bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of the transcription start region of the herpes simplex virus type 1 latency-associated transcript promoter on expression of productively infected neurons in vivo
Authors: Farrell M J , Margolis T P , Gomes W A , Feldman L T
Published: January 2020

It has been previously reported that the latency-associated transcript (LAT) promoter contains a DNA sequence at the LAT transcription start site which resembles the ICP4 consensus DNA binding site and that this site allows ICP4-mediated downregulation of the LAT promoter in transient assays (A. H. Batchelor and P. O'Hare, J. Virol. 64:3269-3279, 1990). We have confirmed these data by showing that an ICP4-expressing plasmid will downregulate lacZ expression from a plasmid containing the LAT promoter and transcription start site (pJA1) and does not downregulate lacZ expression from a plasmid in which the start site has been mutagenized (pWAG15). To determine the role of the LAT transcription start site in regulating LAT promoter activity in the context of the virus, two recombinant viruses, KOS-1 and KOS-15, were studied. KOS-1 contains an 863-bp portion of the LAT promoter, including the LAT cap site, fused to the lacZ gene and inserted into the gC locus (T.P. Margolis, F. Sedarati, A.T. Dobson, L.T. Feldman, and J.G. Stevens, Virology 189:150-160, 1992). The second virus (KOS-15) was constructed in identical fashion, using plasmid pWAG-15, which is not downregulated by ICP4. Vero cells productively infected with KOS-15 produce 10-fold more beta-galactosidase than do those infected with KOS-1. In murine dorsal root ganglia acutely infected with KOS-1, only 1.2% of dorsal root ganglion neurons that expressed viral antigen also expressed beta-galactosidase. In contrast, in KOS-15-infected mice, beta-galactosidase was detected in 18% of viral antigen-positive neurons. Similar findings were observed in trigeminal ganglia acutely infected with KOS-1 and KOS-15. Thus, the region encompassing the LAT transcription start site appears to play an important role in repression of the LAT promoter activity not only in vitro but also in acutely infected neurons in vivo. These results suggest that during productive infection with HSV-1, LAT expression is tightly regulated.
Conserved cysteine residues within the E3/19K protein of adenovirus type 2 are essential for binding to major histocompatibility complex antigens
Authors: Sester M , Burgert H G
Published: January 2020

The E3/19K protein of human adenovirus type 2 is a resident transmembrane glycoprotein of the endoplasmic reticulum. Its capacity to associate with class I histocompatibility (MHC) antigens abrogates cell surface expression and the antigen presentation function of MHC antigens. At present, it is unclear exactly which structure of the E3/19K protein mediates binding to MHC molecules. Apart from a stretch of approximately 20 conserved amino acids in front of the transmembrane segment, E3/19K molecules from different adenovirus subgroups (B and C) share little homology. Remarkably, the majority of cysteines are conserved. In this report, we examined the importance of cysteine residues for the structure and function of E3/19K. We show that E3/19K contains intramolecular disulfide bonds. By using site-directed mutagenesis, individual cysteines were replaced by serines and mutant proteins were stably expressed in 293 cells. On the basis of the differential binding of monoclonal antibody Tw1.3 and cyanogen bromide cleavage experiments, a structural model of E3/19K is proposed, in which Cys-11 and Cys-28 as well as Cys-22 and Cys-83 are linked by disulfide bonds. Both disulfide bonds (all four cysteines) are absolutely critical for the interaction with human MHC antigens. This was demonstrated by three criteria: loss of E3/19K coprecipitation, lack of transport inhibition, and normal cell surface expression of MHC molecules. Mutation of the three other cysteines had no effect. This indicates that a conformational determinant based on two disulfide bonds is crucial for the function of the E3/19K molecule, namely, to bind and to inhibit transport of MHC antigens.
Late transcripts of adenovirus type 12 DNA are not translated in hamster cells expressing the E1 region of adenovirus type 5
Authors: Schiedner G , Schmitz B , Doerfler W
Published: January 2020

Hamster cells are completely nonpermissive for the replication of human adenovirus type 12 (Ad12), whereas types 2 and 5 can replicate in hamster cells. The Ad5-transformed hamster cell line BHK297-C131, which carries the left terminal 18.7% of the Ad5 genome and expresses at least the viral E1A region, can somehow complement Ad12 DNA replication and the transcription of the late Ad12 genes. Since the interaction of Ad12 with hamster cells must constitute a significant factor in the induction of Ad12 tumors in neonatal hamsters, we have continued to examine details of this abortive virus infection. The late Ad12 mRNAs in BHK297-C131 cells are polyadenylated but are synthesized in reduced amounts compared with the Ad12 products in Ad12-infected human cells, which are permissive for viral replication. The late mRNA derived from the Ad12 fiber gene has been assessed for its structural properties. By cloning cDNA transcripts from this region and determining their nucleotide sequences, the authenticity of the complete Ad12 fiber sequence and the completeness of the Ad12-typical tripartite leader have been confirmed. Moreover, in Ad12-infected BHK297-C131 cells the Ad12 virus-associated RNA, a virus-encoded translational activator with the correct nucleotide sequence, is synthesized. Nevertheless, the synthesis of detectable amounts of Ad12 virion-specific proteins, and in particular that of the main viral antigens, hexons and fibers, cannot be documented. Cellular factors needed to promote late mRNA translation might be missing, or inhibitory factors might exist in Ad12-infected BHK297-C131 cells.
The receptor for the subgroup A avian leukosis-sarcoma viruses binds to subgroup A but not to subgroup C envelope glycoprotein
Authors: Gilbert J M , Bates P , Varmus H E , White J M
Published: January 2020

