bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure, function, and evolution of adenovirus-associated RNA: a phylogenetic approach
Authors: Ma Y , Mathews M B
Published: January 2020

To explore the structure and function of a small regulatory RNA, we examined the virus-associated (VA) RNA species of all 47 known human adenovirus serotypes and of one simian virus, SA7. The VA RNA gene regions of 43 human adenoviruses were amplified and sequenced, and the structures of 10 representative VA RNAs were probed by nuclease sensitivity analysis. Most human viruses have two VA RNA species, VA RNA, and VA RNAII, but nine viruses (19%) have a single VA RNA gene. Sequence alignments classified the RNAs into eight families, corresponding broadly to the known virus groups, and three superfamilies. One superfamily contains the single VA RNAs of groups A and F and the VA RNAI species of group C; the second contains the VA RNAI species of groups B1, D, and E and the unclassified viruses (adenovirus types 42 to 47), as well as the single VA RNAs of group B2; and the third contains all VA RNAII species. Fourteen regions of homology occur throughout the molecule. The longest of these correspond to transcription signals; most of the others participate in RNA secondary structure. The previously identified tetranucleotide pair, GGGU:ACCC, is nearly invariant, diverging slightly (to GGGU:ACCU) only in the two group F viruses and forming a stem in the central domain that is critical for VA RNA structure and function. Secondary structure models which accommodate the nuclease sensitivity data and sequence variations within each family were generated. The major structural features-the terminal stem, apical stem-loop, and central domain-are conserved in all VA RNAs, but differences exist in the apical stem and central domains, especially of the VA RNAII species. Sequence analysis suggests that an ancestral VA RNA gene underwent duplication during the evolution of viruses containing two VA RNA genes. Although the VA RNAII gene seems to have been lost or inactivated by secondary deletion events in some viruses, the high degree of homology among the VA RNAII species implies that this RNA may play an undiscovered role in virus survival. We speculate that the VA RNA genes originated from cellular sequences containing multiple tRNA genes.
Structural analysis of human immunodeficiency virus type 1 Gag protein interactions, using cysteine-specific reagents
Authors: McDermott J , Farrell L , Ross R , Barklis E
Published: January 2020

We have examined structural interactions of Gag proteins in human immunodeficiency virus type 1 (HIV-1) particles by utilizing cysteine mutagenesis and cysteine-specific modifying reagents. In immature protease-minus but otherwise wild-type (wt) particles, precursor Pr55Gag proteins did not form intermolecular cystines naturally but could be cross-linked at cysteines, and cross-linking appeared to occur across nucleocapsid (NC) domains. Capsid (CA) proteins in wt mature viruses possess cysteines near their carboxy termini at gag codons 330 and 350, but these residues are not involved in natural covalent intermolecular bonds, nor can they be intermolecularly cross-linked by using the membrane-permeable cross-linker bis-maleimido hexane. The cysteine at gag codon 350 (C-350) is highly reactive to thiol-specific modifying reagents, while the one at codon 330 (C-330) appears considerably less reactive, even in the presence of ionic detergent. These results suggest that the HIV-1 CA C terminus forms an unusually stable conformation. Mutagenesis of C-350 to a serine residue in the mutant C350S (C-350 changed to serine) virtually eliminated particle assembly, attesting to the importance of this region. We also examined a C330S mutant, as well as mutants in which cysteines were created midway through the capsid domain or in the C-terminal section of the major homology region. All such mutants appeared wt on the basis of biochemical assays but showed greatly reduced infectivities, indicative of a postassembly, postprocessing replicative block. Interestingly, capsid proteins of mature major homology region mutant particles could be cysteine cross-linked, implying either that these mutations permit cross-linking of the native C-terminal CA cysteines or that major homology regions on neighbor capsid proteins are in close proximity in mature virions.
Species-specific effects of the hcf-1 gene on baculovirus virulence
Authors: Lu A , Miller L K
Published: January 2020

The host cell-specific factor 1 gene (hcf-1) of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) is required for the efficient replication and/or stability of reporter plasmids carrying an AcMNPV-derived origin of DNA replication in a cell-specific manner; hcf-1 is required for reporter plasmid replication or stability in TN-368 cells, a cell line derived from the cabbage looper Trichoplusia ni, but not in IPLB-SF-21 (SF-21) cells, a cell line derived from the fall armyworm Spodoptera frugiperda (A. Lu and L. K. Miller, J. Virol. 69:6265-6272, 1995). To further define the function of hcf-1, recombinant viruses with null mutations in hcf-1 were constructed in SF-21 cells and the phenotype of the mutants was determined in selected cell lines as well as in insect larvae. In S.frugiperda larvae and SF-21 cells, the phenotype of hcf-1 mutants was indistinguishable from that of wild-type AcMNPV. In T. ni larvae as well as T. ni-derived cell lines, hcf-1 mutants exhibited a mutant phenotype. In TN-368 cells, the replication of hcf-1 mutants was extremely impaired; the phenotype included a defect in viral DNA replication, late gene transcription, and virus production as well as a complete cessation of host and viral protein synthesis. In another cell line derived from T. ni, the BTI-TN5B1-4 cell line, the hcf-1 mutants exhibited a less severe phenotype. In T. ni larvae, the infectivity of the budded form of hcf-1 mutants was decreased significantly (50-fold), although no difference in the oral infectivity of the occluded form was observed. T. ni larvae infected with hcf-1 mutants by either oral or hemocoelic routes, however, died 20 to 30% more slowly than those infected with wild-type AcMNPV. These data indicate that there is a host-specific requirement for hcf-1 and that it exerts cell line-specific effects and possibly tissue-specific effects on the rate at which the virus replicates, thereby affecting the virulence of the virus in a species-specific manner.
Woodchuck hepatitis virus X protein is present in chronically infected woodchuck liver and woodchuck hepatocellular carcinomas which are permissive for viral replication
Authors: Dandri M , Schirmacher P , Rogler C E
Published: January 2020

