bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclic AMP-responsive element-dependent activation of Epstein-Barr virus zebra promoter by human herpesvirus 6
Authors: Flamand L , Menezes J
Published: January 2020

We have recently shown that infection of Epstein-Barr virus (EBV) genome-positive B cells by human herpesvirus 6 (HHV-6) results in the expression of the immediate-early EBV Zebra gene, followed by virus replication (L. Flamand, I. Stefanescu, D. V. Ablashi, and J. Menezes, J. Virol. 67:6768-6777, 1993). Here we show that HHV-6 upregulates Zebra gene transcription through a cyclic AMP-responsive element (CRE) located within the Zebra promoter (Zp). Using human B- or T-cell lines transfected with ZpCat reporter gene constructs, we demonstrate that a region designated the ZII domain of Zp is the target of HHV-6 transactivation. Mutation of the consensus AP-1/CRE site within ZII abolished the inducibility of Zp by HHV-6, whereas positioning of the ZII domain upstream of the beta-globin minimal promoter conferred responsiveness following HHV-6 infection. Binding of these factors to ZII was prevented by oligonucleotides containing CRE but not by AP-1 consensus sequences. Antibodies against CRE-binding (CREB) protein but not against c-Fos or c-Jun were able to supershift the DNA-protein complex, identifying the nature of the transcription factor which binds to ZII as a member of the CREB family of proteins. Finally, transfection of CREB protein and protein kinase A expression vectors were found to activate Zp in Jurkat cells, suggesting that phosphorylated form of CREB protein can play a determining role in the EBV reactivation process.
Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers
Authors: Weitzman M D , Fisher K J , Wilson J M
Published: January 2020

Replication of a human parvovirus, adeno-associated virus (AAV), is facilitated by coinfection with adeno-virus to provide essential helper functions. We have used the techniques of in situ hybridization and immunocytochemistry to characterize the localization of AAV replication within infected cells, Previous studies have shown that adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur. Our studies indicate that AAV is colocalized with the adenovirus replication centers, where it may utilize adenovirus and cellular proteins for its own replication. Expression of the AAV Rep protein inhibits the normal maturation of the adenovirus centers. Similar experiments were performed with recombinant AAV (rAAV) to establish a relationship between intranuclear localization and rAAV transduction. rAAV efficiently entered the cell, and its genome was faintly detectable in a perinuclear distribution and was mobilized to replication centers when the cell was infected with adenovirus. The recruitment of the replication-defective genome into the intranuclear adenovirus domains resulted in enhanced transduction. These studies illustrate the importance of intracellular compartmentalization for such complex interactions as the relationship between AAV and adenovirus.
The herpes simplex virus type 1 VP5 promoter contains a cis-acting element near the cap site which interacts with a cellular protein
Authors: Huang C J , Petroski M D , Pande N T , Rice M K , Wagner E K
Published: January 2020

The promoter controlling the expression of the transcript encoding the major herpes simplex virus type 1 capsid protein (VP5, UL19) extends only 60 bases or so from a functional Sp1 site at --48 to include a cis-acting element 3' of the transcript start site. In the present communication, we report the generation of recombinant viruses bearing mutations between --6 and + 8 relative to the cap site in the VP5 promoter controlling expression of a reporter gene. Analysis of the effects of these mutations upon reporter gene expression along with the results of protein binding assays demonstrates that this cap transcription element functionally interacts with a cellular protein of a normal size of 40 kDa. Thus, like the strict late UL38 promoter characterized earlier, the late VP5 promoter has the essential properties of a cellular promoter.
Herpes simplex ICP27 mutant viruses exhibit reduced expression of specific DNA replication genes
Authors: Uprichard S L , Knipe D M
Published: January 2020

Herpes simplex virus type 1 mutants with certain lesions in the ICP27 gene show a 5- to 10-fold reduction in viral DNA synthesis. To determine how ICP27 promotes amplification of viral DNA, we examined the synthesis, accumulation, and stability of the essential viral replication proteins and steady-state levels of the replication gene transcripts throughout the course of ICP27 mutant virus infections. These studies reveal that in the absence of ICP27, expression of the UL5, UL8, UL52, UL9, UL42, and UL30 genes is significantly reduced at the level of mRNA accumulation. In contrast to that of these beta genes, ICP8 expression is unaltered in mutant virus-infected cells, indicating that ICP27 selectively stimulates only a subset of herpes simplex virus beta genes. Analysis of multiple ICP27 mutant viruses indicates a quantitative correlation between the ability of these mutants to replicate viral DNA and the level of replication proteins produced by each mutant. Therefore, we conclude that the primary defect responsible for restricted viral DNA synthesis in cells infected with ICP27 mutants is insufficient expression of most of the essential replication genes. Of further interest, this analysis also provides new information about the structure of the UL52 gene transcripts.
Cellular pathways involved in the ex vivo expression of bovine leukemia virus
Authors: Kerkhofs P , Adam E , Droogmans L , Portetelle D , Mammerickx M , Burny A , Kettmann R , Willems L
Published: January 2020