The putative subgroup A avian leukosis-sarcoma virus (ALSV) receptor (Tva) was recently cloned by gene transfer (P. Bates, J. A. Young, and H. E. Varmus, Cell 74:1043-1051, 1993; J. A. T. Young, P. Bates, and H. E. Varmus, J. Virol. 67:1811-1816, 1993). Susceptibility to infection by subgroup A ALSV is conferred on cells upon transfection with cDNAs encoding tva. The hypothesis that tva encodes a specific receptor for subgroup A ALSV predicts that the Tva protein should bind to subgroup A, but not to subgroup C, envelope glycoprotein. In this study, we examined this prediction by using several biochemical assays. We established stable NIH 3T3 cell lines expressing either Tva, the subgroup A envelope glycoprotein (Env-A), or the subgroup C envelop glycoprotein (Env-C) and used them in conjunction with soluble forms of these molecules to demonstrate specific binding. When cell lysates containing Tva were mixed with lysates of either Env-A or Env-C, an immunoprecipitable complex formed between Tva and Env-A but not between Tva and Env-C. A soluble, oligomeric form, of Env-A, not Env-C, binds to cells expressing Tva. Reciprocally, a secreted form of Tva can bind to cells expressing Env-A but not to cells expressing Env-C. A specific and stable complex formed between soluble Env-A and secreted Tva as demonstrated by sucrose density gradient centrifugation. Thus, by three kinds of assays, Tva appears to bind specifically to Env-A, which is consistent with genetic evidence that it serves as the cell surface receptor of subgroup A ALSV and the main determinant of subgroup specificity.
The ability of human papillomavirus E6 proteins to target p53 for degradation in vivo correlates with their ability to abrogate actinomycin D-induced growth arrest
Authors: Foster S A , Demers G W , Etscheid B G , Galloway D A
Published: January 2020

Functional p53 protein is associated with the ability of cells to arrest in G1 after DNA damage. The E6 protein of cancer-associated human papillomavirus type 16 (HPV-16) binds to p53 and targets its degradation through the ubiquitin pathway. To determine whether the ability of E6 to interact with p53 leads to a disruption of cell cycle control, mutated E6 proteins were tested for p53 binding and p53 degradation targeting in vitro, the ability to reduce intracellular p53 levels in vivo, and the ability to abrogate actinomycin D-induced growth arrest in human keratinocytes. Mutations scattered throughout the amino terminus, either zinc finger or the central region but not the carboxy terminus, severely reduced the ability of E6 to interact with p53. Expression of HPV-16 E6 or mutated E6 proteins that bound and targeted p53 for degradation in vitro sharply reduced the level of intracellular p53 induced by actinomycin D in human keratinocytes. A perfect correlation between the ability of E6 proteins to reduce the level of intracellular p53 and their ability to block actinomycin D-induced cellular growth arrest was observed. These results suggest that interaction with p53 is important for the ability of HPV E6 proteins to circumvent growth arrest.
Construction of avian hepadnavirus variants with enhanced replication and cytopathicity in primary hepatocytes
Authors: Lenhoff R J , Summers J
Published: January 2020

Hepadnaviruses cause persistent noncytopathic infections of hepatocytes in humans and other animals. Virus replication depends on the pool of viral covalently closed circular DNA (cccDNA) molecules, which serve as transcriptional templates in the nuclei of infected cells. The size of this pool of cccDNA molecules is regulated by the ability of the large envelope protein of the virus to direct newly synthesized viral DNAs into a pathway for viral secretion and thereby inhibit their utilization for viral cccDNA synthesis. In this study, we showed that single amino acid changes in the large envelope protein could cause profound changes in cccDNA levels in transfected permissive cells or in infected cultured hepatocytes. While defects in cccDNA regulation were accompanied by a decrease of enveloped virus production in transfected cells, primary hepatocytes infected by such mutant viruses transiently produced wild-type or higher levels of enveloped virus. Moreover, high levels of cccDNA were always associated with cytopathic effects in the infected hepatocytes. The results demonstrate that the large envelope protein promotes persistent noncytopathic infection of hepatocytes by acting as an overall repressor of virus replication.
Contributions of DNA polymerase subdomains to the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase
Authors: Smith J S , Gritsman K , Roth M J
Published: January 2020

Previous studies showed that an isolated human immunodeficiency virus type 1 (HIV-1) RNase H domain expressed as a fusion protein is highly active in Mn2+, but activity was dependent on a hexahistidine tag located at either the carboxyl or amino terminus of the fusion protein (J. Smith and M. Roth, J. Virol. 67:4037-4049, 1993). It was postulated that a histidine tag can somehow provide a function normally associated with the DNA polymerase domain of HIV-1 reverse transcriptase. To determine the contributions of the DNA polymerase subdomains of HIV-1 reverse transcriptase to its RNase H activity, we have characterized the activity of isolated RNase H domains which include either portions of the connection, the entire connection, or both the thumb and connection as N-terminal extensions. Including increasing lengths of these domains at the N terminus of the RNase H resulted in a progressive increase in Mn(2+)-dependent RNase H activity that was independent of a histidine tag. Activity of the isolated RNase H domains was also stimulated by the addition of independently purified polymerase subdomains. Further, this stimulation was shown to be a result of direct physical interactions between the thumb, connection, and RNase H domains. The connection and thumb subdomains were shown to contribute to substrate binding. The fingers and palm subdomains were found to be essential for Mg(2+)-dependent RNase H activity.
Human cytomegalovirus infection induces transcription and secretion of transforming growth factor beta 1
Authors: Michelson S , Alcami J , Kim S J , Danielpour D , Bachelerie F , Picard L , Bessia C , Paya C , Virelizier J L
Published: January 2020

