by employing an established procedure to generate antigen-binding Fab libraries in phagemid vector pComb3. Molecular and immunochemical analysis indicated that the expected chimeric Fab fragments expressing the HIV-1 epitopes were correctly cloned and expressed in Escherichia coli and retained the binding specificity of the native (hybridoma-derived) MAb. The chimeric Fab fragments targeted the linked HIV-1-derived antigenic determinants to the surface of human APCs in vitro, as evidenced by fluorescence-activated cell sorter analysis. Furthermore, such recombinant immunotargeted HIV-1 peptide antigens demonstrated improved immunogenicity over equivalent nonimmunotargeted control antigens, as shown by their ability to stimulate interleukin-2 production by CD4+ T-helper cells from human donors exposed to HIV-1 antigens. These data suggest that immunotargeting of recombinant peptide antigens via the attached Fab fragments facilitates uptake by human APCs with subsequent access to the MHC class II processing pathway, thereby validating the immunotargeting concept for such recombinant subunit vaccines in an in vitro human system.
Inhibition of apoptosis in human immunodeficiency virus-infected cells enhances virus production and facilitates persistent infection
Authors: Antoni B A , Sabbatini P , Rabson A B , White E
Published: January 2020

Apoptosis is one of several mechanisms by which human immunodeficiency virus type 1 (HIV-1) exerts its cytopathic effects. CD4+ Jurkat T-cell lines overexpressing the adenovirus E1B 19K protein, a potent inhibitor of apoptosis, were used to examine the consequences of inhibition of apoptosis during acute and chronic HIV-1 infections. E1B 19K protein expression inhibited HIV-induced apoptosis, enhanced virus production, and established high levels of persistent viral infection. One E1B 19K-expressing line appeared to undergo HIV-induced death via a nonapoptotic mechanism, illustrating that HIV infection results in lymphocyte depletion through multiple pathways. Increased virus production associated with sustained cell viability suggests that therapeutic approaches involving inhibition of HIV-induced programmed cell death may be problematic.
Conservation of an intact human immunodeficiency virus type 1 vif gene in vitro and in vivo
Authors: Sova P , van Ranst M , Gupta P , Balachandran R , Chao W , Itescu S , McKinley G , Volsky D J
Published: January 2020

Replication of vif-negative human immunodeficiency virus type 1 (HIV-1) is attenuated in certain cell lines and highly impaired in peripheral blood lymphocytes in vitro. To determine whether intact vif is positively selected during natural HIV-1 infection and to determine vif sequence variability, we employed PCR amplification, cloning, and sequencing to investigate the vif region of replicating virus in short-term-passage HIV-1 primary isolates from five asymptomatic individuals and from five persons with AIDS. A total of 46 vif clones were obtained and analyzed. Recombinant proviruses were constructed from selected vif clones from one patient and found to be fully infectious. We found that 38 of the 46 clones sequenced carried open vif reading frames and that there was a low degree of heterogeneity of vif genes within isolates from the same individual and among isolates from different donors. The cysteines previously found to be essential for vif protein function were conserved in all clones. A phylogenetic tree constructed from all available vif nucleotide sequences resulted in a virus grouping similar to those of gag and env. Direct sequencing of vif amplified by PCR from uncultured lymphocytes of 15 individuals at various stages of progression toward AIDS demonstrated vif open reading frames in 13 of 15 samples tested. There was no obvious correlation between disease status and the presence of an intact vif within this sample group at the time of sample procurement. The conservation of the vif open reading frame in vitro and in vivo and its limited variability following virus transmission in vitro are consistent with a role for vif in natural HIV-1 infection.
Delineating minimal protein domains and promoter elements for transcriptional activation by lentivirus Tat proteins
Authors: Southgate C D , Green M R
Published: January 2020