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis. The virus adopts a strategy based on the lack of viral expression in vivo; only very rare BLV-infected B lymphocytes express viral information. When the cells are isolated from animals in persistent lymphocytosis and cultivated ex vivo, a tremendous increase in viral expression occurs. To gain insight into this mechanism, we employed a general approach using chemicals that interfere specifically with cellular pathways involved in signal transduction from the cell membrane to the nucleus. Our data demonstrate that BLV expression is not correlated with the activity of protein kinase A (PKA) and is even inhibited by cyclic AMP (cAMP). The cAMP/PKA pathway is thus apparently not involved in ex vivo viral expression. In contrast, PKC appears to play a key role in this process. Phorbol myristate acetate can directly activate viral expression in B cells (in the absence of T cells). Furthermore, calphostin C, a highly specific inhibitor of PKC, partly decreases ex vivo BLV expression. Our data further demonstrate that calmodulin and calcineurin, a calmodulin-dependent phosphatase, play a key role in the induction of viral expression. The involvement of this calmodulin-dependent pathway could explain the induction of expression that cannot be assigned to PKC. Furthermore, it appears that the activation of viral expression requires a calmodulin but not a PKA-dependent pathway. These data highlight major differences between transient transfection and ex vivo experiments. Finally, despite their homologies, BLV and human T-cell leukemia virus appear to use different signal transduction pathways to induce viral expression.
Possible role of splice acceptor site in expression of unspliced gag-containing message of Moloney murine leukemia virus
Authors: Oshima M , Odawara T , Matano T , Sakahira H , Kuchino Y , Iwamoto A , Yoshikura H
Published: January 2020

Moloney murine leukemia virus (MLV) having the gag coding region alone, G3.6, produced a low level of mRNA (1/10 of the wild-type level). Ligation of 441 nucleotides (nt) containing a splice acceptor (SA) site to the downstream portion of the remaining gag region restored the level of the unspliced message, simultaneously activating a cryptic splice donor (SD) site in the middle of the p30 coding region (between nt 1596 and 1597). Ligation of the 441 nt in the same site in the inverted orientation also increased the level of the unspliced message, activating the same SD site (between nt 1596 and 1597) and a new SA site just in front of the inserted 441 nt (between nt 4770 and 4771). Deletion or inversion of the 441-nt SA sequence from the wild-type MLV or from int in-frame deletion or int frameshift mutant MLVs of nearly full size resulted in the loss of spliced mRNA and concomitantly in a severe reduction of the unspliced mRNA, particularly at 37 degrees C. Deletion of the 5' SD site did not result in the reduction of the unspliced-mRNA level. When the gag region in G3.6 was replaced with a Neo(r) coding region, the level of expression was high. The data taken together suggest that the presence of an SA signal is necessary for high-level expression of unspliced mRNA encoding Gag or Gag-Pol.
Influenza A virus RNA-dependent RNA polymerase: analysis of RNA synthesis in vitro
Authors: Galarza J M , Peng Q , Shi L , Summers D F
Published: January 2020

Influenza A virus RNA-dependent RNA polymerase, purified from virion ribonucleoprotein particles and from which endogenous genomic RNA (vRNA) has been depleted by treatment with micrococcal nuclease, was used to study transcription initiation, elongation, and termination in vitro. Templates that contained either minus- or plus-sense influenza virus nucleoprotein minigenes with conserved 5' and 3' termini and the uridylate tract were constructed. The dinucleotide ApG and alfalfa mosaic virus RNA4 (AlMV4) were used as primers. ApG primed the synthesis of full-length positive-strand or cRNA products and shorter transcripts, depending upon the molar ratio between the nucleoprotein and the vRNA template. Sequence analysis of the ends of these transcripts demonstrated that the 5' termini of both transcripts and the 3' terminus of the full-length product were complementary to the 3' and 5' termini of the vRNA template, respectively, whereas the 3' terminus of the incomplete product corresponded to a sequence located 40 bases downstream from the 5' terminus of the template and was about 20 nucleotides downstream from the uridylate tract, which is the putative signal for polyadenylation. Binding of the cap structure of AlMV4 by the polymerase activated RNA synthesis by ligation-elongation of small genomic RNA fragments which were likely derived from a genome segment protected by the polymerase from micrococcal nuclease digestion. The sequence of these fragments mapped to a region 14 to 28 nucleotides upstream of the 3' terminus of the viral genome. Polymerase subunit involvement in transcription initiation with ApG or AlMV4 was characterized by studying the effect of purified polyclonal antisubunit immunoglobulins of the G class (IgGs) in transcription assays. These results showed that anti-PB2 IgG inhibited transcription initiation in both ApG- and AlMV4-primed reactions, whereas anti-PB1 antibodies also blocked transcription initiated with AlMV4. The differences observed in product size, product sequence, and differential inhibition by antisubunit IgGs are discussed. These observations would support the notion that the influenza virus RNA-dependent RNA polymerase undergoes a conformational change after the binding of the cap structure of host cell heterogeneous nuclear RNA by PB2, which then usually leads to endonucleolytic cleavage of the capped primer 13 nucleotides downstream from the cap.
A recombinant adenovirus expressing an Epstein-Barr virus (EBV) target antigen can selectively reactivate rare components of EBV cytotoxic T-lymphocyte memory in vitro
Authors: Morgan S M , Wilkinson G W , Floettmann E , Blake N , Rickinson A B
Published: January 2020