Human cytomegalovirus (CMV) infection can elicit a transitory, but profound, immunodepression in immunocompetent individuals. Cytopathogenic destruction of CMV-infected leukocytes alone does not seem sufficient to explain this phenomenon, which suggests that immune system mediators (cytokines) may play a role in amplifying local modifications wrought by CMV infection. We reported previously that transforming growth factor beta 1 (TGF-beta 1) stimulates CMV replication (J. Alcami, C. V. Paya, J. L. Virelizier, and S. Michelson, J. Gen. Virol. 74:269-274, 1993). Since TGF-beta 1 can have profound negative effects on cell growth and immune responses, we investigated the induction of TGF-beta 1 following CMV infection of permissive fibroblasts. TGF-beta 1 promoter was activated by immediate-early (IE) proteins in the absence of infection and transactivated at 5 and 9 h after infection. TGF-beta 1 mRNA increased during the early phase of infection, suggesting that this phenomenon is due to enhanced transcription of the TGF-beta 1 gene. A comparative study of the influence of CMV infection and IE protein expression on TGF-beta 1 promoter function in permissive cells pointed to a possible cooperative role between IE proteins and protein(s) expressed during the early phase of viral infection. Induction of TGF-beta 1 by CMV infection could modify infected cells individually, surrounding tissues, and systemic immune reactions to the advantage of virus replication by both upregulating CMV replication and downregulating host immune responses.
ATPase and GTPase activities associated with Semliki Forest virus nonstructural protein nsP2
Authors: Rikkonen M , Peränen J , Kääriäinen L
Published: January 2020

The replication of Semliki Forest virus requires four nonstructural proteins (nsP1 to nsP4), all derived from the same polyprotein. One of these, nsP2, is a multifunctional protein needed in RNA replication and in the processing of the nonstructural polyprotein. On the basis of amino acid sequence homologies, nsP2 was predicted to possess nucleoside triphosphatase and RNA helicase activities. Here, we report the engineered expression in Escherichia coli of nsP2 and of an amino-terminal fragment of it by use of the highly efficient T7 expression system. Both polypeptides were produced as fusion proteins with a histidine tag at the amino terminus and purified by immobilized-metal affinity chromatography. The two recombinant proteins exhibited ATPase and GTPase activities, which were further stimulated by the presence of single-stranded RNA. The activities were not found in similarly prepared fractions from uninduced control cells or cells expressing an unrelated polypeptide. Radiolabeled ribonucleoside triphosphates could be cross-linked to both the full-length and the carboxy-terminally truncated nsP2 protein, and both polypeptides had RNA-binding capacity. We also expressed and purified an nsP2 variant which had a single amino acid substitution in the nucleotide-binding motif (Lys-192-->Asn). No nucleoside triphosphatase activity was associated with this mutant protein.
Induction of feline immunodeficiency virus-specific cytotoxic T cells in vivo with carrier-free synthetic peptide
Authors: Flynn J N , Cannon C A , Beatty J A , Mackett M , Rigby M A , Neil J C , Jarrett C
Published: January 2020

The role of cellular immunity in the establishment and progression of immunosuppressive lentivirus infection remains equivocal. To develop a model system with which these aspects of the host immune response can be studied experimentally, we examined the response of cats to a hybrid peptide containing predicted T-and B-cell epitopes from the gag and env genes of feline immunodeficiency virus (FIV). Cats were immunized with an unmodified 17-residue peptide incorporating residues 196 to 208 (from gag capsid protein p24) and 395 to 398 (from env glycoprotein gp120) of the FIV Glasgow-8 strain by using Quil A as an adjuvant. Virus-specific lymphocytotoxicity was measured by chromium-51 release assays. The target cells were autologous or allogeneic skin fibroblasts either infected with recombinant FIV gag vaccinia virus or pulsed with FIV peptides. Effector cells were either fresh peripheral blood mononuclear cells or T-cell lines stimulated with FIV peptides in vitro. Cytotoxic effector cells from immunized cats lysed autologous, but not allogeneic, target cells when they were either infected with recombinant FIV gag vaccinia virus or pulsed with synthetic peptides comprising residues 196 to 205 or 200 to 208 plus 395. Depletion of CD8+ T cells, from the effector cell population abrogated the lymphocytotoxicity. Immunized cats developed an antibody response to the 17-residue peptide immunogen and to recombinant p24. However, no antibodies which recognized smaller constituent peptides could be detected. This response correlated with peptide-induced T-cell proliferation in vitro. This study demonstrates that cytotoxic T lymphocytes specific for FIV can be induced following immunization with an unmodified short synthetic peptide and defines a system in which the protective or pathological role of such responses can be examined.
Identification of an H-2 Kb-presented Moloney murine leukemia virus cytotoxic T-lymphocyte epitope that displays enhanced recognition in H-2 Db mutant bm13 mice
Authors: Sijts A J , De Bruijn M L , Ressing M E , Nieland J D , Mengedé E A , Boog C J , Ossendorp F , Kast W M , Melief C J
Published: January 2020