The woodchuck hepatitis virus (WHV) X gene (WHx) is required for infectivity of WHV in woodchucks, and the gene encodes a broadly acting transcription factor. Several lines of evidence from cell culture and transgenic mice suggest that X proteins can promote hepatocarcinogenesis. To determine whether WHx-encoded proteins are present during persistent infection and hepatocellular carcinoma (HCC) in woodchucks, we surveyed livers and HCCs from a panel of WHV carrier woodchucks for the presence of WHx by utilizing an immunoprecipitation-Western blot (immunoblot) procedure. We detected a single 15.5-kDa WHx gene product in 100% of the persistently infected livers but not in livers from animals which had recovered from acute infection or in those of uninfected woodchucks. Analysis of HCCs revealed that all of the tumors which contained WHV replication intermediates were also positive for WHx. In contrast, WHx was undetectable in HCCs which did not contain replicative intermediates. Subcellular localization studies detected WHx in the cytoplasm but not in the nuclei of primary woodchuck hepatocytes. Comparative immunoprecipitation experiments revealed that there were 4 X 10(4) to 8 X 10(4) molecules of WHx per primary woodchuck hepatocyte. Four lines of WHx transgenic mice did not develop HCC spontaneously. However, when one line was treated with diethylnitrosamine, the occurrence of precancerous lesions was enhanced compared with that in diethylnitrosamine-treated nontransgenic controls. The apparent absence of WHx in some woodchuck HCCs indicates that WHx may not be required to maintain the tumor phenotype, whereas its presence in all persistently infected livers leaves open the possibility that it plays a role in hepatocarcinogenesis.
Cytomegalovirus assemblin: the amino and carboxyl domains of the proteinase form active enzyme when separately cloned and coexpressed in eukaryotic cells
Authors: Hall M R , Gibson W
Published: January 2020

The cytomegalovirus (CMV) serine proteinase assemblin is synthesized as a precursor that undergoes three principal autoproteolytic cleavages. Two of these are common to the assemblin homologs of all herpes group viruses: one at the maturational site near the carboxyl end of the precursor and another at the release site near the midpoint of the precursor. Release-site cleavage frees the proteolytic amino domain, assemblin, from the nonproteolytic carboxyl domain of the precursor. In CMV, a third autoproteolytic cleavage at an internal site divides assemblin into an amino subunit (An) and a carboxyl subunit (Ac) of approximately the same size that remain associated as an active "two-chain" enzyme. We have cloned the sequences encoding An and Ac as separate genes and expressed them by transfecting human cells with recombinant plasmids and by infecting insect cells with recombinant baculoviruses. When An and Ac from either simian CMV or human CMV were coexpressed in human or insect cells, active two-chain assemblin was formed. This finding demonstrates that An and Ac do not require synthesis as single-chain assemblin to fold and associate correctly in these eukaryotic systems, and it suggests that they may be structurally, if not functionally, distinct domains. An interaction between the independently expressed An and Ac subunits was demonstrated by coimmunoprecipitation experiments, and efforts to disrupt the complex indicate that the subunit interaction is hydrophobic. Cell-based cleavage assays of the two-chain assemblin formed from independently expressed An and Ac also indicate that (i) its specificity for both CMV and herpes simplex virus native substrates is similar to that of single-chain assemblin, (ii) R-site cleavage is not essential for the activity of two-chain recombinant assemblin, and (iii) the human CMV and simian CMV An and Ac recombinant subunits are functionally interchangeable.
Disulfide bond structure determination and biochemical analysis of glycoprotein C from herpes simplex virus
Authors: Rux A H , Moore W T , Lambris J D , Abrams W R , Peng C , Friedman H M , Cohen G H , Eisenberg R J
Published: January 2020