While the bulk of a virus-induced cytotoxic T-lymphocyte (CTL) response may focus on a few immunodominant viral antigens, in certain tumor virus systems the detectability of clones recognizing other, subdominant antigens can assume particular importance. By using the human CTL response to Epstein-Barr virus (EBV) as a model system, here we show that even rare components of virus-specific memory can be selectively reactivated in vitro when the relevant target antigen is expressed in autologous stimulator cells from a recombinant adenovirus (RAd) vector. We generated a replication-deficient adenovirus, RAd-E3C, which in skin fibroblast cultures expressed the EBV nuclear antigen EBNA3C at a 10- to 100-fold-higher level than that naturally present in EBV-transformed lymphoblastoid cell lines (LCLs). Initial experiments with a donor whose polyclonal CTL response to LCL stimulation contained a strong EBNA3C-specific component showed that these CTLs could be efficiently reactivated by in vitro stimulation either with RAd-E3C-infected fibroblasts or with RAd-E3C-infected peripheral blood mononuclear cells. Then we studied donors whose responses to LCL stimulation contained little if any detectable EBNA3C reactivity but were dominated by clones recognizing other EBV target antigens; in vitro stimulation with RAd-E3C-infected peripheral blood mononuclear cells selectively reactivated EBNA3C-specific CTL clones from these individuals, with the epitope specificities of responses subsequently identified at the peptide level. This RAd-based approach could be applied more generally to screen for human CTL responses against any candidate target antigen expressed by tumor cells.
E5 oncoprotein transmembrane mutants dissociate fibroblast transforming activity from 16-kilodalton protein binding and platelet-derived growth factor receptor binding and phosphorylation
Authors: Sparkowski J , Mense M , Anders J , Schlegel R
Published: January 2020

The E5 oncoprotein of bovine papillomavirus type 1 is a 44-amino-acid, hydrophobic polypeptide which localizes predominantly in Golgi membranes and appears to transform cells through the activation of tyrosine kinase growth factor receptors. In fibroblasts, E5 interacts with both the 16-kilodalton vacuolar ATPase subunit and the platelet-derived growth factor receptor (PDGF-R) via its hydrophobic transmembrane domain and induces autophosphorylation of the receptor. To further analyze the correlation between E5 biological activity and its ability to bind these cellular proteins, a series of nine E5 transmembrane mutants was evaluated. In 32D mouse hematopoietic cells, there was an incomplete correlation between the abilities of the E5 mutant proteins to associate the PDGF-R and to transform cells. However, all transforming E5 mutant proteins induced PDGF-R tyrosine phosphorylation. In NIH 3T3 and C127 mouse fibroblasts, both transforming and nontransforming E5 mutant proteins were defective for PDGF-R binding. In addition, while most of the transforming E5 proteins induced PDGF-R phosphorylation, one hypertransforming mutant (serine 17) neither bound nor induced receptor autophosphorylation. These findings support the hypothesis that the transformation of fibroblasts by E5 transmembrane mutants can involve alternative cellular targets or potentially independent activities of the E5 protein. In addition, these results underscore the critical role of the transmembrane domain in mediating E5 biological activities.
Analysis of the receptor-binding site of murine coronavirus spike protein
Authors: Suzuki H , Taguchi F
Published: January 2020

It has been found that a domain composed of 330 amino acids of the N terminus of murine coronavirus spike protein [S1N(330)] is involved in receptor-binding activity (H. Kubo, Y.K. Yamada, and F. Taguchi, J. Virol. 68:5403-5410, 1994). To delineate the amino acid sequences involved in receptor-binding activity, we have compared the S1N(330) proteins of seven different mouse hepatitis virus MHV strains that are able to utilize the MHV receptor protein. Three conserved regions (sites I, II, and III) were found to consist of more than 10 identical amino acids, and they were analyzed for receptor-binding activity by site-directed mutagenesis. S1N(330) with a substitution at position 62 from the N terminus of S1 in region I and that with substitutions at positions 212, 214, and 216 in region II showed no receptor-binding activity. The S1N(330) mutants without receptor-binding activity were not able to prevent virus binding to the receptor. These results suggest that the receptor-binding site on S1N(330) is composed of regions located apart from each other in the protein's primary structure, in which Thr at position 62 as well as amino acids located at positions 212, 214, and 216 are particularly important.
Human immunodeficiency virus type 2 glycoprotein enhancement of particle budding: role of the cytoplasmic domain
Authors: Ritter G D , Yamshchikov G , Cohen S J , Mulligan M J
Published: January 2020

Previous studies have shown that the glycoprotein cytoplasmic domains of human immunodeficiency virus type 2 (HIV-2) or simian immunodeficiency virus of macaques modulate biological activities of the viral glycoprotein complex, including syncytium formation, exterior glycoprotein conformation, and glycoprotein incorporation into budding virus particles. We have now utilized a recombinant expression system to study interactions of full-length or truncated HIV-2 glycoproteins with coexpressed HIV-2 Gag proteins which self-assemble and bud as virus-like particles. Interestingly, budding of HIV-2 virus-like particles from cells was enhanced 5- to 24-fold when Gag was coexpressed with the full-length HIV-2 glycoprotein, compared with Gag expressed either alone or with a truncated HIV-2 glycoprotein. The results obtained in this model system indicate that an additional effect of the lengthy cytoplasmic domain of the glycoprotein of HIV-2 is enhancement of particle budding. We speculate that the cytoplasmic domain of the viral glycoprotein of HIV-2 enhances budding by (i) potentiation of Gag structure or function or (ii) membrane modulation.
n-Butyrate, a cell cycle blocker, inhibits early amplification of duck hepatitis B virus covalently closed circular DNA after in vitro infection of duck hepatocytes
Authors: Turin F , Borel C , Benchaib M , Kay A , Jamard C , Guguen-Guillouzo C , Trépo C , Hantz O
Published: January 2020