Upon infection with the Moloney murine sarcoma virus-murine leukemia virus (MuLV) complex, H-2b C57BL/6 (B6) mice respond with a class I Db-restricted cytotoxic T-lymphocyte (CTL) response, which protects against virus-induced tumorigenesis. In the B6-derived Db mutant B6.CH-2bm13 (bm13) strain, part of the class I Db antigen-presenting groove is shaped by a class I Kb-encoded sequence. Like B6 mice, bm13 mice reject Moloney virus-induced tumors, but the protective CTL response is Kb restricted. In this study we show enhanced levels of Moloney MuLV-specific CTLp with a restriction for Kb in bm13 mice. Through the use of CTL clones from Moloney virus-immunized bm13 mice, the class I Kb-presented CTL epitope was identified. The epitope is located in the Moloney virus gp70 envelope protein region (Moloney envelope, amino acids 189 to 196 [Mol env (189-196)]), SSWDFITV and has the Kb allele-specific binding motif. The Dbm13 molecule does not present the env(189 to 196) epitope to Kb-restricted bm13 CTL. In B6 mice, Mol env(189-196)-specific CTL could be induced by peptide vaccination. B6 mice thus have CTL precursors specific for this epitope but at considerably lower levels than do bm13 mice. We hypothesize that additional positive selection of Kb-restricted CTL on the Dbm13 molecule in bm13 mice explains this difference in precursor frequencies. We examined related strains of MuLV for the presence of Mol env(189-196) sequence equivalents. Rauscher, Friend, and AKV MuLV-encoded Mol env(189-196) epitope equivalents were properly recognized in cytotoxicity assays, both as synthetic and as endogenously expressed (Rauscher MuLV) peptides. In contrast, the mink cell focus-forming virus MuLV-encoded epitope equivalent, lacking a Kb anchor residue, was not presented for CTL recognition and hence can be excluded as an important CTL epitope for mink cell focus-forming viruses.
Respiratory virus infection of mice provokes a permanent humoral immune response
Authors: Hyland L , Sangster M , Sealy R , Coleclough C
Published: January 2020

We have observed that respiratory virus infection of mice provokes an extremely persistent humoral immune reaction, due to a long-sustained population of antibody-secreting cells in the bone marrow. Theories of humoral immunity that strongly distinguish primary and secondary reactions thus may not adequately describe the immune response to respiratory viruses.
Papilloma formation by cottontail rabbit papillomavirus requires E1 and E2 regulatory genes in addition to E6 and E7 transforming genes
Authors: Wu X , Xiao W , Brandsma J L
Published: January 2020

The present study used the cottontail rabbit papillomavirus DNA-rabbit system to evaluate whether the regulatory genes E1 and E2 and the transforming gene E6 are required for papilloma formation. Frameshift mutations were generated in the individual genes in the context of a full-length cottontail rabbit papillomavirus genome, and the mutant DNAs were intradermally inoculated into domestic rabbits. None of the mutants induced papillomas. Marker rescue experiments confirmed that the defects were due to mutations that we deliberately introduced. Marker rescue also confirmed our previous report that the upstream region of E7 around position 9 was critical for papilloma induction. These results demonstrate that the E1 and E2 regulatory genes as well as the E6 and E7 transforming genes are each required for papilloma formation. Each gene may provide molecular targets for therapeutic intervention.
RNA binding properties of poliovirus subviral particles
Authors: Nugent C I , Kirkegaard K
Published: January 2020

The mechanism of encapsidation of the RNA genome of poliovirus and other picornaviruses is unknown. To test whether any of the putative assembly intermediates of poliovirus could interact directly with the poliovirus RNA genome, poliovirus RNA was attached to magnetic streptavidin beads and incubated with partially purified extracts containing 35S-labeled 14S pentamer and 75S empty-capsid subviral particles from infected cells. The amount of labeled protein bound to the beads was monitored, thus testing the RNA-binding activities of only the labeled viral proteins in the preparations. In this assay, nonspecific RNA-binding activity was displayed by the 14S pentameric particles and mature virons. 75S empty capsids displayed no propensity to associate with RNA. 14S pentamers were demonstrated to form rapidly sedimenting complexes and to undergo a conformational alteration upon RNA binding. These findings are consistent with a direct role for the 14S pentameric particles in RNA packaging during poliovirus morphogenesis.
CCAAT box-dependent activation of the TATA-less human DNA polymerase alpha promoter by the human cytomegalovirus 72-kilodalton major immediate-early protein
Authors: Hayhurst G P , Bryant L A , Caswell R C , Walker S M , Sinclair J H
Published: January 2020

Human cytomegalovirus (HCMV) immediate-early (IE) proteins are known potent transregulators of viral and cellular gene expression upon HCMV infection. HCMV is known to activate a number of cellular genes intimately associated with the cell cycle and DNA replication by mechanisms involving the viral major IE 86-kDa protein (IE2). We have recently shown that IE2 mediates this activation in a TATA-dependent manner and interacts directly with the TATA-binding protein. However, a number of TATA-less cellular promoters, e.g., DNA polymerase alpha and dihydrofolate reductase, are also activated by HCMV infection. Consequently, we have asked how HCMV mediates this activation. We show that, consistent with its known TATA dependency, IE2 does not activate the DNA polymerase alpha promoter. In contrast, this promoter is strongly activated by the major IE 72-kDa protein (IE1). Whilst deletion of ATF or E2F sites within the DNA polymerase alpha promoter had little effect on IE1-mediated activation, removal of the CCAAT box appeared to abolish high levels of activation by IE1. Consistent with this observation, we also find that IE1 interacts directly with the CCAAT box binding factor CTF1 in vitro and massively augments CTF1-mediated activation of the DNA polymerase alpha promoter in transient transfection assays.
Substrate determinants for cleavage in cis and in trans by the hepatitis C virus NS3 proteinase
Authors: Bartenschlager R , Ahlborn-Laake L , Yasargil K , Mous J , Jacobsen H
Published: January 2020

