bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross-protection of mice against a global spectrum of rabies virus variants
Authors: Lodmell D L , Smith J S , Esposito J J , Ewalt L C
Published: January 2020

Rabies, a continuing worldwide problem, kills tens of thousands of people and millions of animals each year. The problem is most severe in developing countries, where cell culture-derived vaccines are unaffordable and the available nervous tissue-derived vaccines are often of questionable immunogenicity and may produce neurological complications. To determine the feasibility of developing a vaccine with worldwide applicability, we investigated whether recombinant vaccinia viruses expressing either the glycoprotein (G), the nucleoprotein (N), or both the G and N (GN) of the challenge virus strain (CVS) of rabies virus would cross-protect mice against 17 rabies virus isolates representing the spectrum of rabies virus variants found worldwide. The results were compared with the commercially available human diploid cell vaccine (HDCV). Among mice injected with any of the 17 viruses, > or = 95% were protected by vaccination with recombinant viruses expressing G or GN, and > or = 85% of the mice were protected by the HDCV. The recombinant virus expressing N was less protective, protecting against only 11 of the 17 viruses. Antibody prepared against the G of the strains used in the vaccines neutralized all 17 viruses, and sera from mice infected with any one virus variant cross-neutralized all of the other viruses. Thus, no antigenic differences that would potentiate vaccine failures were identified. These studies suggest that a single rabies virus strain or its G would protect globally against wild-type rabies viruses.
Replication of hepatitis delta virus RNA in mice after intramuscular injection of plasmid DNA
Authors: Polo J M , Lim B , Govindarajan S , Lai M M
Published: January 2020

To establish a readily manipulable small-animal system for the study of human hepatitis delta virus (HDV) replication in vivo, plasmid DNAs containing head-to-tail cDNA dimers of HDV were inoculated intramuscularly into mice. Genomic-sense HDV RNA was detected in the injected muscle within 1 week and increased to substantial levels by week 7 postinjection. The intramuscular accumulation of HDV RNA was determined to be the direct result of viral RNA replication by three lines of evidence: (i) injected tissues also accumulated antigenomic-sense HDV RNA, (ii) plasmid DNA that synthesized primary transcripts of antigenomic sense also led to the accumulation of genomic-sense HDV RNA, and (iii) injection of a cDNA dimer defective in antigenomic RNA cleavage failed to produce detectable HDV RNA in muscle. Immunohistochemical analysis of injected muscle demonstrated the presence and nuclear localization of hepatitis delta antigen in myocytes. Finally, sera from DNA-injected mice contained antibodies specific for delta antigen, indicating the induction of an immunological response to the intracellularly expressed antigen. These findings demonstrated the ability of HDV RNA to replicate in skeletal muscle and provide a useful system for the study of HDV replication, delta antigen processing, and its presentation to the immune system in vivo. Furthermore, this system offers an efficiently replicating RNA as a potential vehicle for in vivo gene transfer.
A single amino acid change in the cytoplasmic domain of the simian immunodeficiency virus transmembrane molecule increases envelope glycoprotein expression on infected cells
Authors: LaBranche C C , Sauter M M , Haggarty B S , Vance P J , Romano J , Hart T K , Bugelski P J , Marsh M , Hoxie J A
Published: January 2020

We have described a virus termed CP-MAC, derived from the BK28 molecular clone of simian immunodeficiency virus, that was remarkable for its ability to infect Sup-T1 cells with rapid kinetics, cell fusion, and CD4 down-modulation (C. C. LaBranche, M. M. Sauter, B. S. Haggarty, P. J. Vance, J. Romano, T. K. Hart, P. J. Bugelski, and J. A. Hoxie, J. Virol. 68:5509-5522, 1994 [Erratum 68:7665-7667]). Compared with BK28, CP-MAC exhibited a number of changes in its envelope glycoproteins, including a highly stable association between the external (SU) and transmembrane (TM) molecules, a more rapid electrophoretic mobility of TM, and, of particular interest, a marked increase in the level of envelope protein expression on the surface of infected cells. These changes were shown to be associated with 11 coding mutations in the env gene (5 in SU and 6 in TM). In this report, we demonstrate that a single amino acid mutation of a Tyr to a Cys at position 723 (Y723C) in the TM cytoplasmic domain of CP-MAC is the principal determinant for the increased expression of envelope glycoproteins on the cell surface. When introduced into the env gene of BK28, the Y723C mutation produced up to a 25-fold increase in the levels of SU and TM on chronically infected cells, as determined by fluorescence-activated cell sorter analysis with monoclonal and polyclonal antibodies. A similar effect was observed when a Tyr-to-Cys change was introduced at the analogous position (amino acid 721) in the SIVmac239 molecular clone, which, unlike BK28 does not contain a premature stop codon in its TM cytoplasmic tail. Substituting other amino acids, including Ala, Ile, and Ser, at this position produced increases in surface envelope glycoproteins that were similar to that observed for the Cys substitution, while a Tyr-to-Phe mutation produced a smaller increase. These results could not be accounted for by differences in the kinetics or efficiency of envelope glycoprotein processing or by shedding of SU from infected cells. However, immunoelectron microscopy demonstrated that the Y723C mutation in BK28 produced a striking redistribution of cell surface envelope molecules from localized patches to a diffuse pattern that covered the entire plasma membrane. This finding suggests that mutation of a Tyr residue in the simian immunodeficiency virus TM cytoplasmic domain may disrupt a structural element that can modulate envelope glycoprotein expression on the surface of infected cells.
A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients
Authors: Günther S , Li B C , Miska S , Krüger D H , Meisel H , Will H
Published: January 2020