During chronic hepadnavirus infection, virus persistence depends on the regulation of the pool of covalently closed circular DNA (cccDNA), which is the template for transcription of viral RNA species. The development of in vitro infection of duck hepatocyte primary cultures by duck hepatitis B virus (DHBV) provides a unique opportunity to study the regulation of cccDNA synthesis. After DHBV in vitro infection, cccDNA is detected 1 day later and is amplified to a high copy number after 1 week in culture. We studied whether this amplification occurs during cell cycle progression of duckling hepatocytes. By using [3H]thymidine incorporation, we found that hepatocytes obtained from 3-week-old ducklings spontaneously entered the S phase of the cell cycle when cultured in serum-free medium without added growth factors. Bromodeoxyuridine labeling confirmed that cellular DNA synthesis took place in more than 50% of parenchymal cells. Cytofluorometry analysis revealed the presence of asynchronous populations and polyploidization processes. The addition of a cell cycle blocker, n-butyrate, completely inhibited [3H]thymidine incorporation and blocked duckling hepatocytes in the G1 phase of the cell cycle. Simultaneously, butyrate inhibited cccDNA amplification and allowed the establishment of DHBV infection, as demonstrated by the detection of a basal level of cccDNA in treated hepatocytes. Both effects were reversible since active cell DNA synthesis was restored and cccDNA accumulated after drug withdrawal.
Respiratory synctial virus infection in BALB/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant Th2-like cytokine pattern
Authors: Waris M E , Tsou C , Erdman D D , Zaki S R , Anderson L J
Published: January 2020

Vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) caused excessive disease in infants upon subsequent natural infection with RSV. Recent studies with BALB/c mice have suggested that T cells are important contributors to lung immunopathology during RSV infection. In this study, we investigated vaccine-induced enhanced disease by immunizing BALB/c mice with live RSV intranasally or with FI-RSV intramuscularly. The mice were challenged with RSV 6 weeks later, and the pulmonary inflammatory response was studied by analyzing cells obtained by bronchoalveolar lavage 4 and 8 days after challenge. FI-RSV-immunized mice had an increased number of total cells, granulocytes, eosinophils, and CD4+ cells but a decreased number of CD8+ cells. The immunized mice also had a marked increase in the expression of mRNA for the Th2-type cytokines interleukin-5 (IL-5) and IL-13 as well as some increase in the expression of IL-10 (a Th2-type cytokine) mRNA and some decrease in the expression of IL-12 (a Th1-type cytokine) mRNA. The clear difference in the pulmonary inflammatory response to RSV between FI-RSV- and live-RSV-immunized mice suggests that this model can be used to evaluate the disease-enhancing potential of candidate RSV vaccines and better understand enhanced disease.
Antiviral cytotoxic T-cell memory by vaccination with recombinant Listeria monocytogenes
Authors: Slifka M K , Shen H , Matloubian M , Jensen E R , Miller J F , Ahmed R
Published: January 2020

Listeria monocytogenes is a facultative intracellular bacterium that is able to escape phagocytic vesicles and replicate in the cytoplasm of infected cells. As with viral vectors, this intracytoplasmic life cycle provides a means for introducing foreign proteins into the major histocompatibility complex class I pathway of antigen presentation. Using recombinant L. monocytogenes (rLM) strains expressing the full-length nucleoprotein (NP) or a single cytotoxic T-lymphocyte (CTL) epitope from lymphocytic choriomeningitis virus (LCMV), we analyzed antiviral CTL responses induced by rLM vaccination. After vaccination, rLM was cleared from the host within 7 days while inducing an LCMV-specific ex vivo CD8+ effector CTL response. Virus-specific CTL memory was maintained for 6 months postvaccination, as demonstrated by vigorous secondary CTL responses after in vitro stimulation. A single immunization with rLM that expressed either the full-length NP gene or the CTL epitope alone resulted in LCMV NP-specific CTL precursor frequencies of approximately 1/10(4) CD8+ T cells. A second rLM vaccination resulted in enhanced virus-specific CTL activity and in vitro proliferation. rLM-vaccinated mice were protected against chronic viral infection by an accelerated virus-specific memory CTL response. These mice cleared infectious virus as well as viral antigen, suggesting that sterilizing immunity was achieved. In contrast to mice that received wild-type LM, rLM-vaccinated mice were protected from virally induced immunosuppression and splenic atrophy associated with chronic LCMV infection. The ability to elicit long-term cell-mediated immunity is fundamental in designing vaccines against intracellular pathogens, and these results demonstrate the efficacy of recombinant LM vaccination for inducing protective antiviral CTL memory.
Identification of amino acids involved in recognition by dengue virus NS3-specific, HLA-DR15-restricted cytotoxic CD4+ T-cell clones
Authors: Zeng L , Kurane I , Okamoto Y , Ennis F A , Brinton M A
Published: January 2020