Processing of the hepatitis C virus polyprotein is accomplished by a series of cotranslational and posttranslational cleavages mediated by host cell signalases and two virally encoded proteinases. Of these the NS3 proteinase is essential for processing at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions. Processing between NS3 and NS4A occurs in cis, implying an intramolecular reaction mechanism, whereas cleavage at the other sites can also be mediated in trans. Sequence analysis of the amino termini of mature cleavage products and comparisons of amino acid residues around the scissile bonds of various hepatitis C virus isolates identified amino acid residues which might contribute to substrate specificity and processing efficiency: an acidic amino acid at the P6 position, a Thr or Cys at the P1 position, and a Ser or Ala at the P1' position. To study the importance of these residues for NS3-mediated cleavage we have undertaken a mutational analysis using an NS3'-5B polyprotein expressed by recombinant vaccinia viruses in mammalian cells. For all NS3-dependent cleavage sites P1 substitutions had the most drastic effects on cleavage efficiency, showing that amino acid residues at this position are the most critical substrate determinants. Since less drastic effects were found for substitutions at the P1' position, these residues appear to be less important for proper cleavage. For all cleavage sites the P6 acidic residue was dispensable, suggesting that it is not essential for substrate recognition and subsequent cleavage. Analysis of a series of mutations at the NS3/4A site revealed great flexibility for substitutions compared with more stringent requirements at the trans cleavage sites. On the basis of these results we propose a model in which processing in cis is determined primarily by polyprotein folding, whereas cleavage in trans is governed not only by the structure of the polyprotein but also by specific interactions between the proteinase and the polyprotein substrate at or around the scissile bond.
Identification and characterization of monoclonal antibodies specific for polymorphic antigenic determinants within the V2 region of the human immunodeficiency virus type 1 envelope glycoprotein
Authors: Shotton C , Arnold C , Sattentau Q , Sodroski J , McKeating J A
Published: January 2020

We have identified six monoclonal antibodies (MAbs) mapping to both linear and conformation-dependent epitopes within the V2 region of the human immunodeficiency virus type 1 clone HXB10. Three of the MAbs (12b, 66c, and 66a) were able to neutralize the molecular clones HXB10 and HXB2, with titers in the range of 9.5 to 20.0 micrograms/ml. MAbs mapping to the crown of the V2 loop (12b, 60b, and 74) bound poorly to cell surface-expressed oligomeric gp120, suggesting an explanation for the poor or negligible neutralizing activity of MAbs to this region. In contrast, MAbs 12b and 60b demonstrated good reactivity with recombinant gp120 in an enzyme-linked immunosorbent assay format, suggesting differential epitope exposure between the recombinant and native forms of gp120. Cross-competition analysis of these MAbs and additional V1V2 MAbs for gp120 binding enabled us to assign the MAbs to six groups (A to F). Selection of neutralization escape mutants with MAbs 10/76b and 11/68b, belonging to nonoverlapping competition groups, identified amino acid changes at residues 165 (I to T) and 185 (D to N), respectively. Interestingly, these escape variants remained sensitive to neutralization by the nonselecting V2 MAbs. All MAbs demonstrated good recognition of IIIB viral gp120 yet failed to neutralize nonclonal stocks of IIIB. In addition, MAbs 12b and 62c bound MN and RF viral gp120, respectively, yet failed to neutralize the respective isolates. Cloning and expression of a library of gp120 and V1V2 fragments from IIIB-, MN-, and RF-infected H9 cultures identified a number of polymorphic sites, resulting in antigenic variation and subsequent loss of V2 MAb recognition. In contrast, the V3 region from the clones of the same isolates showed no amino acid changes, suggesting that the V2 region is polymorphic in long-term-passaged laboratory isolates and may account for the reduced antibody recognition observed.
Dynamic organization of splicing factors in adenovirus-infected cells
Authors: Bridge E , Xia D X , Carmo-Fonseca M , Cardinali B , Lamond A I , Pettersson U
Published: January 2020