Current knowledge of hepatitis B virus (HBV) sequence heterogeneity is based mainly on sequencing of amplified subgenomic HBV fragments. Here, we describe a method which allows sensitive amplification and simplified functional analysis of full-length HBV genomes with or without prior cloning. By this method, a large number of HBV genomes were cloned from sera of six immunosuppressed kidney transplant patients. Two size classes of HBV genomes, one 3.2 kb and another about 2.0 kb in size, were found in all patients. The genome population from one serum sample was studied in detail by size analysis of subgenomic PCR fragments and sequencing. Regions with deletions and insertions were mapped in the C gene and pre-S region. Up to 100% of HBV genomes in all other immunosuppressed patients also had deletions in the C gene. Our results demonstrate the potential of the established method for the structural and functional characterization of heterogeneous populations of complete virion-encapsidated HBV DNAs and suggest that HBV genomes with C gene deletions can have a selective advantage in immunosuppressed patients.
Involvement of the V1/V2 variable loop structure in the exposure of human immunodeficiency virus type 1 gp120 epitopes induced by receptor binding
Authors: Wyatt R , Moore J , Accola M , Desjardin E , Robinson J , Sodroski J
Published: January 2020

The binding of human immunodeficiency virus type 1 (HIV-1) to the cellular receptor CD4 has been suggested to induce conformational changes in the viral envelope glycoproteins that promote virus entry. Conserved, discontinuous epitopes on the HIV-1 gp120 glycoprotein recognized by the 17b, 48d, and A32 antibodies are preferentially exposed upon the binding of soluble CD4 (sCD4). The binding of the 17b and 48d antibodies to the gp120 glycoprotein can also be enhanced by the binding of the A32 antibody. Here we constructed HIV-1 gp120 mutants in which the variable segments of the V1/V2 and V3 structures were deleted, individually or in combination, while the 17b, 48d, and A32 epitopes were retained. The effects of the variable loop deletions on the function of the HIV-1 envelope glycoproteins and on the exposure of epitopes induced by sCD4 or A32 binding to the monomeric gp120 glycoprotein were examined. The variable-loop-deleted envelope glycoproteins were able to mediate virus entry, albeit at lower efficiencies than those of the wild-type glycoproteins. Thus, the V1/V2 and V3 variable sequences contribute to the efficiency of HIV-1 entry but are not absolutely required for the process. Neither the V1/V2 nor V3 loops were necessary for the increase in exposure of the 17b/48d epitopes induced by binding of the A32 monoclonal antibody. By contrast, induction of the 17b, 48d, and A32 epitopes by sCD4 binding apparently involves a movement of the V1/V2 loops, which in the absence of CD4 partially mask these epitopes on the native gp120 monomer. The results obtained with a mutant glycoprotein containing a deletion of the V1 loop alone indicated that the contribution of the V2 loop to these phenomena was more significant than that of the V1 sequences. These results suggest that the V1/V2 loops, which have been previously implicated in CD4-modulated, postattachment steps in HIV-1 entry, contribute to CD4-induced gp120 conformational changes detected by the 17b, 48d, and A32 antibodies.
A human recombinant Fab identifies a human immunodeficiency virus type 1-induced conformational change in cell surface-expressed CD4
Authors: Bachelder R E , Bilancieri J , Lin W , Letvin N L
Published: January 2020

To explore the role of the CD4 molecule in human immunodeficiency virus (HIV) infection following initial virus-CD4 binding, we have characterized CD4-specific antibodies raised by immunizing an HIV-1-infected human with human recombinant soluble CD4 (rsCD4). Fabs were selected from a human recombinant Fab library constructed from the bone marrow of this immunized individual. Here, we describe a human rsCD4-specific recombinant Fab clone selected by panning the library over complexes of human rsCD4 and recombinant HIV-1 envelope protein. While this Fab does not bind to CD4-positive T-cell lines or to human T lymphocytes, it recognizes cell surface-expressed CD4 following the incubation of these cells with a recombinant form of HIV-1 gp120 or with HIV-1 virions. The Fab is not HIV-1 envelope specific, since it does not bind to recombinant gp120 or to native cell surface-expressed HIV-1 envelope proteins. As confirmation of its CD4 specificity, we show that this Fab immunoprecipitates a 55-kDa protein, corresponding to the molecular mass of cellular CD4, from an H9 cell lysate. The specificity of this human Fab provides evidence for a virus-induced conformational change in cell surface-expressed on CD4. The characterization of this altered CD4 conformation and its effects on the host cell will be important in defining postbinding events in HIV infection.
Modification of retroviral RNA by double-stranded RNA adenosine deaminase
Authors: Hajjar A M , Linial M L
Published: January 2020

In this report, we describe a recombinant provirus generated during in vitro passage that contains a short region of adenosine-to-guanosine hypermutation. The hypermutated region is restricted to complementary sequences present in the recombinant provirus. We propose that a duplex was formed in the recombinant RNA prior to reverse transcription. This duplex was a substrate for double-stranded RNA adenosine deaminase, an activity found in all cells examined that deaminates A in double-stranded RNA, converting it to inosine, which is further converted to a guanosine by reverse transcription. It appears that cis viral sequences facilitated the A-->G transitions.
Effects of Elevated Temperatures on Mengovirus Ribonucleic Acid Synthesis and Virus Production in Novikoff Rat Hepatoma Cells
Authors: Shea Mary Ann , Plagemann Peter G. W.
Published: January 2020