A biochemical analysis of glycoprotein C (gC of herpes simplex virus was undertaken to further characterize the structure of the glycoprotein and to determine its disulfide bond arrangement. We used three recombinant forms of gC, gC1(457t), gC1(delta33-123t), and gC2(426t), each truncated prior to the transmembrane region. The proteins were expressed and secreted by using a baculovirus expression system and have been shown to bind to monoclonal antibodies which recognize discontinuous epitopes and to complement component C3b in a dose-dependent manner. We confirmed the N-terminal residues of each mature protein by Edman degradation and confirmed the internal deletion in gC1(delta33-123t). The molecular weight and extent of glycosylation of gC1 (457t), gC1(delta33-123t), and gC2(426t) were determined by treating each protein with endoglycosidases and then subjecting it to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis. The data indicate that eight to nine of the predicted N-linked oligosaccharide sites on gC1(457t) are occupied by glycans of approximately 1,000 Da. In addition, O-linked oligosaccharides are present on gC1(457t), primarily localized to the N-terminal region (amino acids [aa] 33 to 123) of the protein. gC2(426t) contains N-linked oligosaccharides, but no O-linked oligosaccharides were detected. To determine the disulfide bond arrangement of the eight cysteines of gC1(457t),the protein was cleaved with cyanogen bromide. SDS-PAGE analysis followed by Edman degradation identified three cysteine-containing fragments which are not connected by disulfide linkages. Chemical modification of cysteines combined with matrix-assisted laser desorption ionization mass spectrometry identified disulfide bonds between cysteine 1 (aa 127) and cysteine 2 (aa 144) and between cysteine 3 (aa 286) and cysteine 4 (aa 347). Further proteolysis of the cyanogen bromide-generated fragment containing cysteine 5 through cysteine 8, combined with mass spectrometry and Edman degradation, showed that disulfide bonds link cysteine 5 (aa 386) to cysteine 8 (aa 442) and cysteine 6 (aa 390) to cysteine 7 (aa 419). A similar disulfide bond arrangement is postulated to exist in gC homologs from other herpesviruses.
Herpes simplex virus gene expression in neurons: viral DNA synthesis is a critical regulatory event in the branch point between the lytic and latent pathways
Authors: Nichol P F , Chang J Y , Johnson E M , Olivo P D
Published: January 2020

Herpes simplex virus establishes a latent infection in peripheral neurons. We examined viral gene expression in rat peripheral neurons in vitro and determined that viral gene expression is attenuated and delayed in these neurons compared with that in Vero cells. In addition, using pharmacologic and genetic blocks to viral DNA synthesis, we found that viral alpha and beta gene expression was upregulated by viral DNA synthesis. Although maximal gene expression in neurons requires viral DNA synthetic activity, activation of viral gene expression was seen even in the presence of herpes simplex virus DNA polymerase inhibitors, but not in the absence of the origin-binding protein. Initiation of viral DNA synthesis is apparently a key regulatory event in the balance between the lytic and latent pathways in peripheral neurons.
Human T-cell leukemia virus type 2 Rex inhibits pre-mRNA splicing in vitro at an early stage of spliceosome formation
Authors: Bakker A , Li X , Ruland C T , Stephens D W , Black A C , Rosenblatt J D
Published: January 2020

The Rex protein is an essential regulator of RNA expression in human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) that promotes the accumulation of full-length and partially spliced viral transcripts in the cytoplasm. Rex-mediated regulation correlates with specific binding to a cognate RNA recognition element which overlaps the 5' splice site in the viral long terminal repeat. It has been unclear whether Rex directly affects splicing or only nuclear-to-cytoplasmic transport of viral mRNA. We demonstrate that HTLV-2 Rex is a potent inhibitor of splicing in vitro at an early step in spliceosome assembly. Inhibition requires phosphorylation of Rex and the ability of Rex to bind to the Rex response element. Direct inhibition of early spliceosome assembly by Rex may account for differential accumulation of unspliced transcripts and represents a novel mechanism of retroviral gene regulation.
FAETL motif required for leukemic transformation by v-Myb
Authors: Fu S L , Lipsick J S
Published: January 2020

The nuclear protein v-Myb, encoded by the avian myeloblastosis virus (AMV), can induce acute monoblastic leukemia in vivo and transform chicken myelomonocytic cells in culture. The N terminus of v-Myb functions as the DNA-binding domain, and multiple central and C-terminal regions of this protein have been reported to function in transcriptional activation of model reporter genes. We showed previously that a C-terminal domain (amino acids 296 to 371) is required for transcriptional activation and transformation of primary chicken myelomonocytic cells. In this study, we have now analyzed a series of C-terminal mutants of v-Myb to further investigate this domain. A strong correlation was observed between transcriptional activation and leukemic transformation by this series of mutants. Furthermore, deletion analyses demonstrate that the C-terminal 41 amino acids of v=MybAMV (amino acids 331 to 371 of the Myb portion) are nonessential whereas further deletion of amino acids 321 to 330 (EFAETLQLID) results in a nonfunctional protein. Hence, we defined a 10-amino-acid subregion (the "FAETL" motif) required for transcriptional activation and oncogenic transformation by v-Myb Amv. The FAETL region is part of a putative leucine zipper structure and lies near a cluster of phosphorylation sites. Our analysis of mutants with substitutions of the zipper leucines or multiple adjacent phosphorylation sites demonstrates that the function of the FAETL motif is not dependent on an intact leucine zipper structure or adjacent phosphorylation sites. The study of GAL4-Myb fusions suggests that this region is important in maintaining a fully functional conformation of v-Myb. The putative leucine zipper structure has previously been proposed to exert inhibitory effects on c-Myb because its mutation caused increased transcriptional transactivation and transformation. Interestingly, our results show that this region is essential for the functions of v-Myb without requiring a heptad leucine repeat.
Evidence for a phosphorylation site in cytomegalovirus glycoprotein gB
Authors: Norais N , Hall J A , Gross L , Tang D , Kaur S , Chamberlain S H , Burke R L , Marcus F
Published: January 2020