Adenovirus infection affects the nuclear distribution of host splicing factors. Late phase-infected cells contain discrete clusters of small nuclear ribonucleoproteins (snRNPs) that are separate from centers containing the viral 72-kilodalton DNA-binding protein (72K protein). In the present study, we demonstrate that these snRNP clusters also contain splicing factors from the SR protein family. We show that a previously described monoclonal antibody, 3C5, detects SR proteins. Furthermore, we demonstrate that late region 3 transcription occurs at a maximal rate in infected cultures in which greater than 90% of the cells contain the snRNP clusters, indicating that such cells are actively transcribing their late genes. During the onset of the late phase, the intranuclear distribution of splicing factors is very different from that seen after the late phase is established. When late viral transcription commences, cells with snRNP clusters are less prevalent than in cultures that are maintaining maximum levels of late transcription. Instead, a cell type which shows snRNPs, concentrated in foci that also contain the viral 72K DNA-binding protein is detected. This cell type disappears from cultures by 18 to 20 h after a high-multiplicity infection. These results suggest a dynamic organization of splicing factors in infected cells that can be correlated to the status of viral gene expression. Our work also provides an explanation for the differing results that have been published concerning the organization of splicing factors in the adenovirus-infected cell nucleus (L. F. Jiménez-García and D. L. Spector, Cell 73:47-59, 1993). During the present study we observed that a monoclonal antibody against the SC-35 protein, which was used by Jiménez-García and Spector to study the localization of the SC-35 splicing factor in adenovirus-infected cells, cross-reacts with the adenovirus 72K DNA-binding protein and is thus unsuitable for this type of study.
Infection of central nervous system cells by ecotropic murine leukemia virus in C58 and AKR mice and in in utero-infected CE/J mice predisposes mice to paralytic infection by lactate dehydrogenase-elevating virus
Authors: Anderson G W , Palmer G A , Rowland R R , Even C , Plagemann P G
Published: January 2020

Certain mouse strains, such as AKR and C58, which possess N-tropic, ecotropic murine leukemia virus (MuLV) proviruses and are homozygous at the Fv-1n locus are specifically susceptible to paralytic infection (age-dependent poliomyelitis [ADPM]) by lactate dehydrogenase-elevating virus (LDV). Our results provide an explanation for this genetic linkage and directly prove that ecotropic MuLV infection of spinal cord cells is responsible for rendering anterior horn neurons susceptible to cytocidal LDV infection, which is the cause of the paralytic disease. Northern (RNA) blot hybridization of total tissue RNA and in situ hybridization of tissue sections demonstrated that only mice harboring central nervous system (CNS) cells that expressed ecotropic MuLV were susceptible to ADPM. Our evidence indicates that the ecotropic MuLV RNA is transcribed in CNS cells from ecotropic MuLV proviruses that have been acquired by infection with exogenous ecotropic MuLV, probably during embryogenesis, the time when germ line proviruses in AKR and C58 mice first become activated. In young mice, MuLV RNA-containing cells were found exclusively in white-matter tracts and therefore were glial cells. An increase in the ADPM susceptibility of the mice with advancing age correlated with the presence of an increased number of ecotropic MuLV RNA-containing cells in the spinal cords which, in turn, correlated with an increase in the number of unmethylated proviruses in the DNA extracted from spinal cords. Studies with AKXD recombinant inbred strains showed that possession of a single replication-competent ecotropic MuLV provirus (emv-11) by Fv-1n/n mice was sufficient to result in ecotropic MuLV infection of CNS cells and ADPM susceptibility. In contrast, no ecotropic MuLV RNA-positive cells were present in the CNSs of mice carrying defective ecotropic MuLV proviruses (emv-3 or emv-13) or in which ecotropic MuLV replication was blocked by the Fv-1n/b or Fv-1b/b phenotype. Such mice were resistant to paralytic LDV infection. In utero infection of CE/J mice, which are devoid of any endogenous ecotropic MuLVs, with the infectious clone of emv-11 (AKR-623) resulted in the infection of CNS cells, and the mice became ADPM susceptible, whereas littermates that had not become infected with ecotropic MuLV remained ADPM resistant.
Reversal of the interferon-sensitive phenotype of a vaccinia virus lacking E3L by expression of the reovirus S4 gene
Authors: Beattie E , Denzler K L , Tartaglia J , Perkus M E , Paoletti E , Jacobs B L
Published: January 2020

The vaccinia virus (VV) E3L gene, which encodes a potent inhibitor of the interferon (IFN)-induced, double-stranded RNA (dsRNA)-dependent protein kinase, PKR, is thought to be involved in the IFN-resistant phenotype of VV. The E3L gene products, p25 and p20, act as inhibitors of PKR, presumably by binding and sequestering activator dsRNA from the kinase. In this study we demonstrate that VV with the E3L gene specifically deleted (vP1080) was sensitive to the antiviral effects of IFN and debilitated in its ability to rescue vesicular stomatitis virus from the antiviral effects of IFN. Infection of L929 cells with E3L-minus virus led to rRNA degradation typical of activation of the 2'-5'-oligoadenylate synthetase/RNase L system, and extracts of infected cells lacked the PKR-inhibitory activity characteristic of wild-type VV. The reovirus S4 gene, which encodes a dsRNA-binding protein (sigma 3) that can also inhibit PKR activation by binding and sequestering activator dsRNA, was inserted into vP1080. The resultant virus (vP1112) was partially resistant to the antiviral effects of IFN in comparison with vP1080. Further studies demonstrated that transient expression of the reovirus sigma 3 protein rescued E3L-minus VV replication in HeLa cells. In these studies, rescue by sigma 3 mutants correlated with their ability to bind dsRNA. Finally, vP112 was also able to rescue the replication of the IFN-sensitive virus vesicular stomatitis virus in a manner similar to that of wild-type VV. Together, these results suggest that the reovirus S4 gene can replace the VV E3L gene with respect to interference with the IFN-induced antiviral activity.
Functional analysis of the two alternative translation initiation sites of foot-and-mouth disease virus
Authors: Cao X , Bergmann I E , Füllkrug R , Beck E
Published: January 2020