The production of mengovirus in Novikoff rat hepatoma cells is progressively reduced with an increase in incubation temperature of the cells from 34 to 40 C, in spite of the fact that about the same amounts of single-stranded and double-stranded viral ribonucleic acid (RNA) are synthesized at 34, 37, and 40 C; the rate of overall protein synthesis is as high at 40 C as at 37 C. At 40 C, progeny viral RNA accumulates in an undegraded form without being incorporated into virus particles. The results suggest that virus maturation is preferentially inhibited at supraoptimal temperatures. At 42 C, on the other hand, no viral RNA is produced and no viral RNA polymerase activity is detectable in cell lysates. Failure of infected cells to form viral RNA polymerase at 42 C is probably due to an impairment of protein synthesis since most of the polyribosomes are rapidly lost during incubation at 42 C and the rate of amino acid incorporation into protein is 70% lower at 42 C than at 37 C. When infected cells are shifted from 37 to 42 C during the period of active viral RNA synthesis, viral RNA polymerase activity is rapidly lost from the cells, and viral RNA synthesis ceases within 45 min. In contrast, the RNA polymerase is as active in vitro at 42 C as at 37 C, and the activity is relatively stable at 42 C.
Proteins and Glycoproteins of Paramyxoviruses: a Comparison of Simian Virus 5, Newcastle Disease Virus, and Sendai Virus
Authors: Mountcastle Walter E. , Compans Richard W. , Choppin Purnell W.
Published: January 2020

The polypeptides of three paramyxoviruses (simian virus 5, Newcastle disease virus, and Sendai virus) were separated by polyacrylamide gel electrophoresis. Glycoproteins were identified by the use of radioactive glucosamine as a carbohydrate precursor. The protein patterns reveal similarities among the three viruses. Each virus contains at least five or six proteins, two of which are glycoproteins. Four of the proteins found in each virus share common features with corresponding proteins in the other two viruses, including similar molecular weights. These four proteins are the nucleocapsid protein (molecular weight 56,000 to 61,000), a larger glycoprotein (molecular weight 65,000 to 74,000), a smaller glycoprotein (molecular weight 53,000 to 56,000), and a major protein which is the smallest protein in each virion (molecular weight 38,000 to 41,000).
Cells Persistently Infected with Newcastle Disease Virus III. Thermal Stability of Hemagglutinin and Neuraminidase of a Mutant Isolated from Persistently Infected L Cells
Authors: Thacore Harshad R. , Youngner Julius S.
Published: January 2020

Data were obtained which indicated the possible cause of the defective elution from erythrocytes of the mutant virus (NDV
pi
) isolated from L cells persistently infected with the Herts strain of Newcastle disease virus (NDV
o
). The chicken erythrocyte receptors for the mutant and wild-type viruses were equally sensitive to the action of
Vibrio cholera
filtrate neuraminidase; this suggests that the failure of NDV
pi
to elute from chicken erythrocytes is not due to a specific neuraminidase-resistant receptor for this virus on the erythrocyte membrane. There was no difference in the enzyme content of the intact virions of NDV
o
and NDV
pi
when tested with a soluble substrate, indicating that the inefficient elution of NDV
pi
was not due to a reduced enzyme content. The neuraminidase activity of intact NDV
pi
virions was significantly more stable at 55 C than the enzyme of NDV
o
virions, whereas the dissociated enzymes of the two viruses were inactivated at the same rate. On the basis of these findings, it seems likely there is a structural difference between the two viruses. The neuraminidase protein of the mutant NDV
pi
may be incorporated into the viral envelope in such a manner that it is prevented from reacting with the substrate in the erythrocyte membrane, although it can react with a soluble substrate. The hemagglutinin activity of both intact and disrupted NDV
pi
was significantly more resistant to thermal inactivation than that of the wild-type NDV
o
. This finding suggests a genetic difference in the hemagglutinin protein of the two viruses.
Deoxyribonucleic Acid Polymerase Activity Associated with Strain MC29 Tumor Virus
Authors: Weber G. H. , Kiessling A. A. , Beaudreau G. S.
Published: January 2020

Chick embryo cells infected with strain MC29 tumor virus yielded progeny virus that contained detectable deoxyribonucleic acid (DNA) polymerase within the first 48 hr after infection. The noninfected culture fluids displayed no such enzyme activity when examined in an identical manner. Enzyme activity was greatly stimulated by adding DNA template to the reaction mixture and required all four deoxyribonucleoside triphosphates for full activity. When calf thymus DNA was used to direct synthesis, the DNA polymerase from the MC29 virus catalyzed the formation of DNA product having a higher buoyant density in CsCl. DNA product formed in the reaction directed by
Micrococcus lysodeikticus
DNA had the same buoyant density as the template DNA.
Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses III. Base Composition, Molecular Weight, and Conformation of the Ad2
+
ND
1
Genome
Authors: Crumpacker Clyde S. , Henry Patrick H. , Kakefuda Tuyoski , Rowe Wallace P. , Levin Myron J. , Lewis Andrew M.
Published: January 2020

The nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2
+
ND
1
, differs from the defective Ad-SV40 hybrid populations previously described, in that this hybrid virus can replicate without the aid of nonhybrid adenovirus helper. Consequently, the deoxyribonucleic acid (DNA) from this virus, which can be obtained free of nonhybrid adenovirus DNA, is well suited for biophysical studies on Ad-SV40 hybrid DNA. Such studies have been performed and demonstrate Ad2
+
ND
1
DNA to have a buoyant density (1.715 g/cm
3
) and thermal denaturation profile (T
m
= 75.1 C) almost identical with nonhybrid Ad2 DNA. Furthermore, its molecular weight, as determined by analytical zone sedimentation and electron microscopy, was 22 × 10
6
to 25 × 10
6
daltons, which is also very similar to that determined for Ad2. Electron micrographs showed all of the hybrid molecules to be double-stranded and linear. By using this determination of the molecular weight of Ad2
+
ND
1
DNA and assuming that 1% of this molecule consists of covalently linked SV40 DNA (
see companion paper
), we calculate that the hybrid DNA molecule contains 220 × 10
3
to 250 × 10
3
daltons of SV40 DNA, or the equivalent of one-tenth of the SV40 genome.
Absence of Interference During High-Multiplicity Infection by Clonally Purified Vesicular Stomatitis Virus
Authors: Stampfer Martha , Baltimore David , Huang Alice S.
Published: January 2020

Stocks of vesicular stomatitis virus free of defective interfering particles were produced by serial clonal isolation. High-multiplicity infections with these stocks led to no interference or formation of defective interfering particles. Defective interfering particles were generated by three successive passages at high multiplicity.
Genetic Map of Bacteriophage φX174
Authors: Benbow R. M. , Hutchison C. A. , Fabricant J. D. , Sinsheimer R. L.
Published: January 2020

Bacteriophage φX174 temperature-sensitive and nonsense mutations in eight cistrons were mapped by using two-, three-, and four-factor genetic crosses. The genetic map is circular with a total length of 24 × 10
−4
wt
recombinants per progeny phage. The cistron order is D-E-F-G-H-A-B-C. High negative interference is seen, consistent with a small closed circular deoxyribonucleic acid molecule as a genome.
Comparison of Rous Sarcoma Virus-Specific Deoxyribonucleic Acid Polymerases in Virions of Rous Sarcoma Virus and in Rous Sarcoma Virus-Infected Chicken Cells
Authors: Coffin John M. , Temin Howard M.
Published: January 2020

Labeled virions of Rous sarcoma virus (RSV) were disrupted with detergent and analyzed on equilibrium sucrose density gradients. A core fraction at a density of approximately 1.24 g/cc contained all of the
3
H-uridine label and about 30% of the
3
H-leucine label from the virions. Endogenous viral deoxyribonucleic acid (DNA) polymerase activity was only found in the same location. Additional ribonucleic acid (RNA)- and DNA-dependent DNA polymerase activities were found at the top of the gradients. RNA-dependent and DNA-dependent DNA polymerase activities were also found in RSV-converted chicken cells. Particles containing these activities were released from cells by detergent and were shown to contain viral RNA. These particles were analyzed on equilibrium sucrose density gradients and were found to have densities different from virion cores.
Structural Polypeptides of Simian Virus 40
Authors: Estes Mary K. , Huang Eng-Shang , Pagano Joseph S.
Published: January 2020

To determine the number and molecular weights of the structural polypeptides of simian virus 40, we have analyzed purified virus by electrophoresis on 14% polyacrylamide gels containing sodium dodecyl sulfate. Full virus purified by several different methods showed six distinct bands with molecular weights of approximately 43,000 (VP1, containing 70% of virion protein), 32,000 (VP2, 9%), 23,000 (VP3, 10%), 14,000 (VP4, 6%), 12,500 (VP5, 4%), and 11,000 (VP6, 3%) both by analysis of radioactively labeled virions and by visualization of the polypeptide bands after staining. “Empty” virions contain decreased amounts of VP4, 5, and 6. The approximate molecular ratios of the polypeptides were 6.0, 1.0, 1.5, 1.5, 1.1, and 1.0. When virus degraded in an alkaline buffer was analyzed by velocity centrifugation in sucrose gradients, the two larger polypeptides (VP1 and VP2) remained at the top of the gradient, whereas the three smallest polypeptides (VP4, 5, and 6) sedimented as a complex with the viral deoxyribonucleic acid. VP3 was found in association with either VP1 and 2 or VP4, 5, and 6, depending on the conditions of degradation. Presumably, VP1 and VP2, comprising about 80% of the protein, form the capsid of the virus. VP4, 5, and 6 may form a nucleoprotein in the virion, and VP3 may serve as an intermediate structural component.
Virus Interference by Cellular Double-Stranded Ribonucleic Acid
Authors: Kimball P. C. , Duesberg P. H.
Published: January 2020