The majority of T-cell clones derived from a donor who experienced dengue illness following receipt of a live experimental dengue virus type 3 (DEN3) vaccine cross-reacted with all four serotypes of dengue virus, but some were serotype specific or only partially cross-reactive. The nonstructural protein, NS3, was immuno-dominant in the CD4+ T-cell response of this donor. The epitopes of four NS3-specific T-cell clones were analyzed. JK15 and JK13 recognized only DEN3 NS3, while JK44 recognized DEN1, DEN2, and DEN3 NS3 and JK5 recognized DEN1, DEN3, and West Nile virus NS3. The epitopes recognized by these clones on the DEN3 NS3 protein were localized with recombinant vaccinia viruses expressing truncated regions of the NS3 gene, and then the minimal recognition sequence was mapped with synthetic peptides. Amino acids critical for T-cell recognition were assessed by using peptides with amino acid substitutions. One of the serotype-specific clones (JK13) and the subcomplex- and flavivirus-cross-reactive clone (JK5) recognized the same core epitope, WITDFVGKTVW. The amino acid at the sixth position of this epitope is critical for recognition by both clones. Sequence analysis of the T-cell receptors of these two clones showed that they utilize different VP chains. The core epitopes for the four HLA-DR15-restricted CD4+ CTL clones studied do not contain motifs similar to those proposed by previous studies on endogenous peptides eluted from HLA-DR15 molecules. However, the majority of these dengue virus NS3 core epitopes have a positive amino acid (K or R) at position 8 or 9. Our results indicate that a single epitope can induce T cells with different virus specificities despite the restriction of these T cells by the same HLA-DR15 allele. This finding suggests a previously unappreciated level of complexity for interactions between human T-cell receptors and viral epitopes with very similar sequences on infected cells.
Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors
Authors: Ferrari F K , Samulski T , Shenk T , Samulski R J
Published: January 2020

The ability of recombinant adeno-associated virus (AAV) to transduce cells with a marker gene in vitro was found to be substantially increased by the presence of adenovirus. Transfection experiments with adenovirus genomic DNA suggest that this increase is not facilitated by adenovirus-mediated viral uptake but is instead dependent on adenovirus gene expression. Using various adenovirus mutants, we were able to map this function to early-region E4 open reading frame 6. Plasmid expression of open reading frame 6 protein in cells infected with recombinant AAV increased transduction between 100- and 1,000-fold. The increase in transduction was not dependent on the recombinant AAV gene cassette but instead appeared to involve an immediate early step of the AAV life cycle. Chemical and physical agents that have been shown to induce helper-free replication of wild-type AAV were also able to stimulate recombinant AAV transduction, suggesting that the phenomenon might affect AAV DNA replication. Further experiments showed that viral uncoating was not affected and that the rate-limiting step involved synthesis of a second strand on the single-stranded genomic AAV DNA. These data suggest that the adenovirus E4 region, as well as chemical and physical agents, can play an essential role in an immediate-early step of the AAV life cycle, specifically in second-strand synthesis, and have important implications for the use of AAV vectors in gene therapy protocols.
A novel form of hepatitis delta antigen
Authors: Bichko V V , Khudyakov Y E , Taylor J M
Published: January 2020

Hepatitis delta virus (HDV) is known to express a protein termed the small delta antigen, a structural protein which is also essential for genome replication. During replication, posttranscriptional RNA editing specifically modifies some of the HDV RNA, leading to the production of an elongated form of the delta antigen, the large form, which is essential for virus assembly. The present study showed that yet another form of HDV protein is expressed during genome replication. This novel form is not produced in all infected cells, but it arises during replication in transfected cells and in infected woodchucks, and as was previously reported, patients infected with HDV do make antibodies directed against it. These findings are an indicator of the complexity of gene expression during HDV infection and replication.
Assessment of the serological relatedness of genital human papillomaviruses by hemagglutination inhibition
Authors: Roden R B , Hubbert N L , Kirnbauer R , Christensen N D , Lowy D R , Schiller J T
Published: January 2020

To assess the potential for cross-protection among genital human papillomavirus (HPV) types in virus-like particle (VLP)-based vaccinations, inhibition of HPV VLP-mediated hemagglutination by rabbit antisera raised against HPV type 6b (HPV-6b), HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, and HPV-45 was analyzed. Only highly homologous types (HPV-6b and HPV-11, and HPV-18 and HPV-45) exhibited detectable serological cross-reaction for the class of antibodies that inhibit virion-to-cell surface binding. However, analysis of neutralizing monoclonal antibodies to several animal and human papillomaviruses indicated that over half of these antibodies do not prevent cell surface binding, but these latter antibodies do not appear to be more cross-reactive in enzyme-linked immunosorbent assays than those that mediate inhibition of hemagglutination. The data strongly suggest that while there may be limited cross-protection between highly (>85% L1 amino acid identity) homologous types, protection by HPV VLP-based vaccines will be predominantly type specific.
Serine 3 is critical for phosphorylation at the N-terminal end of the nucleoprotein of influenza virus A/Victoria/3/75
Authors: Arrese M , Portela A
Published: January 2020

