bluuebunny/crossref_metadata_2025 huggingface dataset:
Complementation of a primer binding site-impaired murine leukemia virus-derived retroviral vector by a genetically engineered tRNA-like primer
Authors: Lund A H , Duch M , Lovmand J , Jørgensen P , Pedersen F S
Published: January 2020

Reverse transcription of retroviral genomes is primed by a tRNA annealed to an 18-nucleotide primer binding site. Here, we present a primer complementation system to study molecular interaction of the replication machinery with the primer and primer binding site in vivo. Introduction of eight base substitutions into the primer binding site of a murine leukemia virus-based vector allowed efficient RNA encapsidation but resulted in severely reduced vector replication capacity. Replication was restored upon complementation with a synthetic gene designed to encode a complementary tRNA-like primer, but not with a noncomplementary tRNA-like molecule. The engineered primer was shown to be involved in both the initiation of first-strand synthesis and second-strand transfer. These results provide an in vivo demonstration that the retroviral replication machinery may recognize sequence complementarity rather than actual primer binding site and 3' primer sequences. Use of mutated primer binding site vectors replicating via engineered primers may add additional control features to retroviral gene transfer technology.
Second-site proviral enhancer alterations in lymphomas induced by enhancer mutants of SL3-3 murine leukemia virus: negative effect of nuclear factor 1 binding site
Authors: Ethelberg S , Hallberg B , Lovmand J , Schmidt J , Luz A , Grundström T , Pedersen F S
Published: January 2020

SL3-3 is a highly T-lymphomagenic murine retrovirus. Previously, mutation of binding sites in the U3 repeat region for the AML1 transcription factor family (also known as core binding factor [CBF], polyomavirus enhancer binding protein 2 [PEBP2], and SL3-3 enhancer factor 1 [SEF1]) were found to strongly reduce the pathogenicity of SL3-3 (B. Hallberg, J. Schmidt, A. Luz, F. S. Pedersen, and T. Grundström, J. Virol. 65:4177-4181, 1991). We have now examined the few cases in which tumors developed harboring proviruses that besides the AML1 (core) site mutations carried second-site alterations in their U3 repeat structures. In three distinct cases we observed the same type of alteration which involved deletions of regions known to contain binding sites for nuclear factor 1 (NF1) and the addition of extra enhancer repeat elements. In transient-expression experiments in T-lymphoid cells, these new U3 regions acted as stronger enhancers than the U3 regions of the original viruses. This suggests that the altered proviruses represent more-pathogenic variants selected for in the process of tumor formation. To analyze the proviral alterations, we generated a series of different enhancer-promoter reporter constructs. These constructs showed that the additional repeat elements are not critical for enhancer strength, whereas the NF1 sites down-regulate the level of transcription in T-lymphoid cells whether or not the AML1 (core) sites are functional. We therefore also tested SL3-3 viruses with mutated NF1 sites. These viruses have unimpaired pathogenic properties and thereby distinguish SL3-3 from Moloney murine leukemia virus.
Investigation of the dynamics of the spread of human immunodeficiency virus to brain and other tissues by evolutionary analysis of sequences from the p17gag and env genes
Authors: Hughes E S , Bell J E , Simmonds P
Published: January 2020

The time of spread of human immunodeficiency virus type 1 (HIV-1) from lymphoid to nonlymphoid tissues in the course of infection was investigated by sequence comparisons of variants infecting a range of lymphoid and nonlymphoid tissues from three individuals with AIDS in the pl7gag gene and regions flanking the V1/V2 hypervariable regions. Phylogenetic analysis in both regions revealed several lineages in each individual that contained sequences from both lymphoid and nonlymphoid tissues such as the brain. This observation contrasts strongly with the previously described organ-specific sequences in the V3 region in this study population and other investigations. Although individual pairwise comparisons of relatively short sequences such as p17gag are subject to considerable stochastic error, we found that the diversity of gag sequences in variants from lymphoid tissue was consistently lower than that found among variants amplified from the brain. By estimating mean synonymous pairwise distances in the p17gag region, we were able to make an approximate calculation of the ages of populations in different tissues. Those from lymphoid tissue ranged from 2.65 to 5.6 years in the three study subjects, compared with 4.1 to 6.2 years for variants in the brain. Indeed, variants infecting the brain were no more closely related to each other than they were to variants infecting other tissues in the body. In two of the three individuals, these times of divergence indicate that infection of the brain may have occurred as an early event in the progression to disease, preceding the onset of AIDS by several years. This study is the first in which it was possible to estimate times of diversification in different tissues in vivo and is of importance in understanding the dynamics of the spread of HIV-1 into nonlymphoid tissues and its possible adaptation for replication in different cell types.
Marek's disease virus latency-associated transcripts belong to a family of spliced RNAs that are antisense to the ICP4 homolog gene
Authors: Cantello J L , Parcells M S , Anderson A S , Morgan R W
Published: January 2020