As part of our vaccine program, we have purified a recombinant form of human cytomegalovirus glycoprotein B that is able to induce high titers of virus-neutralizing antibodies. The isolated protein was found to be phosphorylated at a serine residue in position -7 from the C terminus of the protein. The corresponding synthetic peptide, HLKDSDEEENV, was an efficient in vitro substrate of casein kinase II.
The rev-responsive element negatively regulates human immunodeficiency virus type 1 env mRNA expression in primate cells
Authors: Churchill M J , Moore J L , Rosenberg M , Brighty D W
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) Rev protein mediates the accumulation of unspliced and singly spliced viral transcripts within the cytoplasm of infected cells, late in the infection cycle, leading to the expression of the viral structural proteins, Gag, Pol, and Env. Rev binds to a complex RNA structure, the Rev-responsive element (RRE), present in all Rev-responsive viral transcripts, relieving their nuclear sequestration. The precise mechanism by which RRE-containing transcripts are retained within the nucleus in the absence of Rev protein is not well understood. We previously demonstrated that the RRE alone plays a crucial role in the nuclear retention of RRE-containing env transcripts in stably transfected Drosophila cells. Here we extend our previous observations and demonstrate that the RRE is a principal determinant of nuclear retention for envelope transcripts in primate cells and, in particular, human CD4+ T cells.
Conditional human immunodeficiency virus type 1 protease mutants show no role for the viral protease early in virus replication
Authors: Kaplan A H , Manchester M , Smith T , Yang Y L , Swanstrom R
Published: January 2020

The human immunodeficiency virus type 1 protease plays a critical role in the proteolytic processing of precursor polyproteins during virion maturation. Contradictory evidence has been obtained for a possible role for the protease early after infection, i.e., during DNA synthesis and/or integration. We have reexamined this question by using conditional mutants of the protease. In one set of experiments, protease mutants that confer a temperature-sensitive phenotype for processing were used to assess the need for protease activity early after infection. No significant difference from results with wild-type virus was seen when infections were carried out at either 35 or 40 degrees C. In a separate set of experiments, infections were carried out in the presence of a protease inhibitor. In this case, both wild-type virus and a drug-resistant variant were used, the latter as a control to ensure a specific effect of the inhibitor. Infection with either virus was not inhibited at drug concentrations that were up to 10-fold higher than those needed to inhibit intracellular processing by the viral protease. The results obtained by both of these experimental protocols provide evidence that the human immunodeficiency virus type 1 protease does not play a role early after infection.
Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication
Authors: Buckwold V E , Xu Z , Chen M , Yen T S , Ou J H
Published: January 2020

The basal core promoter (BCP) of hepatitis B virus (HBV) controls the transcription of both the precore RNA and the core RNA. The precore RNA codes for the secreted e antigen, while the core RNA codes for the major core protein and the DNA polymerase and also is the pregenomic RNA. The double mutation of nucleotides 1762 and 1764 in the BCP from A and G to T and A, respectively, is frequently observed in HBV sequences isolated from chronic patients. Several papers have reported conflicting results regarding whether this double mutation is important for e antigen expression. In order to address this issue, we have introduced this double mutation into the HBV genome and studied its effects on HBV gene expression and replication. Our results indicate that the mutated BCP can no longer bind a liver-enriched transcription factor(s) and that the transcription of only precore RNA and, consequently, the expression of e antigen were reduced. The reduction of precore gene expression was accompanied by an increase in progeny virus production. This increase was found to occur at or immediately prior to the encapsidation of the pregenomic RNA. Thus, the results of our in vitro study resolve the discrepancy of previous clinical observations and indicate that this double mutation suppresses but does not abolish the e antigen phenotype. The implications of these findings in the pathogenesis of HBV are discussed.
Epstein-Barr virus EBNA3A and EBNA3C proteins both repress RBP-J kappa-EBNA2-activated transcription by inhibiting the binding of RBP-J kappa to DNA
Authors: Waltzer L , Perricaudet M , Sergeant A , Manet E
Published: January 2020

Following infection by Epstein-Barr virus (EBV), the production of viral nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C and the viral membrane protein LMP1 is essential for the permanent proliferation of primary B lymphocytes to occur. Among these, the transcription factor EBNA2 is central to the immortalizing process, since it activates not only the transcription of all the EBNA proteins and LMP1, TP1, and TP2 but also certain cellular genes. EBNA2 is targeted to its DNA-responsive elements through direct interaction with the DNA-binding cellular repressor RBP-J kappa. In a transient-expression assay, the EBNA2-activated transcription was found to be downregulated by EBNA3A, EBNA3B, and EBNA3C. However, since it has been reported that EBNA3C, but not EBNA3A, directly contacts RBP-J kappa in vitro, these proteins appear to repress through different mechanisms. Here, we report for the first time that EBNA3A and EBNA3C both stably interact with RBP-J kappa and most probably repress EBNA2-activated transcription by destabilizing the binding of RBP-J kappa to DNA.
Full-length sequence and mosaic structure of a human immunodeficiency virus type 1 isolate from Thailand
Authors: Carr J K , Salminen M O , Koch C , Gotte D , Artenstein A W , Hegerich P A , St Louis D , Burke D S , McCutchan F E
Published: January 2020