Ribonuclease-resistant ribonucleic acid (RNA) was isolated from uridine-labeled cultures of rabbit kidney, chicken embryo, and HeLa cells. This RNA, regardless of its source, was found to induce interference with virus growth in either rabbit kidney or chicken embryo cultures. Nuclease-treated cellular nucleic acids exhibited interference-inducing activity which eluted with a small fraction of RNA in the exclusion volume of a 6% agarose gel column. Besides resistance to ribonucleases, the interference inducer and RNA isolated from partially digested nucleic acids have in common two properties of double-stranded RNA: (i) similar sharp melting profiles were obtained for inducer and ribonuclease-resistant RNA, with T
m
dependent on NaCl concentration; (ii) ribonuclease-resistant inducer and RNA banded together in Cs
2
SO
4
density gradients at a density characteristic of known double-stranded RNA. After melting at low ionic strength, the labeled RNA shifted to a higher density and its capacity to inhibit virus replication was lost. Velocity sedimentation analysis of the cellular ribonuclease-resistant RNA indicated that the majority sedimented between 7 and 11
S
, but only RNA sedimenting at ≧8 to 20
S
had a high specific activity of interference induction. Without prior ribonuclease treatment, the ribonuclease-resistant RNA can be precipitated with 2
m
LiCl and thus appears to exist in purified cellular nucleic acids as part of molecular complexes with both single- and double-stranded regions of RNA. The biosynthesis of cellular double-stranded RNA is inhibited by actinomycin D.
Growth of Murine Cytomegalovirus in Various Cell Lines
Authors: Kim Kwang Soo , Carp Richard I.
Published: January 2020

Murine cytomegalovirus (MCMV) was capable of infecting and replicating in both primary and continuous cell lines obtained from various species. In African green monkey kidney (BSC-1) cells, primary rabbit kidney cells, and baby hamster kidney (BHK-21) cells, there were cytopathic effects (CPE) and virus replication upon initial exposure of cells to virus. In primary fetal sheep brain (FSB) cells, L cells, and rabbit kidney (RK-13) cells, it was necessary to subculture the infected cells one or more times before appearance of CPE and replication of virus. In the case of the infected FSB cultures, it was found that the virus effect could be induced if subculturing were accomplished by trypsinization but did not occur if cells were subcultured by scraping. FSB-grown virus replicated better in FSB than in mouse embryo fibroblast (MEF) cells. The CPE produced in all of the above cell lines was similar to that observed in MEF infected with MCMV. The virus grown in different cell lines was completely neutralized when mixed with several reference sera prepared in rabbits or mice. The populations of virions released from infected MEF and FSB cells were compared by isopycnic centrifugation in potassium tartrate, and no differences were revealed in the buoyant densities of the populations. Human embryonic brain cells, human embryonic kidney cells, a human lung fibroblast cell strain (WI-38), HeLa, and Hep-2 were not susceptible to MCMV.
Synthesis of the Major Bacteriophage f1 Coat Protein
Authors: Smilowitz Henry , Lodish Harvey , Robbins Phillips W.
Published: January 2020

A method which allows one to follow the synthesis of the major f1 coat protein in normal, unirradiated f1-infected cells is reported. The
N
-terminal tryptic peptide of this protein, labeled with
14
C-lysine, has a negative charge at
p
H 4.5 and is readily separated from the contaminating peptides of host cell proteins. This technique was used to study several aspects of the synthesis of the major f1 coat protein in infected cells.
Sindbis virus DNA-based expression vectors: utility for in vitro and in vivo gene transfer
Authors: Dubensky T W , Driver D A , Polo J M , Belli B A , Latham E M , Ibanez C E , Chada S , Brumm D , Banks T A , Mento S J , Jolly D J , Chang S M
Published: January 2020

Several DNA-based Sindbis virus vectors were constructed to investigate the feasibility and potential applications for initiating the virus life cycle in cells transfected directly with plasmid DNA. These vectors, when transfected into mammalian cells, have been used to produce virus, to express heterologous genes, and to produce infectious vector particles. This approach involved the conversion of a self-replicating vector RNA (replicon) into a layered DNA-based expression system. The first layer includes a eukaryotic RNA polymerase II expression cassette that initiates nuclear transcription of an RNA which corresponds to the Sindbis virus vector replicon. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the vector, proceeds according to the Sindbis virus replication cycle and results in expression of the heterologous gene. The Sindbis virus DNA vectors expressed reporter genes in transfected cells at levels that were comparable to those of in vitro-transcribed RNA replicons and were approximately 10-fold higher than the levels produced by conventional RNA polymerase II-dependent plasmids in which the promoter and reporter gene were linked directly. Reporter gene expression was also observed in rodent muscle following injection with Sindbis virus DNA vectors. In a second application, packaged vector particles were produced in cells cotransfected with complementing replicon and defective helper DNAs. The Sindbis virus-derived DNA vectors described here increase the utility of alphavirus-based vector systems in general and also provide a vector with broad potential applications for genetic immunization.
The v-erbB oncogene confers enhanced cellular susceptibility to reovirus infection
Authors: Strong J E , Lee P W
Published: January 2020