The influenza A virus nucleoprotein (NP) is a phosphoprotein that encapsidates the viral genomic RNA. To map the in vivo phosphorylation site(s) of this protein, 32P-labeled NP was purified from cell cultures infected with influenza virus A/Victoria/3/75 by immunoaffinity chromatography. The purified protein was then subjected to chemical digestion with formic acid, which cleaves proteins at Asp-Pro bonds, and the resulting products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two of the phosphorylated products obtained were identified as fragments corresponding to the N-terminal 88 amino acids and to the C-terminal 196 residues of the NP. To identify the phosphate acceptor site(s) at the N-terminal phosphorylated region of NP, each of the seven serines within this region was individually changed to alanine by site-directed mutagenesis. The mutant proteins were then transiently expressed in mammalian cells and analyzed for their phosphorylation state. It was observed that the S-to-A mutation at position 3 drastically reduced the amount of 32P label incorporated into NP, whereas the other substitutions did not have a discernible effect on the phosphorylation level of the protein. In addition, all serine-altered proteins were tested for their functionality in an artificial system in which expression of a synthetic chloramphenicol acetyl-transferase RNA molecule is driven by influenza virus proteins synthesized from cloned genes. The results obtained demonstrate that all mutant proteins were competent to cooperate with the subunits of the viral polymerase for expression of the synthetic virus-like chloramphenicol acetyltransferase RNA in vivo. These data are discussed regarding the possible roles of NP phosphorylation for the viral replicative cycle.
Sequences regulating tropism of human immunodeficiency virus type 1 for brain capillary endothelial cells map to a unique region on the viral genome
Authors: Moses A V , Stenglein S G , Strussenberg J G , Wehrly K , Chesebro B , Nelson J A
Published: January 2020

Two infectious molecular clones of human immunodeficiency virus type 1, NL4-3 and JR-CSF, differ in their abilities to productively infect human brain capillary endothelial (HBCE) cells. The phenotypes of recombinants between these two molecular strains were examined to identify viral sequences responsible for the difference in HBCE cell tropism between the two parental strains. Our results indicate that HBCE cell tropism maps to a region that encompasses the C1 region of env and includes overlapping reading frames for the accessory genes vpr, vpu, tat, and rev. This region was unique for HBCE cell tropism and did not cosegregate with either macrophage or T-cell line tropism. However, several recombinant clones displayed dual tropism for both HBCE cells and macrophages. These endothelial cell- and macrophage-tropic strains may have a unique pathogenic advantage by entering the brain via HBCE cells and subsequently infecting microglial cells with high efficiency, leading to the induction of human immunodeficiency virus dementia.
Longitudinal studies of viral sequence, viral phenotype, and immunologic parameters of human immunodeficiency virus type 1 infection in perinatally infected twins with discordant disease courses
Authors: Hutto C , Zhou Y , He J , Geffin R , Hill M , Scott W , Wood C
Published: January 2020

Perinatal human immunodeficiency virus type 1 (HIV-1) infections cause a broad spectrum of clinical disease and are variable in both the age of the patient at onset of serious disease and the progression of the clinical course. Heterozygotic perinatally infected twins with a marked difference in their clinical courses were monitored during the first 2 years of life. Twin B, the second-born twin, developed AIDS by 6 months of age and died at 22 months of age, while twin A remained minimally symptomatic through the first 2 years. Sequential blood specimens were obtained from the twins in order to characterize the immunologic properties of the children and the phenotypes and genotypes of the HIV-1 isolates at various times. Twin A developed neutralizing antibodies and a high-level antibody-mediated cellular cytotoxicity (ADCC) response, while twin B had no neutralizing antibody and a much lower ADCC response. The virus isolates obtained from the two children at various time points proliferated equally well in peripheral blood mononuclear cells, were nonsyncytium inducing, and could not infect established T-cell lines. They differed in their ability to infect primary macrophages. In parallel to the biological studies, the HIV-1 tat and part of the env gene sequences of the longitudinal isolates at four time points were determined. Sequences of virus from both twins at different time points were highly conserved; the viruses evolved at a similar rate until the last analyzed time point, at which there was a dramatic increase in sequence diversity for the sicker child, especially in the tat gene. Our results show that the viruses isolated at different times do not have significant changes in growth properties. The absence or low levels of neutralizing antibodies may correlate with disease progression in the twins.
Inhibitory activity of the equine infectious anemia virus major 5' splice site in the absence of Rev
Authors: Tan W , Schalling M , Zhao C , Luukkonen M , Nilsson M , Fenyö E M , Pavlakis G N , Schwartz S
Published: January 2020

The major 5' splice site of equine infectious anemia virus (EIAV) conforms to the consensus 5' splice site in eight consecutive positions and is located immediately upstream of the gag AUG. Our results show that the presence of this 5' splice site on the EIAV gag mRNA decreases Gag production 30- to 60-fold. This is caused by inefficient nuclear mRNA export and inefficient mRNA utilization. Inhibition could be overcome by providing human immunodeficiency virus type 1 Rev/Rev-responsive element, human T-cell leukemia virus type 1 Rex/Rex-responsive element, or simian retrovirus type 1 constitutive transport element. In addition, inhibition could be abolished by introducing single point mutations in the 5' splice site or by moving the 5' splice site away from its natural position immediately upstream of the gag AUG. This demonstrates that both maintenance of a perfect consensus 5' splice site and its proper location on the mRNA are important for inhibitory activity of the EIAV major 5' splice site.
Synthesis of Semliki Forest virus RNA polymerase components nsP1 through nsP4 in Saccharomyces cerevisiae by expression of cDNA encoding the nonstructural polyprotein
Authors: Russo P , Laakkonen P , Ahola T , Kääriäinen L
Published: January 2020