Human immunodeficiency virus type 1 isolates of envelope genotype E are contributing substantially to the global pandemic. These strains appear to be mosaics, with the gag gene from clade A and the envelope from clade E; the parental clade E strain has not been found. Here we report the first full genomic sequence of one such mosaic virus, isolate CM240 from Thailand. Multiple breakpoints between the two parental genotypes have been found in a CM240 virus. The entire gag-pol region and most, if not all, of the accessory genes vif, vpr, tat, rev, and vpu appear to derive from clade A. The genotype switches to E shortly after the signal peptide of the envelope and back to clade A near the middle of gp41; thus, the portion of the envelope that lies on the cytoplasmic side of the membrane appears to be principally derived not from clade E, as previously thought, but from clade A. Another small segment not belonging to any recognized clade and presumably also contributed by the parental E strain has been found in the long terminal repeat. It may be significant that the implied virion structure resembles a pseudotype virus with the matrix and core from one clade and the outer envelope from another. In the long terminal repeat, differences were observed between CM240 and other clades in the number of NF-kappa B binding sites, the sequence of the TATA box, and the putative secondary structure of the transactivation response region stem-loop. The mosaic structure of a CM240 virion is suggestive of phenotypic differences which might have contributed to the emergence of this variant.
The posttranscriptional control element of the simian retrovirus type 1 forms an extensive RNA secondary structure necessary for its function
Authors: Tabernero C , Zolotukhin A S , Valentin A , Pavlakis G N , Felber B K
Published: January 2020

It was previously shown that a 240-nucleotide (nt) RNA element (cis-acting transactivation element [CTE]) located between the env gene and the 3' long terminal repeat of simian retrovirus type 1 (SRV-1) can functionally replace posttranscriptional activation directed by Rev and the Rev-responsive element (RRE) when inserted into a Rev- and RRE-deficient molecular clone of human immunodeficiency virus type 1, resulting in efficient virus replication. Here, we analyze the molecular and structural requirements for function of this RNA element. Deletion mutagenesis demonstrated that the core element spans 173 nt. SRV-2 and Mason-Pfizer monkey virus have highly homologous elements, which function similarly when inserted into the Rev/RRE-deficient human immunodeficiency virus type 1. Computer prediction indicated that the core CTEs of all three viruses have similar extensive secondary structures. Mutagenesis of the SRV-1 CTE revealed that both sequence and secondary structure are essential for function. Nuclease probing of the SRV-1 CTE further supported the genetic analysis and confirmed the predicted structural features of the RNA element. Sequence analysis of the 240-nt SRV-1 CTE, after continuous long-term propagation of the Rev-independent viruses, revealed that the genetically defined core element remained unchanged, while regions outside the core CTE underwent deletions or duplications. These data further support our in vitro mutagenesis data and demonstrate the importance of the sequence and structure of the SRV-1 CTE for appropriate function.
A reevaluation of the higher taxonomy of viruses based on RNA polymerases
Authors: Zanotto P M , Gibbs M J , Gould E A , Holmes E C
Published: January 2020

In order to assess the validity of classifications of RNA viruses, published alignments and phylogenies of RNA-dependent RNA and DNA polymerase sequences were reevaluated by a Monte Carlo randomization procedure, bootstrap resampling, and phylogenetic signal analysis. Although clear relationships between some viral taxa were identified, overall the sequence similarities and phylogenetic signals were insufficient to support many of the proposed evolutionary groupings of RNA viruses. Likewise, no support for the common ancestry of RNA-dependent RNA polymerases and reverse transcriptases was found.
Influenza virus nucleoprotein-specific immunoglobulin G subclass and cytokine responses elicited by DNA vaccination are dependent on the route of vector DNA delivery
Authors: Pertmer T M , Roberts T R , Haynes J R
Published: January 2020

Endpoint immunoglobulin G (IgG) titers and cytotoxic T-lymphocyte (CTL) activities were identical between mice immunized via the intramuscular and epidermal (gene gun) routes with 100 and 1 micrograms, respectively, of an influenza virus nucleoprotein (NP) expression vector. However, examination of the relative levels of two IgG subclasses demonstrated that muscle inoculation resulted in predominantly IgG2a responses, whereas gene gun immunization yielded a preponderance of IgG1 antibodies. Inasmuch as these data suggested that muscle inoculation and gene gun delivery elicited Th1-like and Th2-like responses, respectively, gamma interferon release profiles from antigen-stimulated splenocytes were remarkably similar between these groups. Interleukin-4 (IL-4) production assays, on the other hand, revealed qualitative differences that could be correlated with the divergent IgG subclass data. Waning gamma interferon production in gene gun-immunized animals was countered by a marked increase in IL-4 production following the third immunization, as was the case in control animals immunized with inactivated influenza virus formulated with Freund's adjuvant. In contrast, significant levels of IL-4 production were not observed in the intramuscular DNA inoculation group, despite similar decreases in gamma interferon production with increasing immunizations. These data show that intramuscular inoculation leads to Th1-like responses due to elevated IgG2a levels, production of gamma interferon, CTL activity, and lack of IL-4. However, gene gun responses are more difficult to categorize because of the presence of significant gamma interferon and CTL activity on the one hand and elevated IgG1 antibodies and increasing IL-4 production with successive immunizations on the other. In addition, there was a lack of correlation between IgG isotype ratios and cytokine production in all of the NP DNA-immunized animals, in that IgG subclass ratios remained fixed while cytokine production patterns fluctuated with successive immunizations. These data are consistent with the idea that the types of responses elicited following DNA immunization. are dependent on both the identity of the antigen and the route of DNA administration.
Amino acids essential for RNase H activity of hepadnaviruses are also required for efficient elongation of minus-strand viral DNA
Authors: Chen Y , Marion P L
Published: January 2020