We have previously demonstrated that two mouse cell lines that are poorly infectible by reovirus become highly susceptible upon transfection with the gene encoding the epidermal growth factor receptor (EGFR) (J. E. Strong, D. Tang, and P. W. K. Lee, Virology 197:405-411, 1993). This enhancement of infection efficiency requires a functional EGFR, since such an enhancement is not observed in cells expressing a mutated (kinase-inactive) EGFR. The additional finding that reovirus is capable of directly binding to the N-terminal ectodomain of the EGFR (D. Tang, J. E. Strong, and P. W. K. Lee, Virology 197:412-414, 1993) has led us to question whether this interaction is required for the activation of a signalling cascade that somehow augments the ensuing infection process. In the present study, we address this question, using cells transfected with the v-erbB oncogene, which encodes a protein structurally related to the EGFR but lacking a large portion of the N-terminal ligand-binding domain. The v-erbB protein also possesses ligand-independent, constitutive tyrosine kinase activity. Control NIH 3T3 cells, which are poorly infectible by reovirus (serotype 3, strain Dearing), and NIH 3T3 cells transfected with the v-erbB oncogene (THC-11) were assayed for their susceptibilities to reovirus infection. Infectivity was determined by immunofluorescent detection of viral proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of radiolabeled cells, and plaque titration. All three assays demonstrated a drastically higher degree of susceptibility to infection in the THC-11 cell line. This enhanced susceptibility was found to be abrogated by treatment of the cells with genistein, an inhibitor of tyrosine protein kinases, but only partially by treatment with daidzein, an inactive analog of genistein. We propose that the mechanism of enhancement of infection efficiency conferred by EGFR and v-erbB is through the opportunistic utilization by the virus of an already activated signal transduction pathway.
Induction of susceptibility to tumor necrosis factor by E1A is dependent on binding to either p300 or p105-Rb and induction of DNA synthesis
Authors: Shisler J , Duerksen-Hughes P , Hermiston T M , Wold W S , Gooding L R
Published: January 2020

The introduction of the adenovirus early region 1A (E1A) gene products into normal cells sensitizes these cells to the cytotoxic effects of tumor necrosis factor (TNF). Previous studies have shown that the region of E1A responsible for susceptibility is CR1, a conserved region within E1A which binds the cellular proteins p300 and p105-Rb at nonoverlapping sites. Binding of these and other cellular proteins by E1A results in the induction of E1A-associated activities such as transformation, immortalization, DNA synthesis, and apoptosis. To investigate the mechanism by which E1A induces susceptibility to TNF, the NIH 3T3 mouse fibroblast cell line was infected with viruses containing mutations within E1A which abrogate binding of some or all of the cellular proteins to E1A. The results show that TNF susceptibility is induced by E1A binding to either p300 or p105-Rb. E1A mutants that bind neither p300 nor p105-Rb do not induce susceptibility to TNF. Experiments with stable cell lines created by transfection with either wild-type or mutant E1A lead to these same conclusions. In addition, a correlation between induction of DNA synthesis and induction of TNF sensitivity is seen. Only viruses which induce DNA synthesis can induce TNF sensitivity. Those viruses which do not induce DNA synthesis also do not induce TNF sensitivity. These data suggest that the mechanisms underlying induction of susceptibility to TNF by E1A are intimately connected to E1A's capacity to override cell cycle controls.
YY1 represses human papillomavirus type 16 transcription by quenching AP-1 activity
Authors: O'Connor M J , Tan S H , Tan C H , Bernard H U
Published: January 2020

YY1 is a multifunctional transcription factor that has been shown to regulate the expression of a number of cellular and viral genes, including the human papillomavirus (HPV) oncogenes E6 and E7. In this study, we have analyzed the YY1-mediated repression of the HPV type 16 (HPV-16) E6-E7 promoter. A systematic analysis to identify YY1 sites present in the HPV-16 long control region showed that of 30 potential YY1 binding motifs, 24 bound purified recombinant YY1 protein, but only 10 of these were able to bind YY1 when nuclear extracts of HeLa cells were used. Of these, only a cluster of five sites, located in the vicinity of an AP-1 motif, were found to be responsible for repressing the HPV-16 P97 promoter. All five sites were required for repression, the mutation of any one site giving rise to a four- to sixfold increase in transcriptional activity. The target for YY1-mediated repression was identified as being a highly conserved AP-1 site, and we propose that AP-1 may represent a common target for YY1 repression. We also provide data demonstrating that YY1 can bind the transcriptional coactivator CREB-binding protein and propose a potentially novel mechanism by which YY1 represses AP-1 activity as a result of this YY1-CREB-binding protein interaction.
Simian virus 40 small t antigen activates the carboxyl-terminal transforming p53-binding domain of large T antigen
Authors: Zerrahn J , Tiemann F , Deppert W
Published: January 2020

Expression of the simian virus 40 large T antigen (large T) in F111 rat fibroblasts generated only minimal transformants (e.g., F5 cells). Interestingly, F111-derived cells expressing only an amino-terminal fragment of large T spanning amino acids 1 to 147 (e.g., FR3 cells), revealed the same minimal transformed phenotype as F111 cells expressing full-length large T. This suggested that in F5 cells the transforming domain of large T contained within the C-terminal half of the large T molecule, and spanning the p53 binding domain, was not active. Progression to a more transformed phenotype by coexpression of small t antigen (small t) could be achieved in F5 cells but not in FR3 cells. Small-t-induced progression of F5 cells correlated with metabolic stabilization of p53 in complex with large T: whereas in F5 cells the half-life of p53 in complex with large T was only slightly elevated compared with that of (uncomplexed) p53 in parental F111 cells or that in FR3 cells, coexpression of small t in F5 cells led to metabolic stabilization and to high-level accumulation of p53 complexed to large T. In contrast, coexpression of small t had no effect on p53 stabilization or accumulation in FR3 cells. This finding strongly supports the assumption that the mere physical interaction of large T with p53, and thus p53 inactivation, in F5 cells expressing large T only does not reflect the main transforming activity of the C-terminal transforming domain of large T. In contrast, we assume that the transforming potential of this domain requires activation by a cellular function(s) which is mediated by small t and correlates with metabolic stabilization of p53.
Domains required for assembly of adenovirus type 2 fiber trimers
Authors: Hong J S , Engler J A
Published: January 2020