A Semliki Forest virus nonstructural polyprotein, P1234, expressed in the yeast Saccharomyces cerevisiae in the absence of a replicative RNA template appeared to be properly cleaved into nsP1 to nsP4. All nsPs were membrane associated, and nsP2 was also transported to the nucleus. The membrane fraction containing nsPs showed guanine-7-methyltransferase and guanylyltransferase-like activities, typical for Semliki Forest virus nsP1.
Oral inoculation with herpes simplex virus type 1 infects enteric neuron and mucosal nerve fibers within the gastrointestinal tract in mice
Authors: Gesser R M , Koo S C
Published: January 2020

Herpes simplex virus type 1 (HSV-1) is commonly encountered first during childhood as an oral infection. After this initial infection resolves, the virus remains in a latent form within innervating sensory ganglia for the life of the host. We have previously shown, using a murine model, that HSV-1 placed within the lumen of the esophagus gains access to nerves within the gut wall and establishes a latent infection in sensory ganglia (nodose ganglia) of the tenth cranial nerve (R. M. Gesser, T. Valyi-Nagy, S. M. Altschuler, and N. W. Fraser, J. Gen. Virol. 75:2379-2386, 1994). Peripheral processes of neurons in these ganglia travel through the vagus nerve and function as primary sensory receptors in most of the gastrointestinal tract, relaying information from the gut wall and mucosal surface to secondary neurons within the brain stem. In the work described here, we further examined the spread of HSV-1 through the enteric nervous system after oral inoculation. By immunohistochemistry, HSV-1 was found to infect myenteric ganglia in Auerbach's plexus between the inner and outer muscle layers of the gut wall, submucosal ganglia (Meisner's plexus), and periglandular ganglion plexuses surrounding submucosal glands. Virus-infected nerve fibers were also seen projecting through the mucosal layer to interact directly with surface epithelial cells. These intramucosal nerve fibers may be a conduit by which intraluminal virus is able to gain access to the enteric nervous system from the gastrointestinal lumen.
Induction of interleukin-12 (IL-12) by recombinant glycoprotein gp120 of human immunodeficiency virus type 1 in human monocytes/macrophages: requirement of gamma interferon for IL-12 secretion
Authors: Fantuzzi L , Gessani S , Borghi P , Varano B , Conti L , Puddu P , Belardelli F
Published: January 2020

We studied the effects of the gp120 glycoprotein of human immunodeficiency virus type 1 on the expression of interleukin-12 (IL-12) in human monocytes and in monocyte-derived macrophages. Induction of the mRNA for both the p35 and p40 subunits of IL-12 was observed in both cell types after gp120 treatment. We then evaluated cytokine secretion by using an enzyme-linked immunosorbent assay which recognizes only the IL-12 heterodimer. No IL-12 was detected in monocytes/macrophages treated with gp120 alone. A consistent IL-12 secretion was found in macrophages primed with gamma interferon (IFN-gamma) and subsequently treated with gp120. Low levels of IL-12 were occasionally observed in IFN-gamma-primed monocytes stimulated with gp120. The greater response of macrophages than of monocytes to the priming effect of IFN-gamma was consistent with the finding that IFN-gamma induced a much stronger antiviral state to vesicular stomatitis virus in macrophages than in monocytes. These data indicate that gp120 is an inducer of IL-12 expression in monocytes/macrophages and that IFN-gamma is an essential cofactor for IL-12 secretion, especially in differentiated macrophages.
Requirements for lymphocyte activation by unusual strains of simian immunodeficiency virus
Authors: Du Z , Ilyinskii P O , Sasseville V G , Newstein M , Lackner A A , Desrosiers R C
Published: January 2020

When residues 17 and 18 in nef of simian immunodeficiency virus strain SIVmac239 were changed from RQ to YE, the resultant virus was able to replicate in peripheral blood mononuclear cell cultures without prior lymphocyte activation and without the addition of exogenous interleukin-2, caused extensive lymphocyte activation in these cultures, and produced an acute disease in rhesus and pigtail macaques (Z. Du, S. M. Lang, V. G. Sasseville, A. A. Lackner, P. 0. Ilyinskii, M. D. Daniel, J. U. Jung, and R. C. Desrosiers, Cell 82:665-674, 1995). These properties are similar to those of the acutely lethal pathogen SIVpbj14 but dissimilar to those of the parental SIVmac239. We show here that the single change of R to Y at position 17 in nef of SIVmac239 is sufficient to confer the full, unusual phenotype. Conversely, the lymphocyte-activating properties of SIVpbj14 were lost by the single change of Y to R at position 17 of nef. The change of R17F or Q18E in SIVmac239 nef did not confer the unusual in vitro properties. Since SIVpbj14 has a duplication of the NF-kappaB binding sequence in the transcriptional control region, we also constructed and tested strains of SIVmac239/Rl7Y with zero, one, and two NF-kappaB binding elements. We found no difference in the properties of SIVmac239/R17Y, either in cell culture or in vivo, whether zero, one, or two NF-kappaB binding sites were present. Thus, tyrosine at position 17 of nef is absolutely necessary for the unusual phenotype of SIVpbj14 and is sufficient to convert SIVmac239 to a virus with a phenotype like that of SIVpbjl4. Multiple NF-kappaB binding sites are not required for the in vitro properties or for acute disease.
Monkeys immunized with intertypic chimeric dengue viruses are protected against wild-type virus challenge
Authors: Bray M , Men R , Lai C J
Published: January 2020