The effect of deletion of each of the two authentic polyprotein translation initiation sites of foot-and-mouth disease virus on viral protein synthesis and replication was analyzed. Deletion of either the first or the second initiation site led to the expression of only one form of the leader protein, L or L', respectively, but in vitro processing of the viral polyprotein and cleavage of eIF-4 gamma were not affected by either deletion. Whereas RNA in which the first translation initiation site had been deleted led to the production of viruses in transfected BHK cells, deletion of the second translation initiation site abolished virus replication.
Expression of the human immunodeficiency virus type 1 (HIV-1) nef gene during HIV-1 production increases progeny particle infectivity independently of gp160 or viral entry
Authors: Miller M D , Warmerdam M T , Page K A , Feinberg M B , Greene W C
Published: January 2020

The nef gene product of human immunodeficiency virus type 1 (HIV-1) promotes more-rapid kinetics of viral replication in primary peripheral blood mononuclear cells. We have previously shown that these enhancing effects of Nef on HIV-1 replication reflect an increase in viral infectivity detectable both in limiting dilution assays and through a single-cycle infection of the HeLa-CD4-long terminal repeat-beta-galactosidase indicator cell line. We now demonstrate that nef-defective HIV-1 can be rescued to near wild-type levels of infectivity by coexpressing Nef in trans in the cell line producing the virus. This observation indicates that HIV-1 virions produced in the presence of Nef are intrinsically different. However, we show that the major viral structural proteins are quantitatively similar in purified viral preparations. We also demonstrate the functional equivalence of the gp120-gp41 envelope glycoprotein complexes of Nef+ and Nef- HIV-1 through an assay for viral entry. Finally, we show that env-defective Nef+ HIV-1 pseudotyped with an amphotropic envelope is also more infectious than similarly pseudotyped Nef- HIV-1. Thus, the production of HIV-1 in the presence of Nef results in viral particles that are more infectious, and this increased infectivity is manifested at a stage after viral entry but prior to or coincident with HIV-1 gene expression.
Unique insertion sequence and pattern of CD4 expression in variants selected with immunotoxins from human immunodeficiency virus type 1-infected T cells
Authors: Fang H , Pincus S H
Published: January 2020

To study the variability of human immunodeficiency virus type 1 (HIV-1), we used immunotoxins to select for variants within a population of H9 cells persistently infected with a molecular clone of HIV-1 designated NL4-3. Chimeric immunotoxin CD4-PE40 (a chimeric fusion protein consisting of the amino-terminal two domains of CD4 and the carboxy-terminal domains of Pseudomonas exotoxin A) was used to select for cells lacking cell surface expression of HIV Env (envelope proteins gp160, gp120, and gp41). The cells described here (A1, A7, C9, and E9) fail to express HIV proteins because they have markedly diminished transcription of the integrated provirus (A1, A7, and E9) or no HIV provirus (C9). Analysis demonstrated that two different cloned variants, A1 and E9, contain the complementary sequence of tRNA(3Lys) (45 bp) inserted 3' to the primer-binding site, following by a 169-bp deletion through the start of the gag gene. No HIV mRNA was detected by Northern (RNA) blotting, but PCR demonstrated the presence of the viral message. These variants were found very infrequently in the unselected H9/NL4-3 cell population, and they contained proviruses distinct from that found in the dominant population. In addition, all of these variants had similar patterns of CD4 surface expression that allowed them to escape reinfection within the tissue culture. The data are discussed with regard to mechanisms and errors of HIV reverse transcription, as well as the evolution of mutants within a population of persistently infected cells.
Functional interaction between the human cytomegalovirus 86-kilodalton IE2 protein and the cellular transcription factor CREB
Authors: Lang D , Gebert S , Arlt H , Stamminger T
Published: January 2020

The 86-kDa IE2 protein (IE86) of human cytomegalovirus (HCMV) has been described as a promiscuous transactivator of viral, as well as cellular, gene expression. Investigation of the mechanism used by IE86 to activate gene expression from the early UL112/113 promoter of HCMV revealed the existence of three binding sites for IE86 located between nucleotides -290 and -120 relative to the transcriptional start site (H. Arlt, D. Lang, S. Gebert, and T. Stamminger, J. Virol. 68:4117-4125, 1994). As shown previously, deletion of these target sites resulted in a reduction of IE86-mediated transactivation by approximately 70%. The remaining promoter, however, could still be stimulated about 40-fold, indicating the presence of an additional responsive element within these sequences. Here, we provide evidence that a binding site for the cellular transcription factor CREB can also act as a target for IE86 transactivation. By DNase I protection analysis, a binding sequence for CREB could be detected between nucleotides -78 and -56 within the respective promoter region. After in vitro mutagenesis of this CREB-binding site within the context of the entire UL112/113 promoter, a marked reduction in transactivation levels was evident. Moreover, when individual CREB-binding sites were positioned upstream of a minimal, TATA box-containing UL112/113 promoter, they were able to confer strong IE86 responsiveness, whereas a mutated sequence did not exert any effect. In far Western blot and pull-down experiments, a direct interaction of IE86 with the cellular transcription factor CREB could be observed. The in vivo relevance of this in vitro interaction was confirmed by using various GAL4 fusion proteins in the presence or absence of IE86 which revealed a strong activation only in the presence of both a GAL4-CREB fusion and IE86. This shows that at least one specific member of the ATF/CREB family of transcription factors is involved in mediating transactivation by the HCMV IE86 protein.
Genetic analysis of human immunodeficiency virus type 1 strains from patients in Cyprus: identification of a new subtype designated subtype I
Authors: Kostrikis L G , Bagdades E , Cao Y , Zhang L , Dimitriou D , Ho D D
Published: January 2020