Marek's disease virus (MDV) latency-associated transcripts include at least two MDV small RNAs (MSRs) and a 10-kb RNA which map antisense to the ICP4 homolog gene and are relatively abundant in MDV-transformed lymphoblastoid cells. This report further describes the biological and structural properties of these RNAs. First, these RNAs were detected in primary lymphomas isolated from chickens infected with several oncogenic MDV strains. Second, the MSRs are nonpolyadenylated, whereas, the 10-kb RNA is predominantly polyadenylated. Third, MSRs localize to the nuclei of both lymphoblastoid cells and cytolytically infected chicken embryo fibroblasts. Fourth, the 3'-region splice junctions of the MSRs during latent and productive infection were determined by sequencing RNA-PCR products generated with primers that flank the 3' splice region. The MSRs contain at least three introns, the largest of which overlaps the ICP4 putative translational start site. Fifth, the 5' end of the MSRs initiates approximately 5 kb upstream from the main body of the RNA. The extreme 5' exon is approximately 251 nucleotides (nt) long and is joined to the main body of the transcript upon removal of a 4,852-nt intron. Finally, the 10-kb RNA lies entirely within the repeats flanking the unique short region of the genome. We believe that the MSRs and 10-kb RNA belong to a family of spliced RNAs that map antisense to the ICP4 gene and comprise a complex transcriptional unit expressed during MDV-induced T-cell transformation.
The N terminus of hamster polyomavirus middle T antigen carries a determinant for specific activation of p59c-Fyn
Authors: Goutebroze L , Dunant N M , Ballmer-Hofer K , Feunteun J
Published: January 2020

Transformation by rodent polyomaviruses is mediated primarily by middle T antigen, a membrane-bound protein that does not carry an intrinsic enzymatic activity but interacts and subverts the activity of cellular regulators of proliferation. The multiple protein partners of murine polyomavirus (Py) middle T antigen include the tyrosine kinases c-Src and, to a lesser extent, c-Fyn and c-Yes. By contrast, the hamster polyomavirus (HaPV) middle T antigen selectively activates the c-Fyn gene product. This difference may account for the contrasting tumor patterns induced by the two viruses. The sequences of the respective N-terminal and C-terminal functional domains of murine Py and HaPV middle T antigens are highly conserved whereas the intervening stretches are clearly divergent, leading to the speculation that this divergence may direct the specificity for tyrosine kinase activation. We have addressed this issue by constructing a chimera middle T antigen molecule carrying the N-terminal domain from HaPV (exon 1) in phase with the other two domains from murine Py (exon 2). The biological properties of this chimera molecule are indistinguishable from those of HaPV middle T antigen; it specifically activates p59c-Fyn and carries the transforming phenotype of the HaPV middle T antigen on rat fibroblasts.
Late expression of a beta chemokine homolog by murine cytomegalovirus
Authors: MacDonald M R , Li X Y , Virgin H W
Published: January 2020

A gene (HJ1) present in murine cytomegalovirus (MCMV) encodes an open reading frame (ORF) whose predicted amino acid sequence shows homology to the beta (or C-C) class of chemotactic cytokines known as chemokines. The region of homology is located in the carboxyl-terminal portion of the 116-amino-acid ORF. A 0.9-kb RNA transcript corresponding to the HJ1 gene is expressed in MCMV-infected fibroblasts and macrophages as a member of the late kinetic class of virally encoded transcripts. A transcriptional start site is located between the third and fourth methionine residues in the ORF, predicting a primary amino acid structure, starting at the fourth methionine residue, which includes a possible signal peptide as well as conserved cysteine residues typical for mammalian beta chemokines. The RNA transcript is polyadenylated, suggesting that it can undergo translation within the cytoplasm of an MCMV-infected cell. We speculate that expression of the chemokine homolog at late times in the MCMV replication cycle plays a role in MCMV pathogenesis, possibly by action as a chemotactic agonist or antagonist or by alteration of the activation or differentiation state of a susceptible cell such as a macrophage.
Rhodanine resistance and dependence of echovirus 12: a possible consequence of capsid flexibility
Authors: Kraus W , Zimmermann H , Eggers H J , Nelsen-Salz B
Published: January 2020

Recombinant viruses of echovirus 12 carrying mutations of a rhodanine-resistant or -dependent variant, were investigated, and five single mutations each inducing a rhodanine-resistant or -dependent phenotype were defined. Four mutations are localized in the capsid protein VP1, and the fifth exchange is in VP4. All original and recombinant viruses were shown to be stabilized by the antiviral drug rhodanine against heat inactivation. Hence, resistant and dependent variants still seem able to bind rhodanine, and apparently none of the exchanges affects the putative drug binding site. We hypothesize that drug resistance and dependence are consequences of an increased flexibility of the virus capsid.
Analysis of the 2-kilobase latency-associated transcript expressed in PC12 cells productively infected with herpes simplex virus type 1: evidence for a stable, nonlinear structure
Authors: Rødahl E , Haarr L
Published: January 2020