Dengue epidemics caused by the four dengue virus serotypes continue to pose a major public health problem in most tropical and subtropical regions. A safe and effective vaccine against dengue is still not available. The current strategy for dengue immunization favors the use of a vaccine containing each of the four serotypes. We previously employed full-length dengue type 4 virus (DEN4) cDNA to construct a viable intertypic dengue virus of type 1 or type 2 antigenic specificity that contained the genes for the capsid-premembrane-envelope (C-pre-M-E) structural proteins of DEN1 or pre-M and E structural proteins of DEN2 substituting for the corresponding DEN4 genes. Chimeras DEN1/DEN4 and DEN2/DEN4, which express the nonstructural proteins of DEN4 and the C-pre-M-E structural proteins of DEN1 or the pre-M-E structural proteins of DEN2, and therefore the antigenicity of type 1 or type 2, were used to immunize rhesus monkeys. Other monkeys were inoculated with parental DEN1, DEN2, or cDNA-derived DEN4. Three of four monkeys immunized with DEN1/DEN4 developed neutralizing antibodies against DEN1 and were protected against subsequent DEN1 challenge. All four monkeys immunized with DEN2/DEN4 developed antibodies against DEN2 and were protected against subsequent DEN2 challenge. DEN1- and DEN2-immunized monkeys were protected against homologous virus challenge, but DEN4-immunized animals became viremic on cross-challenge with DEN1 or DEN2. In a second experiment, eight monkeys were immunized with equal mixtures of DEN1/DEN4 and DEN2/DEN4. Each of these monkeys developed neutralizing antibodies against both DEN1 and DEN2 and were protected against subsequent challenge with DEN1 or DEN2. Chimeric dengue viruses similar to those described here could be used to express serotype-specific antigens in a live attenuated tetravalent human vaccine.
EBNA-2 and EBNA-3C extensively and mutually exclusively associate with RBPJkappa in Epstein-Barr virus-transformed B lymphocytes
Authors: Johannsen E , Miller C L , Grossman S R , Kieff E
Published: January 2020

Although genetic and biochemical data indicate that the cell protein RBPJkappa is a mediator of EBNA-2 and EBNA-3C effects on transcriptional regulatory elements, the extent of association of these Epstein-Barr virus nuclear proteins with RBPJkappa in transformed B lymphocytes has not been determined. We now report that most of the EBNA-2 and at least 20% of the EBNA-3C coimmunoprecipitated with RBPJkappa from extracts of transformed B lymphocytes that contained most of the cellular EBNA-2 and EBNA3C. Both proteins are associated preferentially with the smaller of the two RBPJkappa isoforms. EBNA-2-RBPJkappa complexes do not contain EBNA-3C, and EBNA-3C-RBPJkappa complexes do not contain EBNA-2. Although EBNA-2 and EBNA-3C are extensively associated with RBPJkappa, a fraction of RBPJkappa appears to be free of EBNAs after repeated immunoprecipitations with anti-EBNA, Epstein-Barr virus-immune, human antibody. Promoters with RBPJkappa sites in their regulatory elements are likely to be differentially regulated by these RBPJkappa-EBNA-2 and RBPJkappa-EBNA-3 complexes.
Transduction of human CD34+ hematopoietic progenitor cells by a retroviral vector expressing an RRE decoy inhibits human immunodeficiency virus type 1 replication in myelomonocytic cells produced in long-term culture
Authors: Bahner I , Kearns K , Hao Q L , Smogorzewska E M , Kohn D B
Published: January 2020

Genetic modification of hematopoietic stem cells with a synthetic "anti-human immunodeficiency virus type 1 (HIV-1) gene" which inhibits replication of HIV-1 may allow production of mature lymphoid and monocytic cells resistant to HIV-1 growth after autologous transplantation. Because productive HIV-1 replication requires binding of the Rev protein to the Rev-responsive element (RRE) within the viral transcripts for the HIV-1 structural proteins, anti-HIV-1 gene products which interfere with Rev-RRE interactions may inhibit HIV-1 replication. One such strategy involves overexpression of the RRE sequences in transcripts derived from retroviral vectors to act as decoys to sequester Rev protein and prevent its binding to the RRE element in HIV-1 transcripts. We developed an in vitro model to test the efficacy of this gene therapy approach in primary human hematopoietic cells. Human CD34+ hematopoietic progenitor cells from normal bone marrow or umbilical cord blood were transduced with retroviral vectors carrying RRE decoy sequences as part of a long terminal repeat-directed transcript expressing the neo gene (L-RRE-neo) or with a control vector expressing only the neo gene (LN). The transduced progenitors were allowed to differentiate into mature myelomonocytic cells which were able to support vigorous growth of the monocytotropic isolate of HIV-1, JR-FL. HIV-1 replication was measured in unselected cell populations and following G418 selection to obtain uniformly transduced cell populations. Inhibition of HIV-1 replication in the unselected cell cultures was between 50.2 and 76.7% and was highly effective (99.4 to 99.9%) in the G418-selected cultures. Progenitors transduced by either the L-RRE-neo vector or the control LN vector were identical with respect to hematopoietic growth and differentiation. These findings demonstrate the ability of an RRE decoy strategy to inhibit HIV-1 replication in primary human myelomonocytic cells after transduction of CD34+ progenitor cells, without adverse effects on hematopoietic cell function.