DNA sequences encoding the C2 to V3 region of envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) were amplified by PCR from uncultured peripheral blood mononuclear cells obtained from 24 of 25 HIV-1-seropositive patients from Cyprus. By using a heteroduplex mobility assay (HMA), all amplified products were studied genetically and compared with 16 previously characterized HIV-1 strains belonging to subtypes A through F. HMA results revealed that HIV-1 gp120 sequences from 15 of our patients were of subtype B of HIV-1, whereas one isolate was of subtype C. However, gp120 sequences from eight patients had no obvious similarities to the known subtypes as defined by HMA. DNA sequencing and phylogenetic analyses of molecular clones confirmed the HMA results and placed the eight undefined HIV-1 isolates into three distinct genetic clusters. On the basis of branch topology and lengths of the phylogenetic tree, we conclude that one group consisting of three clones from two patients represents a new HIV-1 env subtype, which we have termed subtype I. The remaining two sequence clusters, consisting of five sequences from four patients and two sequences from two other patients, are distally related to subtypes A and F. These data demonstrate the extensive heterogeneity of HIV-1 in Cyprus, including the presence of new subtype.
Investigation of the control of coronavirus subgenomic mRNA transcription by using T7-generated negative-sense RNA transcripts
Authors: Hiscox J A , Mawditt K L , Cavanagh D , Britton P
Published: January 2020

The subgenomic mRNAs of the coronavirus transmissible gastroenteritis virus (TGEV) are not produced in equimolar amounts. We have developed a reporter gene system to investigate the control of this differential subgenomic mRNA synthesis. Transcription of mRNAs by the TGEV polymerase was obtained from negative-sense RNA templates generated in situ from DNA containing a T7 promoter. A series of gene cassettes was produced; these cassettes comprised the reporter chloramphenicol acetyltransferase (CAT) gene downstream of transcription-associated sequences (TASs) (also referred to as intergenic sequences and promoters) believed to be involved in the synthesis of TGEV subgenomic mRNAs 6 and 7. The gene cassettes were designed so that negative-sense RNA copies of the CAT gene with sequences complementary to the TGEV TASs, or modified versions, at the 3' end would be synthesized in situ by T7 RNA polymerase. Using this system, we have demonstrated that CAT was expressed from mRNAs derived from the T7-generated negative-sense RNA transcripts only in TGEV-infected cells and only from transcripts possessing a TGEV negative-sense TAS. Analysis of the CAT mRNAs showed the presence of the TGEV leader RNA sequence at the 5' end, in keeping with observations that all coronavirus mRNAs have a 5' leader sequence corresponding to the 5' end of the genomic RNA. Our results indicated that the CAT mRNAs were transcribed from the in situ-synthesized negative-sense RNA templates without the requirement of TGEV genomic 5' or 3' sequences on the T7-generated negative-sense transcripts (3'-TAS-CAT-5'). Modification of the TGEV TASs indicated (i) that the degree of potential base pairing between the 3' end of the leader RNA and the TGEV negative-sense TAS was not the sole determinant of the amount of subgenomic mRNA transcribed and (ii) that other factors, including nucleotides flanking the TAS, are involved in the regulation of transcription of TGEV subgenomic mRNAs.
An internal ribosomal entry signal in the rat VL30 region of the Harvey murine sarcoma virus leader and its use in dicistronic retroviral vectors
Authors: Berlioz C , Torrent C , Darlix J L
Published: January 2020

The genetic organization of the 5' genomic RNA domain of the highly oncogenic Harvey murine sarcoma virus appears to be unusual in that a multifunctional untranslated leader precedes the v-ras oncogene. This 5' leader is 1,076 nucleotides in length and is formed of independent regions involved in key steps of the viral life cycle: (i) the Moloney murine leukemia virus 5' repeat, untranslated 5' region, and primer binding site sequences necessary for the first steps of proviral DNA synthesis, (ii) the virus-like 30S (VL30)-derived sequence containing a functional dimerization-packaging signal (E/DLS) directing viral RNA dimerization and packaging into MLV virions, and (iii) an Alu-like sequence preceding the 5' untranslated sequence of v-rasH which contains the initiation codon of the p21ras oncoprotein. These functional features, the unusual length of this leader (1,076 nucleotides), and the presence of stable secondary structures between the cap and the v-ras initiation codon might well cause a premature stop of the scanning ribosomes and thus inhibit v-ras translation. In order to understand how Harvey murine sarcoma virus achieves a high level of expression of the ras oncogene, we asked whether the rat VL30 sequence, 5' to v-ras, could contribute to an efficient synthesis of the ras oncoprotein. The implications of the VL30 sequence in the translation initiation of Ha-ras were investigated in the rabbit reticulocyte lysate system and in murine cells. Results show that the rat VL30 sequence allows a cap-independent translation of a downstream reporter gene both in vitro and in murine cells. Additional experiments performed with dicistronic neo.VL30.lacZ mRNAs indicate that the 5' VL30 sequence (positions 380 to 794) contains an internal ribosomal entry signal. This finding led us to construct a new dicistronic retroviral vector with which the rat VL30 sequence was able to direct the efficient expression of a 3' cistron and packaging of recombinant dicistronic RNA into murine leukemia virus virions.