The major latency-associated transcript (LAT) expressed in PC12 cells productively infected with herpes simplex virus type 1 is a 2-kb, nonpolyadenylated RNA molecule that accumulates in the nuclei of infected cells. In actinomycin D-treated cells, the 2-kb LAT gene transcript has a half-life considerably greater than 12 h. After polyacrylamide gel electrophoresis, two species of the transcript were observed, a major species that was retarded in the gel and a minor species that migrated as a 1.96-kb RNA molecule. RNase H digestion after hybridization of the RNA with an oligonucleotide complementary to positions -80 to -101 relative to the 3' end of the 2-kb LAT gene transcript changed the mobility of the retarded species into that of the rapidly migrating species. Our data indicate that the 2-kb LAT gene transcript expressed in productively infected PC12 cells is present in a stable, nonlinear form.
Dominant-negative inhibitors of EBNA-1 of Epstein-Barr virus
Authors: Kirchmaier A L , Sugden B
Published: January 2020

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is required in trans to support replication of the EBV genome once per cell cycle via the latent origin of replication, oriP. EBNA-1 can also activate transcription on binding to the family of repeats of oriP to enhance some heterologous as well as native EBV promoters. We have made and screened derivatives of EBNA-1 for the ability to act as inhibitors of wild-type EBNA-1. These derivatives lack the linking or the retention functions of EBNA-1 and were analyzed for the residual ability to activate transcription and replication. We have identified derivatives of EBNA-1 that can inhibit up to 98% of wild-type EBNA-1's activities. We have also identified one derivative of EBNA-1 with only two of EBNA-1's three linking domains which can support transcription and replication inefficiently.
Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line
Authors: Malik P , McQuiston S A , Yu X J , Pepper K A , Krall W J , Podsakoff G M , Kurtzman G J , Kohn D B
Published: January 2020

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population.
Characterization of early stages in vaccinia virus membrane biogenesis: implications of the 21-kilodalton protein and a newly identified 15-kilodalton envelope protein
Authors: Rodríguez J R , Risco C , Carrascosa J L , Esteban M , Rodríguez D
Published: January 2020

Vaccinia virus (VV) membrane biogenesis is a poorly understood process. It has been proposed that cellular membranes derived from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) are incorporated in the early stages of virion assembly. We have recently shown that the VV 21-kDa (A17L gene) envelope protein is essential for the formation of viral membranes. In the present work, we identify a 15-kDa VV membrane protein encoded by the A14L gene. This protein is phosphorylated and myristylated during infection and is incorporated into the virion envelope. Both the 21- and 15-kDa proteins are found associated with cellular tubulovesicular elements related to the ERGIC, suggesting that these proteins are transported in these membranes to the nascent viral factories. When synthesis of the 21-kDa protein is repressed, organized membranes are not formed but numerous ERGIC-derived tubulovesicular structures containing the 15-kDa protein accumulate in the boundaries of the precursors of the viral factories. These data suggest that the 21-kDa protein is involved in organizing the recruited viral membranes, while the 15-kDa protein appears to be one of the viral elements participating in the membrane recruitment process from the ERGIC, to initiate virus formation.
The presence of host-derived HLA-DR1 on human immunodeficiency virus type 1 increases viral infectivity
Authors: Cantin R , Fortin J F , Lamontagne G , Tremblay M
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) incorporates several host cell components when budding out of the infected cell. One of the most abundant host-derived molecules acquired by HIV-1 is the HLA-DR determinant of the major histocompatibility complex class II (MHC-II) molecules. The fact that CD4 is the natural ligand of MHC-II prompted us to determine if such virally embedded cellular components can affect the biology of the virus. Herein, we report for the first time that the incorporation of cellular HLA-DR1 within HIV-1 enhances its infectivity. This observation was made possible with virions bearing or not bearing on their surfaces host-derived HLA-DR1 glycoproteins. Such virus stocks were prepared by a transient-expression system based on transfection of 293T cells with a recombinant luciferase-encoding HIV-1 molecular clone along with plasmids encoding the alpha and beta chains of HLA-DR1. Cell-free virions recovered from transfected cells were shown to have efficiently incorporated host-derived HLA-DR1 glycoproteins. Infectivity was increased by a factor of 1.6 to 2.3 for virions bearing on their surfaces host-derived HLA-DR1. The observed enhancement of HIV-1 infectivity was independent of the virus stocks used and was seen in several T-lymphoid cell lines, in a premonocytoid cell line, and in primary peripheral blood mononuclear cells. Finally, we determined that the presence of virion-bound cellular HLA-DR1 is associated with faster kinetics of virus infection. Taken together, these results suggest that HLA-DR-1-bearing HIV-1 particles had a greater infectivity per picogram of viral p24 protein than HLA-DR1-free virions.
Human T-cell leukemia virus type 1 Tax releases cell cycle arrest induced by p16INK4a
Authors: Low K G , Dorner L F , Fernando D B , Grossman J , Jeang K T , Comb M J
Published: January 2020