Lentivirus Tat proteins comprise a novel class of RNA-binding transcriptional activators that are essential for viral replication. In this study, we performed a series of protein fusion experiments to delineate the minimal protein domains and promoter elements required for Tat action. We show that a 15-amino-acid region of equine infectious anemia virus (EIAV) Tat protein, when fused to the GAL4 or LexA DNA binding domain, can activate transcription in appropriate promoter contexts. In the natural human immunodeficiency virus type 1 long terminal repeat, activation by Tat is dependent on multiple binding sites for the cellular transcription factor SP1. We delineate a 114-amino-acid region of the SP1 glutamine-rich activation domain that when fused to the GAL4 DNA binding domain can support transcription activation by Tat. Using these Tat and SP1 derivatives, we show that Tat activation can be reconstructed on a completely synthetic promoter lacking all cis-acting elements unique to the human immunodeficiency virus long terminal repeat. Our results indicate that lentivirus Tat proteins have essential properties of typical cellular transcriptional activators and define useful reagents for studying the detailed mechanism of Tat action.
Chromosome mapping of Rfv3, a host resistance gene to Friend murine retrovirus
Authors: Hasenkrug K J , Valenzuela A , Letts V A , Nishio J , Chesebro B , Frankel W N
Published: January 2020

Inoculation of adult mice with Friend virus complex usually induces rapid viremia and erythroleukemia, resulting in death in 1 to 3 months. In certain mouse strains, a single host gene, Rfv3, controls the ability to mount a virus-specific neutralizing antibody response which results in elimination of viremia. In this study, microsatellite markers were used to localize the Rfv3 gene to a 20-centimorgan region of mouse chromosome 15 unlinked to immunoglobulin loci, T-cell receptor loci, or the major histocompatibility complex. Potential candidate genes for Rfv3 are several genes expressed in cells of the immune system and previously mapped to the same region, including a T-cell antigen gene, Ly6, and three cytokine receptor genes, IL2rb, IL3rb1, and IL3rb2.
Regulatory function of the equine herpesvirus 1 ICP27 gene product
Authors: Zhao Y , Holden V R , Smith R H , O'Callaghan D J
Published: January 2020

The UL3 protein of equine herpesvirus 1 (EHV-1) KyA strain is a homolog of the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1) and the ORF 4 protein of varicella-zoster virus. To characterize the regulatory function of the UL3 gene product, a UL3 gene expression vector (pSVUL3) and a vector expressing a truncated version of the UL3 gene (pSVUL3P) were generated. These effector plasmids, in combination with an EHV-1 immediate-early (IE) gene expression vector (pSVIE) and chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs, were used in transient transfection assays. These assays demonstrated that the EHV-1 UL3 gene product is a regulatory protein that can independently trans activate the EHV-1 IE promoter; however, this effect can be inhibited by the repressive function of the IE gene product on the IE promoter (R. H. Smith, G. B. Caughman, and D. J. O'Callaghan, J. Virol. 66:936-945, 1992). In the presence of the IE gene product, the UL3 gene product significantly augments gene expression directed by the promoters of three EHV-1 early genes (thymidine kinase; IR4, which is the homolog of HSV-1 ICP22; and UL3 [ICP27]) and the promoter of the EHV-1 late gene IR5, which is the homolog of HSV-1 US10. Sequences located at nucleotides -123 to +20 of the UL3 promoter harbor a TATA box, SP1 binding site, CAAT box, and octamer binding site and, when linked to the CAT reporter gene, are trans activated to maximal levels by the pSVIE construct in transient expression assays. Results from CAT assays also suggest that the first 11 amino acids of the UL3 protein are not essential for the regulatory function of the UL3 gene product.
Interaction of eukaryotic initiation factor eIF-4B with a picornavirus internal translation initiation site
Authors: Meyer K , Petersen A , Niepmann M , Beck E
Published: January 2020