The hepadnavirus P gene contains amino acid sequences which share homology with all known RNases H. In this study, we made four mutants in which single amino acids of the duck hepatitis B virus (DHBV) RNase H region were altered. In two of them, amino acids at locations comprising the putative catalytic site were changed, while the remaining mutants had alterations at amino acids conserved among hepadnaviruses. Transfection of these mutant genomes into permissive cells resulted in synthesis of several discrete viral nucleic acid species, ranging in apparent sizes from approximately 500 to 3,000 bp, numbered I, II, III, IV, and V. While the locations of the species were similar in all mutants, the proportions of the species varied among the mutants. Analysis of the nucleic acid species revealed that they were hybrid molecules of RNA and minus-strand DNA, indicating that the RNase H activity was missing or greatly reduced in these mutants. Primer extension experiments showed that the mutant viruses initiated minus-strand viral DNA synthesis normally. The 3' termini of minus-strand DNA in species II, III, and IV were mapped just downstream of nucleotides 1659, 1220, and 721, respectively. Species V contained essentially full-length minus-strand viral DNA. A parallel amino acid change in the putative catalytic site of the HBV RNase H domain resulted in accumulation of low-molecular-weight hybrid molecules consisting of RNA and minus-strand DNA and similar in size and pattern to those seen with DHBV. These studies demonstrate experimentally the involvement of the C-terminal portion of the P gene in RNase H activity in both DHBV and human hepatitis B virus and indicate that the amino acids essential for RNase H activity of hepadnavirus P protein are also important for the efficient elongation of minus-strand viral DNA.
Peripheral blood mononuclear cells from sheep infected with a variant of bovine leukemia virus synthesize envelope glycoproteins but fail to induce syncytia in culture
Authors: Johnston E R , Powers M A , Kidd L C , Radke K
Published: January 2020

Peripheral blood mononuclear cells (PBMCs) infected with the oncogenic retrovirus bovine leukemia virus (BLV) produce virus when cultured briefly. BLV can be transmitted in cocultures to adherent susceptible cells, which become infected, express viral proteins, and fuse into multinucleated syncytia several days later. PBMCs from 3 of 10 BLV-infected sheep displayed a lifelong deficiency in induction of syncytium formation among indicator cells in culture, although large numbers of PBMCs synthesized viral transcripts or capsid protein. Since the infected, syncytium-deficient PBMCs were > or = 97% B cells, the deficiency could not be attributed to altered host cell tropism. The syncytium-deficient phenotype was recapitulated in newly infected sheep, demonstrating that this property is regulated by the viral genotype. The alteration in the BLV genome delayed but did not prohibit the establishment of BLV infection in vivo. Envelope glycoproteins were synthesized in syncytium-deficient PBMCs, translocated to the cell surface, and incorporated into virions. However, monoclonal antibodies specific for the BLV surface glycoprotein did not stain fixed PBMCs of the syncytium-deficient phenotype. Moreover, an animal with syncytium-deficient PBMCs had lower titers of neutralizing antibodies throughout the first 5 years of infection than an animal with similar numbers of infected PBMCs of the syncytium-inducing phenotype. The syncytium-deficient variant productively infected indicator cells at greatly reduced efficiency, showing that the alteration affects an early step in viral entry or replication. These results suggest that the alteration maps in the env gene or in a gene whose product affects the maturation or conformation, and consequently the function, of the envelope protein complex.
Activation of the human thymidine kinase (TK) promoter by simian virus 40 large T antigen requires both the T antigen pRb family-binding domain and TK promoter sequences resembling E2F-binding sites
Authors: Anderson M M , Chen J , Cole C N , Conrad S E
Published: January 2020

Infection of quiescent cells with the DNA tumor virus simian virus 40 induces expression of the cellular thymidine kinase (TK) gene a minimum of 10- to 20-fold, and this induction depends upon the viral protein large T antigen (T-Ag). To define both human TK promoter elements and T-Ag functional domains required for transcriptional induction, we have established a system in which stable Rat-1 transfectants harboring TK promoter-luciferase hybrid genes are infected with recombinant adenoviruses expressing either wild-type or mutant forms of T-Ag and luciferase expression is measured as an indicator of promoter activity. The results show that (i) a 135-bp TK promoter fragment is activated 10- to 15-fold by viral infection; (ii) this activation is the result of both T-Ag-dependent and -independent mechanisms; (iii) the T-Ag pRb family-binding domain, but not the p53-binding, helicase, or ATPase domain, is required for activation; and (iv) activation is severely diminished with a TK promoter fragment in which E2F-like-binding sites have been removed. These data demonstrate a requirement for both an E2F-related factor and a pRb family member in activation of the TK promoter by T-Ag. This contrasts with the promiscuous activation of many cellular and viral genes by T-Ag, which is independent of its ability to bind pRb.
Human adenovirus-specific CD8+ T-cell responses are not inhibited by E3-19K in the presence of gamma interferon
Authors: Flomenberg P , Piaskowski V , Truitt R L , Casper J T
Published: January 2020