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein causes cellular transformation by deregulating important cellular processes such as DNA repair, transcription, signal transduction, proliferation, and growth. Although it is clear that normal cell cycle control is deregulated during HTLV-1-induced cellular transformation, the effects of Tax on cell cycle control are not well understood. Flow cytometric analyses of human T cells indicate that cell cycle arrest in late G1, at or before the G1/S restriction point, by p16INK4a is relieved by Tax. Furthermore, Tax-dependent stimulation of 5-bromo-2'-deoxyuridine incorporation and transcriptional activation is inhibited by p16INK4a. This result suggests that p16INK4a is able to block Tax-dependent stimulation of DNA synthesis and cell cycle progression into S phase. In vitro binding assays with recombinant glutathione S-transferase fusion proteins and [35S]methionine-labeled proteins indicate that Tax binds specifically with p16INK4a but not with either p21cip1 or p27kip1. Furthermore, sequential immunoprecipitation assays with specific antisera and [35S]methionine-labeled cell lysates subsequent to coexpression with Tax and p16INK4a indicate that the two proteins form complexes in vivo. Immunocomplex kinase assays with cyclin-dependent kinase 4 antiserum indicate that Tax blocks the inhibition of cdk4 kinase activity by p16INK4a. This study identifies p16INK4a as a novel cellular target for Tax and suggests that the inactivation of p16INK4a function is a mechanism of cell cycle deregulation by Tax.
Functional dissection of the Moloney murine leukemia virus envelope protein gp70
Authors: Bae Y , Kingsman S M , Kingsman A J
Published: January 2020

The envelope protein of Moloney murine leukemia virus (Mo-MLV) is a complex glycoprotein that mediates receptor binding and entry via fusion with cell membranes. By using a series of substitution mutations and truncations in the Mo-MLV external envelope surface protein gp70, we have identified regions important for these processes. Firstly, truncations of gp70 revealed that the minimal continuous receptor-binding region is amino acids 9 to 230, in broad agreement with other studies. Secondly, within this region there are two key basic amino acids, Arg-83 and Arg-95, that are essential for receptor binding and may interact with a negatively charged residue(s) or with the pi electrons of the aromatic ring on a hydrophobic residue(s) in the basic amino acid transporter protein that is the Mo-MLV ecotropic receptor. Finally, we showed that outside the minimal receptor-binding region at amino acids 2 to 8, there is a region that is essential for postbinding fusion events.
Two Microplitis demolitor polydnavirus mRNAs expressed in hemocytes of Pseudoplusia includens contain a common cysteine-rich domain
Authors: Strand M R , Witherell R A , Trudeau D
Published: January 2020

Microplitis demolitor is a polydnavirus-carrying wasp that parasitizes the larval stage of Pseudoplusia includens. A previous study indicated that M. demolitor polydnavirus (MdPDV) infects primarily hemocytes in parasitized hosts. Thereafter, several alterations that compromise the immune response of P. includens toward the developing parasitoid occur in hemocytes. In this study, we identified two MdPDV mRNAs (1.0 and 1.5 kb) expressed in P. includens hemocytes that have homology to the viral genomic clone pMd-2. Corresponding 1.0- and 1.5-kb cDNA clones (MdPi455 and MdPi59) were isolated from an MdPDV-infected hemocyte cDNA library. Nucleotide sequence analysis of the cDNA clones confirmed that the 1.5- and 1.0-kb mRNAs have significant regions of homology. Sequence alignment revealed that the gene, OMd1.0, encoding the 1.0-kb mRNA is present in pMd-2. This gene contains two introns and three exons that agree with the sequence for MdPi455. In contrast, the 1.5-kb mRNA is likely encoded by a related gene located on the same MdPDV genomic DNA as is OMd1.0. The predicted peptide sequences for the 1.0- and 1.5-kb transcripts contain a cysteine-rich region at their 5' ends that have some similarity with epidermal growth factor-like motifs. Hybridization studies revealed that both mRNAs are expressed in granular cells and plasmatocytes, the primary classes of hemocytes involved in defense against M. demolitor and other parasites.
Two proline residues are essential in the calcium-binding activity of rotavirus VP7 outer capsid protein
Authors: Gajardo R , Vende P , Poncet D , Cohen J
Published: January 2020

Rotavirus maturation and stability of the outer capsid are calcium-dependent processes. It has been shown previously that the concentration of Ca2+-solubilizing outer capsid proteins from rotavirus particles is dependent on the virus strain. This property of viral particles has been associated with the gene coding for VP7 (gene 9). In this study the correlation between VP7 and resistance to low [Ca2+] was confirmed by analyzing the origin of gene 9 from reassortant viruses prepared under the selective pressure of low [Ca2+]. After chemical mutagenesis, we selected mutant viruses of the bovine strain RF that are more resistant to low [Ca2+]. The genes coding for the VP7 proteins of these independent mutants have been sequenced. Sequence analysis confirmed that these mutants are independent and revealed that all mutant VP7 proteins have proline 75 changed to leucine and have an outer capsid that solubilized at low [Ca2+]. The mutation of proline 279 to serine is found in all but two mutants. The phenotype of mutants having a single proline change can be distinguished from the phenotype of mutants having two proline changes. Sequence analysis showed that position 75 is in a region (amino acids 65 to 78) of great variability and that proline 75 is present in most of the bovine strains. In contrast, proline 279 is in a conserved region and is conserved in all the VP7 sequences in data banks. This region is rich in oxygenated residues that are correctly allocated in the metal-coordinating positions of the Ca2+-binding EF-hand structure pattern, suggesting that this region is important in the Ca2+ binding of VP7.
Induction of cyclins E and A in response to mitogen removal: a basic alteration associated with the arrest of differentiation of C2 myoblasts transformed by simian virus 40 large T antigen
Authors: Tedesco D , Baron L , Fischer-Fantuzzi L , Vesco C
Published: January 2020