Entry of human adenovirus into cells is a two-step process, mediated in the first step by a specific interaction between the trimeric fiber protein and a specific receptor on the surface of susceptible cells. Because of the interest in human adenovirus as a vector for gene therapy, we have mapped domains in the fiber protein that are important for proper assembly of this trimeric structure and for proper addition of O-linked N-acetylglucosamine (0-GlcNAc). Mutants of adenovirus type 2 fiber in this study were expressed in human cells by use of a recombinant vaccinia virus expression system that yielded protein indistinguishable from the fiber produced during adenovirus infection. The N-terminal half of the protein did not appear to influence fiber trimer formation, since deletions up to 260 amino acids (aa) from the N-terminal end as well as in-frame deletions within the shaft of the molecule still allowed trimerization; internal deletions in the shaft between aa 61 and 260 appeared to alter addition of 0-GlcNAc, as judged by loss of reactivity to a monoclonal antibody specific for this carbohydrate addition. Deletions from the C terminus of the molecule (as small as 2 aa) appeared to prevent trimer formation. Additions of amino acids to the C-terminal end of the fiber showed variable results: a 6-aa addition allowed trimer formation, while a 27-aa addition did not. These trimer-defective mutants were also relatively less stable, as judged kV pulse-chase experiments. Taken together, our results indicate that trimerization of the fiber requires at least two domains, the entire head (aa 400 to 582), and at least the C-terminal-most 15 aa of the shaft.
Serotype-dependent induction of pulmonary neutrophilia and inflammatory cytokine gene expression by reovirus
Authors: Farone A L , Frevert C W , Farone M B , Morin M J , Fields B N , Paulauskis J D , Kobzik L
Published: January 2020

Reovirus type 3 Dearing (T3D) causes a prominent neutrophil influx, substantially greater than seen with reovirus type 1 Lang (T1L) in a rat model of viral pneumonia. We sought to measure reovirus-mediated increases in chemokine mRNA expression in pulmonary cells. We found that the neutrophilia induced by T1L and T3D infection in vivo correlated directly with increased levels of chemokine mRNA expression in T3D-infected compared with those of T1IL-infected lungs. In vitro, reovirus-infected normal alveolar macrophages (AMs) and the rat AM cell line NR8383 expressed greater levels of macrophage inflammatory protein 2, KC, and tumor necrosis factor alpha mRNA. A synergism between reovirus and lipopolysaccharide was also detected for macrophage inflammatory protein 2 and KC mRNA expression. Tumor necrosis factor protein secretion was also increased to a greater extent by T3D than by T1L in primary rat AMs and the NR8383 cells. We conclude that the virus-mediated inflammatory cytokine induction suggests a role for these cytokines in the neutrophil influx observed in the rat reovirus pneumonia model.
Constitutive retinoid receptors expressed from adenovirus vectors that specifically activate chromosomal target genes required for differentiation of promyelocytic leukemia and teratocarcinoma cells
Authors: Lipkin S M , Grider T L , Heyman R A , Glass C K , Gage F H
Published: January 2020

Sufficient knowledge of transcription factor structure and function has accumulated to allow attempts at the rational design of novel transcription factors for the study of gene regulation and potential application in gene therapy. In the present studies, we have systematically evaluated the function of chimeric retinoid receptors generated by fusion with the transactivation domain of VP16 and expression in adenovirus vectors. By varying the location of fusion of the VP16 transactivation domain with the retinoic acid receptor (RAR) or retinoid X receptor (RXR), marked differences in the specificity of gene activation were obtained. Although several chimeric proteins activated both RAR and RXR target genes, fusion of the NT16 transactivation domain to the N terminus of RAR permitted specific activation of reporter genes containing retinoic acid response elements. In contrast, fusion of the VP16 transactivation domain to the C terminus of RXR permitted specific activation of reporter genes containing RXR response elements. When tested for their ability to activate chromosomal targets, the chimera consisting of VP16 linked to the N terminus of PAR was much more active in promoting the differentiation of HL-60 cells and NTera-2 cells than the chimera consisting of VP16 linked to the C terminus of RXR. These observations support the existence of two distinct retinoid signalling pathways predicted on the basis of biochemical and pharmacologic studies and provide direct evidence that the programs of differentiation elicited by retinoic acid in these cells are mediated by a specific subset of binding sites for RAR-RXR heterodimers. VP16-RAR and VP16-RXR fusion proteins should be of further use in dissecting the relative contributions of RARs and RXRs to specific programs of gene expression. Constitutive retinoid receptors may also be considered for use as novel tumor suppressor genes for genetically based treatment of retinoid-responsive cancers.
Broad host range of human T-cell leukemia virus type 1 demonstrated with an improved pseudotyping system
Authors: Sutton R E , Littman D R
Published: January 2020