Adenovirus has considerable potential as a gene therapy vector, but recent animal data suggest that transduced cells are destroyed by adenovirus-specific cytotoxic T-lymphocyte (CTL) responses. Therefore, it will be important to develop strategies to evade adenovirus-specific CTL responses in humans. As a first step, an assay was developed to detect and characterize human CTLs directed against adenovirus. Adenovirus-specific CTL responses were demonstrated to be present in four of five healthy adults by in vitro stimulation of peripheral blood mononuclear cells with autologous fibroblasts infected with the adenovirus type 2 (Ad2) E3 deletion mutant Ad2+ND1. Killing by adenovirus-specific CTLs was major histocompatibility complex class I restricted and was documented to be mediated by CD8+ T cells. Wild-type-Ad2-infected cells were poor CTL targets compared with cells infected with the E3 deletion mutant because of the expression of E3-19K, an early viral glycoprotein which prevents transport of major histocompatibility complex class I antigens out of the endoplasmic reticulum to the cell surface. However, preincubation of targets with gamma interferon resulted in enhanced killing of wild-type-Ad2-infected cells, to levels comparable to those obtained with Ad2+ ND1-infected cells. Radioimmunoprecipitation analysis revealed that gamma interferon not only increased the synthesis of class I antigens but also allowed excess molecules to escape from the endoplasmic reticulum. It is concluded that E3-19K expression in adenovirus-infected cells inhibits human CTL recognition in vitro but that gamma interferon may help overcome the E3-19K effect during acute infection in vivo.
Identification of domains in the simian immunodeficiency virus matrix protein essential for assembly and envelope glycoprotein incorporation
Authors: González S A , Burny A , Affranchino J L
Published: January 2020

The matrix domain (MA) of the simian immunodeficiency virus (SIV) is encoded by the amino-terminal region of the Gag polyprotein precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. To define domains of the SIV MA protein that are involved in virus morphogenesis, deletion and substitution mutations were introduced in this protein in the context of a gag-protease construct and expressed in the vaccinia virus vector system. The MA mutants were characterized with respect to synthesis and processing of the Gag precursor, assembly and release of virus-like particles, and incorporation of the envelope (Env) glycoprotein into particles. We have identified two regions of the SIV MA which are critical for particle formation. Both domains are located in a central hydrophobic alpha-helix of the SIV MA, according to data on the structure of this protein. In addition, we have characterized a domain whose mutation impairs the incorporation of SIV Env glycoproteins with long transmembrane cytoplasmic tails into particles. Interestingly, these mutant particles retained the ability to associate with SIV Env proteins with short cytoplasmic tails.
Differential glycosylation, virion incorporation, and sensitivity to neutralizing antibodies of human immunodeficiency virus type 1 envelope produced from infected primary T-lymphocyte and macrophage cultures
Authors: Willey R L , Shibata R , Freed E O , Cho M W , Martin M A
Published: January 2020

Two primary cell targets for human immunodeficiency virus type 1 (HIV-1) infection in vivo are CD4+ T lymphocytes and monocyte-derived macrophages (MDM). HIV-1 encodes envelope glycoproteins which mediate virus entry into these cells. We have utilized infected and radiolabelled primary peripheral blood mononuclear cell (PBMC) and MDM cultures to examine the biochemical and antigenic properties of the HIV-1 envelope produced in these two cell types. The gp120 produced in MDM migrates as a broad, diffuse band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels compared with that of the more homogeneous gp120 released from PBMCs. Glycosidase analyses indicated that the diffuse appearance of the MDM gp120 is due to the presence of asparagine-linked carbohydrates containing lactosaminoglycans, a modification not observed with the gp120 produced in PBMCs. Neutralization experiments, using isogeneic PBMC and MDM-derived macrophage-tropic HIV-1 isolates, indicate that 8- to 10-fold more neutralizing antibody, directed against the viral envelope, is required to block virus produced from MDM. These results demonstrate that HIV-1 released from infected PBMC and MDM cultures differs in its biochemical and antigenic properties.
The human T-cell leukemia virus type 1 posttranscriptional trans-activator Rex contains a nuclear export signal
Authors: Palmeri D , Malim M H
Published: January 2020

The Rex protein of human T-cell leukemia virus type 1 is required for the nuclear export of unspliced viral mRNA and, therefore, for virus replication. In this manuscript, we demonstrate that Rex shuttles between the nucleus and the cytoplasm and that its activation domain constitutes a nuclear export signal that specifies efficient transport to the cytoplasm. These findings are consistent with a model for Rex-mediated trans-activation in which Rex-viral mRNA complexes are targeted for nuclear export by the direct action of the activation domain.
The Moloney murine sarcoma virus ts110 5' splice site signal contributes to the regulation of splicing efficiency and thermosensitivity
Authors: Ainsworth J R , Rossi L M , Murphy E C
Published: January 2020

The 5' splice site signal (5'ss) in Moloney murine sarcoma virus ts110 (MuSVts110) RNA was found to participate in the regulation of its splicing phenotype. This 5'ss (CAG/GUAGGA) departs from the mammalian consensus (CAG/GURAGU) at positions +4 and +6, both of which base pair with U1 and U6 small nuclear RNAs during splicing. A doubling in splicing efficiency and near elimination of the splicing thermosensitivity characteristic of MuSVts110 were observed in 5'ss mutants containing a U at position +6 (termed 5' A6U), even in those in which U1-5'ss complementarity had been reduced. At the permissive temperature (28 degrees C), the 5' A6U mutation increased the efficiency of the second splicing reaction, while at the nonpermissive temperature (39 degrees C), both splicing reactions were positively affected.
Protection against apoptosis by the vaccinia virus SPI-2 (B13R) gene product
Authors: Dobbelstein M , Shenk T
Published: January 2020