We previously showed that C2 myoblasts transformed by simian virus 40 large T antigen (SVLT) stop the myogenic process after the induction of myogenin and of high Rb levels; the induced Rb, however, becomes notably phosphorylated. We have analyzed the protein levels and activities of cyclin-dependent kinases (cdks) in untransformed C2 cells and in transformants of either SVLT or the cytoplasmic mutant NKT1 (which permits differentiation) upon a shift from growth medium (GM) to mitogen-poor differentiation medium (DM). After the shift, cdk4 levels remained constant and cdk6 levels decreased in all cell types; cdk2 minimally increased only in SVLT cells. Cyclin D1 was downregulated in DM in all cell types, and cyclin D3 was upregulated (albeit less strongly in SVLT cells than in the others). In contrast, a dramatic difference between SVLT cells and the other cells was observed for cyclins E and A, which essentially disappeared (as protein and RNA) in normal C2 and NKT1 cells upon the shift from GM to DM, whereas they increased in SVLT cells. Concurrently, cdk2 activity ceased in C2 and NKT1 cells in DM, whereas it persisted at 20% of the GM level in SVLT cells. cdk4 activity was detectable in all cells only in GM. Cyclin E and A induction thus appeared to sustain enough Rb phosphorylation to interfere with tissue-specific expression, with cdk activity not high enough to activate cyclin self-regulation. In DM, cdk2 complexed to D3 was underphosphorylated in all cells, and SVLT allowed strong inductions of p21 and p27 without affecting their complexes with cdks.
Deletion of the C-terminal 33 amino acids of cucumber mosaic virus movement protein enables a chimeric brome mosaic virus to move from cell to cell
Authors: Nagano H , Okuno T , Mise K , Furusawa I
Published: January 2020

The movement protein (MP) gene of brome mosaic virus (BMV) was precisely replaced with that of cucumber mosaic virus (CMV). Infectivity tests of the chimeric BMV on Chenopodium quinoa, a permissive host for cell-to-cell movement of both BMV and CMV, showed that the chimeric BMV failed to move from cell to cell even though it replicated in protoplasts. A spontaneous mutant of the chimeric BMV that displayed cell-to-cell movement was subsequently obtained from a local lesion during one of the experiments. A cloned cDNA representing the genomic RNA encoding the MP of the chimeric BMV mutant was analyzed and found to contain a mutation in the CMV MP gene resulting in deletion of the C-terminal 33 amino acids of the MP. Directed mutagenesis of the CMV MP gene showed that the C-terminal deletion was responsible for the movement capability of the mutant. When the mutation was introduced into CMV, the CMV mutant moved from cell to cell in C. quinoa, though the movement was less efficient than that of the wild-type CMV. These results indicate that the CMV MP, except the C-terminal 33 amino acids, potentiates cell-to-cell movement of both BMV and CMV in C. quinoa. In addition, since C. quinoa is a common host for both BMV and CMV, these results suggest that the CMV MP has specificity for the viral genomes during cell-to-cell movement of the virus and that the C-terminal 33 amino acids of the CMV MP are involved in that specificity.
Glycoprotein H of herpes simplex virus type 1 requires glycoprotein L for transport to the surfaces of insect cells
Authors: Westra D F , Glazenburg K L , Harmsen M C , Tiran A , Jan Scheffer A , Welling G W , Hauw The T , Welling-Wester S
Published: January 2020

In mammalian cells, formation of heterooligomers consisting of the glycoproteins H and L (gH and gL) of herpes simplex virus type 1 is essential for the cell-to-cell spread of virions and for the penetration of virions into cells. We examined whether formation of gH1/gL1 heterooligomers and cell surface expression of the complex occurs in insect cells. Three recombinant baculoviruses, expressing gL1, gH1, and truncated gH1 (gH1t), which lacks the transmembrane region, were constructed. It was shown that recombinant gH1/gL1 and gH1t/gL1 heterooligomers were produced in insect cells. As in mammalian cells, gH1 and gH1t were not detected on the surfaces of insect cells in the absence of gL1. When coexpressed with gL1, recombinant gH1 was displayed on the surfaces of insect cells. Coexpression of gH1t and gL1 resulted in secretion of the gH1t/gL1 complex into the cell culture medium, indicating that gH1t is also transported to the surfaces of insect cells. Our results indicate that the process of folding and intracellular transport of gH1 and gL1 is comparable in insect cells and mammalian cells and that the baculovirus expression system can be used to examine the complex formation and the intracellular transport of gH1 and gL1. The availability of secreted gH1t/gL1 complex offers the opportunity to further investigate the immunological properties of this complex.
A multivalent minigene vaccine, containing B-cell, cytotoxic T-lymphocyte, and Th epitopes from several microbes, induces appropriate responses in vivo and confers protection against more than one pathogen
Authors: An L L , Whitton J L
Published: January 2020