We studied the interaction of cellular proteins with the internal ribosome entry site (IRES) of foot-and-mouth disease virus by UV cross-linking and observed specific binding of a 80-kDa protein contained in cytosolic HeLa cell extract and in rabbit reticulocyte lysate. Binding of the protein was dependent on the presence of ATP. Immunoprecipitation with eIF-4B antiserum revealed that the protein is identical to the initiation factor eIF-4B. Deletions in the 3' part, but not in the 5' part, of the IRES interfered with UV cross-linking, indicating that the binding site of eIF-4B is located close to the end of the element. Attempts to separate ribosome-associated from non-ribosome-associated protein fractions of cytosolic cell extracts led to the loss of cross-linking activity. This finding suggests that additional protein factors contribute to this interaction of eIF-4B with the IRES of foot-and-mouth disease virus.
Transcriptional activity of core binding factor-alpha (AML1) and beta subunits on murine leukemia virus enhancer cores
Authors: Zaiman A L , Lewis A F , Crute B E , Speck N A , Lenz J
Published: January 2020

Core binding factor (CBF), also known as polyomavirus enhancer-binding protein 2 and SL3 enhancer factor 1, is a mammalian transcription factor that binds to an element termed the core within the enhancers of the murine leukemia virus family of retroviruses. The core elements of the SL3 virus are important genetic determinants of the ability of this virus to induce T-cell lymphomas and the transcriptional activity of the viral long terminal repeat in T lymphocytes. CBF consists of two subunits, a DNA binding subunit, CBF alpha, and a second subunit, CBF beta, that stimulates the DNA binding activity of CBF alpha. One of the genes that encodes a CBF alpha subunit is AML1, also called Cbf alpha 2. This locus is rearranged by chromosomal translocations in human myeloproliferative disorders and leukemias. An exogenously expressed Cbf alpha 2-encoded subunit (CBF alpha 2-451) stimulated transcription from the SL3 enhancer in P19 and HeLa cells. Activity was mediated through the core elements. Three different isoforms of CBF beta were also tested for transcriptional activity on the SL3 enhancer. The longest form, CBF beta-187, increased the transcriptional stimulation by CBF alpha 2-451 twofold in HeLa cells, although it had no effect in P19 cells. Transcriptional activation by CBF beta required binding to the CBF alpha subunit, as a form of CBF beta that lacked binding ability, CBF beta-148, failed to increase activity. These results indicated that at least in certain cell types, the maximum activity of CBF required both subunits. They also provided support for the hypothesis that CBF is a factor in T lymphocytes that is responsible for recognition of the SL3 cores. We also examined whether CBF could distinguish a 1-bp difference between the enhancer core of SL3 and the core of the nonleukemogenic virus, Akv. This difference strongly affects transcription in T cells and leukemogenicity of SL3. However, no combination of CBF alpha and CBF beta subunits that we tested was able to distinguish the 1-bp difference in transcription assays. Thus, a complete understanding of how T cells recognize the SL3 core remains to be elucidated.
In vitro packaging of individual genomic segments of bacteriophage phi 6 RNA: serial dependence relationships
Authors: Qiao X , Casini G , Qiao J , Mindich L
Published: January 2020

Bacteriophage phi 6 has a genome of three segments of double-stranded RNA enclosed in a procapsid composed of four different proteins. The preformed procapsid is capable of packaging plus-strand transcripts of the genomic segments in an in vitro reaction. The packaging of the three segments shows a strong order of dependence in that segment S packages alone, but segment M requires S and and segment L requires S and M for efficient packaging. Packaging of individual segments is dependent on unique packaging sequences of about 200 nucleotides near the 5' ends of the segments. Deletions that invade these regions destroy packaging competence for the particular segment and for the dependent segments as well. In the presence of 2 mM phosphate and at magnesium ion concentrations above 4 mM, packaging becomes progressively more independent and ultimately nonspecific with respect to phi 6 sequences.
The N-terminal region of the adenovirus type 5 E1A proteins can repress expression of cellular genes via two distinct but overlapping domains
Authors: Dorsman J C , Hagmeyer B M , Veenstra J , Elfferich P , Nabben N , Zantema A , van der Eb A J
Published: January 2020