Vaccinia virus contains a gene, termed SPI-2 or B13R, that is closely related in its sequence to a potent inhibitor of apoptosis from cowpox virus (crmA). Infection by vaccinia virus protects HeLa cells against apoptosis that is induced by an immunoglobulin M antibody against the fas receptor or by tumor necrosis factor alpha. This effect is profoundly reduced when the SPI-2 gene is deleted. The SPI-2 gene, when transiently expressed in these cells, can also protect against apoptosis mediated by these agents. Given the similarity to crmA, it seems likely that SPI-2 functions in an analogous fashion, inhibiting the activity of ICE protease family members and blocking the onset of apoptosis.
A gene transfer vector-cell line system for complete functional complementation of adenovirus early regions E1 and E4
Authors: Brough D E , Lizonova A , Hsu C , Kulesa V A , Kovesdi I
Published: January 2020

The improvements to adenovirus necessary for an optimal gene transfer vector include the removal of virus gene expression in transduced cells, increased transgene capacity, complete replication incompetence, and elimination of replication-competent virus that can be produced during the growth of first-generation adenovirus vectors. To achieve these aims, we have developed a vector-cell line system for complete functional complementation of both adenovirus early region 1 (E1) and E4. A library of cell lines that efficiently complement both E1 and E4 was constructed by transforming 293 cells with an inducible E4-ORF6 expression cassette. These 293-ORF6 cell lines were used to construct and propagate viruses with E1 and E4 deleted. While the construction and propagation of AdRSV beta gal.11 (an E1-/E4- vector engineered to contain a deletion of the entire E4 coding region) were possible in 293-ORF6 cells, the yield of purified virus was depressed approximately 30-fold compared with that of E1- vectors. The debilitation in AdRSV beta gal.11 vector growth was found to correlate with reduced fiber protein and mRNA accumulation. AdCFTR.11A, a modified E1-/E4- vector with a spacer sequence placed between late region 5 and the right inverted terminal repeat, efficiently expressed fiber and grew with the same kinetic profile and virus yield as did E1- vectors. Moreover, purified AdCFTR.11A yields were equivalent to E1- vector levels. Since no overlapping sequences exist in the E4 regions of E1-/E4- vectors and 293-ORF6 cell lines, replication-competent virus cannot be generated by homologous recombination. In addition, these second-generation E1-/E4- vectors have increased transgene capacity and have been rendered virus replication incompetent outside of the new complementing cell lines.
Characterization of tumor cell lines derived from murine gammaherpesvirus-68-infected mice
Authors: Usherwood E J , Stewart J P , Nash A A
Published: January 2020

Cell lines were derived from mice with murine gammaherpesvirus-68 (MHV-68)-associated lymphoproliferative disease. Four were of an ambiguous phenotype and were MHV-68 negative. One, S11, was a B lymphocyte that contained MHV-68 genomes in both linear and episomal forms and released virus. The line was clonable and grew into tumors in nude mice. This is the first naturally occurring MHV-68-positive B-cell line to be generated, and it will be an invaluable tool for the study of MHV-68 latency.
Regulation of EBNA gene transcription in lymphoblastoid cell lines: characterization of sequences downstream of BCR2 (Cp)
Authors: Puglielli M T , Desai N , Speck S H
Published: January 2020

During Epstein-Barr virus (EBV) latent infection of B lymphocytes in vitro, six EBV nuclear antigens (EBNAs) are expressed from one of two promoters, Cp and Wp, whose activities are mutually exclusive. Upon infection, Wp is initially active, followed by a switch to Cp for the duration of latency. In this study, the impact on Cp and Wp activity of sequences downstream of the distal EBNA gene promoter, Cp, was assessed in two lymphoblastoid cell lines. Cp activity was detected in constructs extending from just upstream of oriP to the first W1 exon. In contrast, Wp activity required the presence of the next downstream exon, W2. Viral sequences from -2199 to +2680 bp, relative to the Cp transcription start site, were dispensable for Wp activity. Sequences from +155 to +2680 bp, relative to the Cp transcription start site, were dispensable for Cp activity. Deletion of a 200-bp region from +2680 to +2880 bp downstream of Cp decreased both Cp and Wp activity two- to fivefold. Wp activity was also significantly diminished by deletion of the sequences from +2880 to +3000 bp downstream of the Cp transcription initiation site, which encompassed the Wp CCATT box. Based on deletion analyses of 10.3 kb of viral genomic sequence extending from just upstream of oriP to the first Wp, the only deletions which significantly upregulated Wp activity were those which abrogated Cp activity. However, reporter constructs in which the orientation of Cp was reversed displayed Wp activity comparable to that of reporter constructs in which Cp was deleted, even though the steady-state level of Cp-initiated transcripts from the inverted promoter was indistinguishable from that observed with Cp in normal orientation. This is the first direct evidence to support transcriptional interference as the mechanism for the mutually exclusive behavior of Cp and Wp.