The development of safe and effective vaccines remains a major goal in the prevention, and perhaps treatment, of infectious diseases. Ideally, a single vaccine would confer protection against several pathogens and would induce both cellular and humoral arms of the immune response. We originally demonstrated that two virus-specific cytotoxic T-lymphocyte (CTL) epitopes, from the same virus but presented by different major histocompatibility complex alleles, when linked in tandem as minigenes in a recombinant vaccinia virus, could confer complete protection against subsequent viral challenge. In the study, we extended this approach, which we termed string of beads, expanding the immunogenic scope in two ways: first, by introduction of T helper (Th) and B-cell (antibody) epitopes alongside CTL epitopes and second, by including immunogenic sequences from a variety of infectious agents, five viruses and one bacterium. The vaccine (VV-sv) comprises CTL epitopes from Sendai virus, respiratory syncytial virus, and lymphocytic choriomeningitis virus (LCMV); Th epitopes from vesicular stomatitis virus and Mycobacterium tuberculosis; and an antibody epitope from mengovirus. The construct contains a single start codon, and the epitopes are linked directly, without intervening spacer amino acids. There was some concern that the combination of several normally immunodominant epitopes might result in a new hierarchy of dominance, in which certain epitopes predominated and others exhibited reduced immunogenicity. However we show that when analyzed in tissue culture and in vivo, all six epitopes are expressed. CTL and Th cells are induced in vivo, along with neutralizing antibody. The induced immunity is biologically relevant: after VV-sv immunization, the antimengovirus antibody confers protection against mengovirus challenge. Similarly, CTL induced by the LCMV epitope protected mice against challenge with this agent. Thus, a polyvalent, minigene-based vaccine can simultaneously induce several classes of immune response and thereby can confer protection against diverse pathogens.
Oral immunization with recombinant Mycobacterium bovis BCG simian immunodeficiency virus nef induces local and systemic cytotoxic T-lymphocyte responses in mice
Authors: Lagranderie M , Balazuc A M , Gicquel B , Gheorghiu M
Published: January 2020

Recombinant live Mycobacterium bovis BCG vectors (rBCG) induce strong cellular and humoral immune responses against various antigens after either systemic or oral immunization of mice. Cytotoxic T-lymphocyte (CTL) responses may contribute to the control of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infections whose portal of entry is the gastrointestinal or genital mucosa. In this study, we immunized BALB/c mice with a recombinant BCG SIV nef and observed its behavior in oropharyngeal and target organ lymphoid tissues. The cellular immune responses, particularly the intestinal intraepithelial and systemic CTL responses, were investigated. The results showed that rBCG SIV nef translocated the oropharyngeal mucosa and intestinal epithelium. It diffused to and persisted in target lymphoid organs. Specific SIV Nef peptide proliferative responses and cytokine production were observed. Strong systemic and mucosal CTL responses were induced. In particular, we demonstrated direct specific anti-Nef CTL in intestinal intraepithelial CD8beta+ T cells. These findings provide evidence that orally administered rBCG SIV nef may contribute to local defenses against viral invasion. Therefore, rBCG SIV nef could be a candidate vaccine to protect against SIV infection and may be used to develop an oral rBCG HIV nef vaccine.
Size variation within the second hypervariable region of the surface envelope gene of the bovine lentivirus BIV in experimentally and naturally infected cattle
Authors: Suarez D L , Whetstone C A
Published: January 2020

The bovine lentivirus also known as the bovine immunodeficiency-like virus (BIV) has conserved and hypervariable regions in the surface envelope (SU) gene. Size variation between isolates can be as large as 200 bp, mostly occurring in the second hypervariable (V2) gene region of the SU gene. The V2 region was cloned and sequenced from both experimentally and naturally infected cattle. Temporal evaluation of provirus from an experimentally inoculated cow showed two different-sized variants that appeared over time. The variation appeared to result from a recombinational event resulting in an apparent direct repeat. Cloned proviral nucleotide sequence diversity increased over time. Virus that was cultured and then cloned and sequenced showed progressive change from the inoculum virus, but culturing reduced the diversity of the clones as compared with direct amplification of provirus from leukocyte samples from the cow. The quasispecies phenomenon was evident in clones sequenced from a cow naturally infected with BIV. Of 10 clones examined from the V2 region, 6 different-size clones were present with nine different patterns of sequence rearrangement. Sequence length of different clones varied by as much as 43 amino acids (aa), with 21- and 15-aa direct repeats accounting for most of the size variation. Similar to other lentiviruses, BIV appears to mutate rapidly, which may be important in viral persistence and pathogenesis.
Modification of the Sendai virus-specific antibody and CD8+ T-cell responses in mice homozygous for disruption of the interleukin-4 gene
Authors: Mo X Y , Sangster M Y , Tripp R A , Doherty P C
Published: January 2020

Homozygous disruption (-/-) of the interleukin-4 (IL-4) gene did not obviously modify the severity of Sendai virus infection in the highly susceptible 129/J mouse strain. The virus was cleared from the respiratory tract, and potent cytotoxic T lymphocyte (CTL) effectors were present in the cell population recovered by bronchoalveolar lavage. However, the prevalence of virus-specific CTL precursors (p) was consistently diminished in the spleen and regional lymph nodes of the IL-4 -/- mice at day 7 after infection. Also, virus-specific serum immunoglobulin G1 (IgG1) levels were greatly reduced and few IgG1-producing cells were detected in the lymphoid tissue. The effect on IgG1 class switching was to be expected, but the decrease in CTLp numbers has not been observed previously for a virus-specific immune response.
A large-scale evaluation of peptide vaccines against foot-and-mouth disease: lack of solid protection in cattle and isolation of escape mutants
Authors: Taboga O , Tami C , Carrillo E , Núñez J I , Rodríguez A , Saíz J C , Blanco E , Valero M L , Roig X , Camarero J A , Andreu D , Mateu M G , Giralt E , Domingo E , Sobrino F , Palma E L
Published: January 2020