The transforming E1A 12S and E1A 13S proteins of human adenovirus type 5 (Ad5) contain two and three conserved regions, respectively. In the present study, the contribution of sequences in the nonconserved N-terminal region of the E1A proteins to morphological transformation and to down-regulation of a number of mitogen-inducible genes was investigated. As described previously, transformation of NRK cells (an established normal rat kidney cell line) results in denser cell growth and a cuboidal cellular morphology. None of the cells expressing N-terminally mutated E1A proteins, however, show these transformed properties, which suggests an important role for sequences in that domain. The decrease in cyclin D1 levels requires exactly the same sequences. The ability to transform NRK cells and to reduce cyclin D1 levels does not correlate with the presence in the E1A proteins of binding domains for p300, CBP, p107, pRb, cyclin A, or cdk2. In contrast, down-regulation of expression of the JE gene in NRK cells and repression of transcription of the collagenase gene in human HeLa cells does correlate with the presence in the E1A proteins of an intact binding domain for p300 and CBP. The results suggest that the N-terminal domain of the E1A proteins can repress expression of cellular genes by at least two different mechanisms.
Macaques immunized with HLA-DR are protected from challenge with simian immunodeficiency virus
Authors: Arthur L O , Bess J W , Urban R G , Strominger J L , Morton W R , Mann D L , Henderson L E , Benveniste R E
Published: January 2020

Macaques immunized with uninfected human cells have been shown to be protected from challenge with simian immunodeficiency virus (SIV) propagated in human cells. To identify the potential antigens involved in this protection, macaques were immunized with uninfected human cells, sucrose density gradient-purified culture fluid from uninfected human cells (mock virus), beta-2 microglobulin (beta 2M), immunoaffinity-purified HLA class I and class II proteins from these human cells, and adjuvant. Although all macaques immunized with beta 2M and HLA class I developed high antibody titers to beta 2M, these animals were not protected from a subsequent challenge with infectious SIV grown in human cells. In contrast, the macaques immunized with class II protein (HLA-DR) and mock virus developed antibodies to class II protein and were protected from the intravenous infectious virus challenge. The class II protein- and mock virus-immunized animals which were protected from challenge were given boosters of the appropriate antigen and challenged with the same SIV propagated in macaque cells. All animals became infected, indicating that the protection seen with human class II protein did not extend to protection from infection with SIV containing macaque class II proteins. Since the virus released from SIV-infected macaque cells would contain macaque class II proteins, our results suggest that the initial SIV infected was completely prevented. In addition, the lack of protection from challenge with SIV propagated in macaque cells provided strong evidence that the protection was due to an immune response to the cellular proteins and not to epitopes cross-reactive between class II proteins and the viral proteins, since the identical virus proteins were present in both challenge stocks. These results are the first demonstration that immunization with a purified cellular protein can protect from virus infection.
Theiler's virus as a vector for foreign gene delivery
Authors: Zhang L , Sato S , Kim J I , Roos R P
Published: January 2020

DA strain and other strains of the TO subgroup of Theiler's murine encephalomyelitis viruses are members of the Cardiovirus genus of picornaviruses and produce a persistent demyelinating disease in mice. A recent study from our laboratory (W.-P. Kong, G. D. Ghadge, and R. P. Roos, Proc. Natl. Acad. Sci. USA 91:1796-1800, 1994) demonstrated that the leader, which is encoded at the N terminus of the Theiler's murine encephalomyelitis virus polyprotein, can be partially replaced by foreign sequences as well as completely deleted, with no loss of infectivity in BHK-21 cells. In this study, we have inserted up to 724 nucleotides into the leader coding region of an infectious DA clone. Recombinant viruses were produced, and the inserts were shown to be stable for at least three passages in BHK-21 cells.
Attenuated Mengo virus: a new vector for live recombinant vaccines
Authors: Altmeyer R , Girard M , van der Werf S , Mimic V , Seigneur L , Saron M F
Published: January 2020