Studies of human T-cell leukemia virus type 1 (HTLV-1) have been hampered by the difficulty of achieving high cell-free and cell-associated infectious titers. Current retroviral pseudotyping systems using the HTLV-1 envelope generate titers of less than 200 infectious particles per ml. We describe here an improved system for pseudotyping using a defective human immunodeficiency virus (HIV) type 1 genome in combination with HTLV-1 env in 293T producer cells. Introduction of additional copies of rev and treatment of cells with sodium butyrate resulted in a cell-associated titer of 10(5)/ml and cell-free titers of greater than 10(4)/ml . By using this system, we found that the host range of HTLV-1 is even greater than previously suspected. Earlier studies which assigned a chromosomal location for the HTLV-1 receptor may therefore reflect cell-to-cell variation in receptor number rather than the absolute presence or absence of a receptor. The generation of higher-titer HIV(HTLV-1) may facilitate identification of the cellular receptor and investigations of the pathophysiology of HTLV-1 infection.
Mutational analysis of ICP0R, a transrepressor protein created by alternative splicing of the ICP0 gene of herpes simplex virus type 1
Authors: Spatz S J , Nordby E C , Weber P C
Published: January 2020

The immediate-early protein ICP0 (infected-cell polypeptide 0) of herpes simplex virus type 1 (HSV-1) is a promiscuous transactivator of both viral and nonviral promoters in transient expression assays. Failure to splice the second of two introns in the ICP0 gene results in the utilization of an alternate stop codon that generates a truncated form of ICP0 called ICP0R. This protein exists in low levels in HSV-1-infected cells and functions as a dominant negative repressor of ICP0-mediated transactivation in transient expression assays. To conduct a detailed structure-function analysis of ICP0R, a series of insertion and deletion mutants of this protein were generated and analyzed in transfection assays. These studies indicated that segments of ICP0R that were rich in acidic amino acid residues (amino acids 9 to 76 and 233 to 241) or glycine residues (amino acids 242 to 262) were dispensable for the dominant negative phenotype. In contrast, the RING finger domain (amino acids 116 to 156) and surprisingly the sequences carboxy terminal to it (amino acids 157 to 232) were absolutely essential for transdominant repression. Consistent with these findings, the amino acid sequences of these two regions were conserved among other alphaherpesvirus ICP0 homologs. A construct containing only amino acids 76 to 232 inhibited ICP0-mediated transactivation almost as efficiently as wild-type ICP0R and represented the minimal sequences necessary for the dominant negative phenotype. These results demonstrated that the critical functional domain shared by both ICP0R and ICP0 is much more complex than a simple RING finger motif. Western blot (immunoblot) analyses of transfected cell lysates revealed that nearly all of the mutant constructs directed the expression of stable ICP0R proteins of the predicted molecular weight. However, there was a striking inverse correlation between the ability of a mutant construct to mediate transrepression and the amount of protein that it synthesized, indicating that dominant negative inhibition is achieved through the action of very little ICP0R protein.
The RGG box motif of the herpes simplex virus ICP27 protein mediates an RNA-binding activity and determines in vivo methylation
Authors: Mears W E , Rice S A
Published: January 2020

ICP27 is an essential herpes simplex virus type 1 nuclear regulatory protein that is required for efficient viral gene expression. Although the mechanism by which ICP27 regulates genes is unknown, a variety of evidence suggests that it functions posttranscriptionally, and recent studies indicate that it is an RNA-binding protein. Previously, we noted that a short arginine- and glycine-rich sequence in ICP27 (residues 138 to 152) is similar to an RGG box motif, a putative RNA-binding determinant found in a number of cellular proteins (W. Mears, V. Lam, and S. Rice, J. Virol. 69:935-947, 1995). In the present study, we have further investigated ICP27's association with RNA and examined the role of the RGG box in RNA binding. We find that ICP27 binds efficiently to RNA homopolymers composed of poly(G) and weakly to poly(U) RNA homopolymers. Poly(G) binding activity maps to the N-terminal 189 residues of ICP27 and requires the RGG box sequence. Using a northwestern blotting assay, we demonstrate that the RGG box alone (residues 140 to 152) can mediate RNA binding when attached to a heterologous protein. As many cellular RGG box proteins are methylated on arginine residues, we also investigated the in vivo methylation status of ICP27. Our results demonstrate that ICP27 is methylated in herpes simplex virus-infected cells. Methylation is dependent on the presence of the RGG box, suggesting that one or more arginine residues in the RGG box sequence are modified. These data demonstrate that ICP27 displays the characteristics of an RGG box-type RNA-binding protein.
Enhanced transcriptional activation by E2 proteins from the oncogenic human papillomaviruses
Authors: Kovelman R , Bilter G K , Glezer E , Tsou A Y , Barbosa M S
Published: January 2020

A systematic comparison of transcriptional activation by papillomavirus E2 proteins revealed that the E2 proteins from high-risk human papillomaviruses (human papillomavirus type 16 [HPV-16] and HPV-18) are much more active than are the E2 proteins from low-risk HPVs (HPV-6b and HPV-11). Despite the tropism of HPVs for particular epithelial cell types, this difference in transcriptional activation was observed in a number of different epithelial and nonepithelial cells. The enhanced activities of the E2 proteins from high-risk HPVs did not result from higher steady-state levels of protein in vivo, and in vitro DNA-binding assays revealed similar binding properties for these two classes of E2 proteins. These results demonstrate that the E2 proteins from high-risk HPVs have an intrinsically enhanced potential to activate transcription from promoters with E2-responsive elements. We found that there are also substantial differences between the activation properties of the bovine papillomavirus type 1 E2 protein and those of either of the two classes of HPV E2 proteins, especially with regard to requirements for particular configurations of E2 binding sites in the target promoter. Our results indicate that there are at least three distinct functional classes of E2 proteins and that these classes of E2 proteins may perform different roles during the respective viral life cycles.