A large-scale vaccination experiment involving a total of 138 cattle was carried out to evaluate the potential of synthetic peptides as vaccines against foot-and-mouth disease. Four types of peptides representing sequences of foot-and-mouth disease virus (FMDV) C3 Argentina 85 were tested: A, which includes the G-H loop of capsid protein VP1 (site A); AT, in which a T-cell epitope has been added to site A; AC, composed of site A and the carboxy-terminal region of VP1 (site C); and ACT, in which the three previous capsid motifs are colinearly represented. Induction of neutralizing antibodies, lymphoproliferation in response to viral antigens, and protection against challenge with homologous infectious virus were examined. None of the tested peptides, at several doses and vaccination schedules, afforded protection above 40%. Protection showed limited correlation with serum neutralization activity and lymphoproliferation in response to whole virus. In 12 of 29 lesions from vaccinated cattle that were challenged with homologous virus, mutant FMDVs with amino acid substitutions at antigenic site A were identified. This finding suggests the rapid generation and selection of FMDV antigenic variants in vivo. In contrast with previous studies, this large-scale vaccination experiment with an important FMDV host reveals considerable difficulties for vaccines based on synthetic peptides to achieve the required levels of efficacy. Possible modifications of the vaccine formulations to increase protective activity are discussed.
Specific repression of Tax trans-activation by TAR RNA-binding protein TRBP
Authors: Donzeau M , Winnacker E L , Meisterernst M
Published: January 2020

The human T-cell leukemia virus type 1 (HTLV-1)-encoded Tax protein activates transcription from the long terminal repetition via association with host cellular factors. In this study, we searched for cellular proteins that interact with Tax and modulate its activity by using the yeast two-hybrid system. One of the strongest interactors was found to be identical with TRBP, which was previously shown to bind to the RNA encoded by the Tat response element of human immunodeficiency virus type 1. Interactions are demonstrated with Escherichia coli-expressed proteins in vitro and in mammalian cells, using one- and two-hybrid systems, and with antibodies that coprecipitate Tax and TRBP at physiological TRBP concentrations. Moreover, TRBP, when directed into the cytoplasm, is capable of preventing transport of Tax into the nucleus. A 60-amino-acid polypeptide suffices for binding to Tax. TRBP inhibits activation of transcription by both Tax and GAL4-Tax fusion proteins. Inhibition is specific for Tax and is not seen with the other activators tested. Our data are consistent with the interpretation that TRBP inhibits the interplay of Tax with the transcription machinery or accessory factors.
Exon 1 leader sequences downstream of U5 are important for efficient human immunodeficiency virus type 1 gene expression
Authors: Lenz C , Scheid A , Schaal H
Published: January 2020

In previous studies, little attention has been paid to maintaining the native HIV-1 leader sequence in reporter constructs analyzing the human immunodeficiency virus type 1 (HIV-1) promoter activity. To investigate a possible influence of the leader sequence on HIV-1-driven gene expression in the presence as well as in the absence of Tat, an expression vector was designed for transcripts consisting of the native HIV-1 tat 1.4 mRNA leader followed by the open reading frame for the bacterial chloramphenicol acetyltransferase (CAT). Deletion mutants with mutations within the leader sequence downstream of U5 (lsdU5) were constructed, as well as a mutant containing a mutation with a reverse orientation of this region. Quantification of CAT protein in HeLa-T4+ cells transiently transfected with wild-type and mutant leader constructs showed that the exon 1-derived lsdU5 region has an influence on basal as well as Tat-induced protein expression. The dramatic decrease in the level of CAT protein upon deletion of lsdU5 was paralleled by a drop in the steady-state level of CAT mRNA. Deletion of the exon 1-derived lsdU5 region also decreased the expression of mRNAs containing authentic HIV-1 sequences instead of CAT. The effect observed with the reporter constructs was not due to the loss of binding sites for nuclear factors, as could be shown with DBF1 and Sp1 mutant constructs. Nuclear run-on transcription assays showed that the presence or absence of lsdU5 did not influence the rate of transcription. This indicates that the exon 1 lsdU5 element functions at the posttranscriptional level in the processing, nucleocytoplasmic export, or stabilization of HIV-1 transcripts.
Nonstructural protein 3 of hepatitis C virus blocks the distribution of the free catalytic subunit of cyclic AMP-dependent protein kinase
Authors: Borowski P , Oehlmann K , Heiland M , Laufs R
Published: January 2020