Several features make Mengo virus an excellent candidate for use as a vaccine vector. The virus has a wide host range, including rodents, pigs, monkeys, and most likely humans, and expresses its genome exclusively in the cytoplasm of the infected cell. Stable attenuated strains exist which are deleted for part of the 5' noncoding region of the genome. Here we report an attenuated Mengo virus recombinant, vLCMG4, that encodes an immunodominant cytotoxic T-lymphocyte epitope of the lymphocytic choriomeningitis virus (LCMV) nucleo-protein. vLCMG4 induced protective immunity against lethal LCMV infection after a single, low-dose immunization in BALB/c mice and elicited an LCMV-specific CD8+ cytotoxic T lymphocyte response. This demonstrates the potential of recombinant Mengo virus vaccines to confer protection against infectious diseases by the induction of cellular immune responses.
Integration is required for productive infection of monocyte-derived macrophages by human immunodeficiency virus type 1
Authors: Englund G , Theodore T S , Freed E O , Engelman A , Martin M A
Published: January 2020

Certain human immunodeficiency virus type 1 (HIV-1) isolates are able to productively infect nondividing cells of the monocyte/macrophage lineage. We have used a molecular genetic approach to construct two different HIV-1 integrase mutants that were studied in the context of an infectious, macrophage-tropic HIV-1 molecular clone. One mutant, HIV-1 delta D(35)E, containing a 37-residue deletion within the central, catalytic domain of integrase, was noninfectious in both peripheral blood mononuclear cells and monocyte-derived macrophages. The HIV-1 delta D(35)E mutant, however, exhibited defects in the assembly and/or release of progeny virions in transient transfection assays, as well as defects in entry and/or viral DNA synthesis during the early stages of monocyte-derived macrophage infection. The second mutant, HIV-1D116N/8, containing a single Asp-to-Asn substitution at the invariant Asp-116 residue of integrase, was also noninfectious in both peripheral blood mononuclear cells and monocyte-derived macrophages but, in contrast to HIV-1 delta D(35)E, was indistinguishable from wild-type virus in reverse transcriptase production. PCR analysis indicated that HIV-1D116N/8 entered monocyte-derived macrophages efficiently and reverse transcribed its RNA but was unable to complete its replication cycle because of a presumed block to integration. These data are consistent with the hypothesis that integration is an obligate step in productive HIV-1 infection of activated peripheral blood mononuclear cells and primary human macrophage cultures.
The bovine leukemia virus encapsidation signal is discontinuous and extends into the 5' end of the gag gene
Authors: Mansky L M , Krueger A E , Temin H M
Published: January 2020

In order to define bovine leukemia virus (BLV) sequences required for efficient vector replication, a series of mutations were made in a BLV vector. Testing the replication efficiency of the vectors with a helper virus and helper plasmids allowed for separation of the mutant vectors into three groups. The replication efficiency of the first group was reduced by a factor of 7; these mutants contained deletions in the 5' end of the gag gene. The second group of mutants had replication reduced by a factor of 50 and had deletions including the 5' untranslated leader region. The third group of mutants replicated at levels comparable to those of the parental vector and contained deletions of the 3' end of the gag gene, the pol gene, and the env gene. Analysis of cytoplasmic and virion RNA levels indicated that vector RNA expression was not affected but that the vector RNA encapsidation was less efficient for group 1 and group 2 mutants. Additional mutations revealed two regions important for RNA encapsidation. The first region is a 132-nucleotide-base sequence within the gag gene (nucleotides 1015 to 1147 of the proviral DNA) and facilitates efficient RNA encapsidation in the presence of the second region. The second region includes a 147-nucleotide-base sequence downstream of the primer binding site (nucleotide 551) and near the gag gene start codon (nucleotide 698; gag begins at nucleotide 628) and is essential for RNA encapsidation. We conclude that the encapsidation signal is discontinuous; a primary signal, essential for RNA encapsidation, is largely in the untranslated leader region between the primer binding site and near the gag start codon. A secondary signal, which facilitates efficient RNA encapsidation, is in a 132-nucleotide-base region within the 5' end of the gag gene.