Chronic hepatitis resulting from hepatitis C virus (HCV) infection develops into cirrhosis in at least half of infected patients and increases the risk of hepatocellular carcinoma. The pathogenic effects of a number of viruses result from the disturbance of intracellular signal cascades caused by viral antigens. Therefore, we investigated the interaction of nonstructural protein 3 (NS3) of HCV with the cyclic AMP-dependent signal pathway. We found a similarity between the HCV sequence Arg-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg localized in NS3 and the general consensus sequence of protein kinase A (PKA). Consequently, the catalytic (C) subunit of PKA bound to a bacterially expressed fragment of HCV polyprotein containing amino acid residues 1189 to 1525. When this fragment was introduced into cells, it inhibited the translocation of the C subunit into the nucleus after stimulation with forskolin. The result of this inhibition was significantly reduced histone phosphorylation. Therefore, the presence of NS3 in the cytoplasm of infected cells may affect a wide range of PKA functions and contribute to the pathogenesis of the diseases caused by HCV.
Binding of the E1 and E2 proteins to the origin of replication of bovine papillomavirus
Authors: Sedman T , Sedman J , Stenlund A
Published: January 2020

DNA replication of bovine papillomavirus (BPV) requires two viral proteins encoded from the E1 and E2 open reading frames. E1 and E2 are sequence-specific DNA binding proteins that bind to their cognate binding sites in the BPV origin of replication (ori). The E1 and E2 proteins can interact physically with each other, and this interaction results in cooperative binding when binding sites for both proteins are present. We have analyzed the binding of E1 to the ori in the absence and presence of E2, using DNase I footprint analysis, gel mobility shift assays, and interference analysis. We have also generated a large number of point mutations in the E1 binding site and tested them for binding of E1 as well as for activity in DNA replication. Our results demonstrate that E1 binds to the ori in different forms in the absence and presence of E2 and that E2 has both a quantitative and a qualitative effect on the binding of E1. Our results also suggest that the ori contains multiple overlapping individual E1 recognition sequences which together constitute the E1 binding site and that different subsets of these recognition sequences are used for binding of E1 in the presence and absence of E2.
Studies of the susceptibility of nude, CD4 knockout, and SCID mutant mice to the disease induced by the murine AIDS defective virus
Authors: Simard C , Klein S J , Mak T , Jolicoeur P
Published: January 2020

Murine AIDS (MAIDS) is induced by a defective retrovirus that infects lymphocyte cells of the B lineage. To determine whether functional T cells are required for the infection of B cells, T-cell-deficient mice (nude, CD4 knockout, and SCII)) were infected with helper-free stocks of the MAIDS defective virus. Infection of B cells was monitored by Northern blot analysis and in situ hybridization. The C57BL/6 nude mice contained clusters of infected B cells, but less so than did the euthymic mice. In contrast, the (C57BL/6 x BALB/c)F1 nude mice harbored more infected B cells than did their euthymic littermates when maintained in a pathogen-free environment. Clusters of infected B cells were also detected in the MAIDS virus-infected CD4-/- knockout mice despite the total absence of CD4+ T cells in these mice. However, infected cells were not detected in SCID mice (deficient in mature T and B cells) inoculated with the same virus, indicating that precursor B cells are not a target of the virus in the absence of mature CD4+ T cells. These data confirm that the primary event in the development of MAIDS is the infection of relatively mature peripheral B cells and that CD4+ T cells are required to promote the expansion of these infected B cells.
A role for naturally occurring variation of the murine coronavirus spike protein in stabilizing association with the cellular receptor
Authors: Gallagher T M
Published: January 2020

Murine hepatitis virus (MHV), a coronavirus, initiates infection by binding to its cellular receptor (MHVR) via spike (S) proteins projecting from the virion membrane. The structures of these S proteins vary considerably among MHV strains, and this variation is generally considered to be important in determining the strain-specific pathologies of MHV infection, perhaps by affecting the interaction between MHV and the MHVR. To address the relationships between S variation and receptor binding, assays capable of measuring interactions between MHV and MHVR were developed. The assays made use of a novel soluble form of the MHVR, sMHVR-Ig, which comprised the virus-binding immunoglobulin-like domain of MHVR fused to the Fc portion of human immunoglobulin G1. sMHVR-Ig was stably expressed as a disulfide-linked dimer in human 293 EBNA cells and was immobilized to Sepharose-protein G via the Fc domain. The resulting Sepharose beads were used to adsorb radiolabelled MHV particles. At 4 degrees C, the beads specifically adsorbed two prototype MHV strains, MHV JHM (strain 4) and a tissue culture-adapted mutant of MHV JHM, the JHMX strain. A shift to 37 degrees C resulted in elution of JHM but not JHMX. This in vitro observation of JHM (but not JHMX) elution from its receptor at 37 degrees C was paralleled by a corresponding 37 degrees C elution of receptor-associated JHM (but not JHMX) from tissue culture cells. The basis for this difference in maintenance of receptor association was correlated with a large deletion mutation present within the JHMX S protein, as sMHVR-Ig exhibited relatively thermostable binding to vaccinia virus-expressed S proteins containing the deletion. These results indicate that naturally occurring mutations in the coronavirus S protein affect the stability of the initial interaction with the host cell and thus contribute to the likelihood of successful infection by incoming virions. These changes in virus entry features may result in coronaviruses with novel pathogenic properties.