bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of genes introduced into cells by retroviral infection is more efficient than that of genes introduced into cells by DNA transfection
Authors: Hwang L H , Gilboa E
Published: January 2020

Calcium phosphate-mediated DNA transfection and retroviral infection are two alternative gene transfer techniques designed to introduce specific DNA fragments into the chromosomes of recipient cells. To compare the efficiency of expression of genes introduced into cells by either of these two techniques, a retrovirus-derived vector was constructed from the genome of Moloney murine leukemia virus by replacing the coding sequences of the envelope gene with the bacterial Neor gene derived from Tn5, termed rEnv-Neor. Expression of the hybrid Neor gene in NIH 3T3 cells after DNA transfection or retroviral infection was determined by measuring the steady-state levels of the corresponding cytoplasmic polyadeylated RNA species. Cells containing one copy of the integrated rEnv-Neor DNA introduced into cells by retroviral infection expressed 10- to 50-fold-higher levels of vector-specific RNA compared with cells harboring one copy of the same DNA derived by DNA transfection. Analysis of the integrated rEnv-Neor DNA with the methylation-sensitive restriction enzyme SmaI has shown that DNA integrated after DNA transfection but not after viral infection is partially methylated, predominantly in the 5' long terminal repeat, the region involved in initiation of transcription.
Transcription of the hepatitis B surface antigen gene in cultured murine cells initiates within the presurface region
Authors: Standring D N , Rutter W J , Varmus H E , Ganem D
Published: January 2020

Cloned hepatitis B virus (HBV) DNA directs the synthesis of the viral surface antigen (HBsAg) when introduced into mouse L cells by DNA transformation. We have used recombinants between the Rous sarcoma virus long terminal repeat and subgenomic fragments of HBV DNA to localize regions of the HBV genome required for HBsAg expression. Examination of HBV-specific RNA from such transformants indicates that transcription initiates at three distinct sites (153, 163, and 183 nucleotides upstream from the translation initiation codon for mature HBsAg). Thus in these cells, a large segment of the presurface reading frame is not represented in HBsAg mRNA. The termination site of this RNA lies within the coding sequences for the viral core antigen, some 1,094 +/- 10 base pairs downstream from the TAA stop codon for HBsAg. Two additional open reading frames are present in the resultant unspliced HBsAg RNA.
Gamma-T4 hybrid bacteriophage carrying the thymidine kinase gene of bacteriophage T4
Authors: Mileham A J , Murray N E , Revel H R
Published: January 2020

Among 32 lambda-T4 recombinant phages selected for growth on a thymidylate synthetase-deficient (thyA) host, 2 were shown to carry the T4 thymidine kinase (tk) gene. The lambda-T4tk phages contain two T4 HindIII DNA fragments (2.0 and 1.5 kilobases) that hybridize to restriction fragments of T4 DNA, encompassing the tk locus at 60 kilobases on the T4 map. The T4tk insert compensates for the simultaneous host deficiencies of thymidine kinase and thymidylate synthetase in a thymidine kinase-deficient (tdk) host growing in the presence of fluorodeoxyuridine when provided with thymidine and uridine. The lambda-T4tk hybrid phages specified five polypeptides with Mrs of 22,000 (22K), 21K, 14K, 11K, and 9K.
Immunological and genomic analyses of two serotypes of avian paramyxovirus isolated from wild ducks in Japan
Authors: Nerome K , Shibata M , Kobayashi S , Yamaguchi R , Yoshioka Y , Ishida M , Oya A
Published: January 2020

A total of 11 avian paramyxoviruses isolated from migrating feral ducks in Niigata, Japan, were characterized by serological and genomic analyses. Hemagglutination inhibition and immuno-double-diffusion tests with antisera specific for the isolated hemagglutinin-neuraminidase polypeptides of reference strains indicated that, of these, eight isolates possessed hemagglutinin-neuraminidase antigen closely related to that of duck/Hong Kong/D3/75, and the remaining three isolates possessed antigen closely related to that of duck/Hong Kong/199/77. RNA analysis of the eight isolates identified serologically as duck/Hong Kong/D3/75 by oligonucleotide mapping revealed that these isolates were genetically very similar to each other but different from the reference strain and isolates reported previously. The oligonucleotide maps of duck/Hong Kong/199/77-like isolates appeared to be very similar to each other, suggesting the same origin, but not to the duck/Hong Kong/199/77 virus.
Deletion of DNA sequence in a nononcogenic variant of Herpesvirus saimiri
Authors: Koomey J M , Mulder C , Burghoff R L , Fleckenstein B , Desrosiers R C
Published: January 2020

The 110-kilobase-pair stretch of unique sequence DNA of Herpesvirus saimiri is flanked by highly repetitive DNA. Detailed restriction endonuclease mapping has localized the left junction of repetitive and unique DNA to a 100-base-pair region. H. saimiri 11att, a replication competent nononcogenic variant of strain 11, has a deletion of 2.3 kilobase pairs of sequence information that spans this left junction of repetitive and unique DNA.
Virus-induced diabetes mellitus: mengovirus infects pancreatic beta cells in strains of mice resistant to the diabetogenic effect of encephalomyocarditis virus
Authors: Yoon J W , Morishima T , McClintock P R , Austin M , Notkins A L
Published: January 2020

In mice, Mengovirus produces a fatal encephalitis. Plaque purification of the virus resulted in the isolation of a clone (Mengo- 2T ), which in addition to encephalitis caused diabetes. Microscopic examination of pancreases from infected mice revealed necrosis in the islets of Langerhans and infiltration of inflammatory cells. By immunofluorescence viral antigens were found in the islets, and radioimmunoassays demonstrated a substantial decrease in pancreatic immunoreactive insulin. Studies on susceptibility among inbred strains of mice showed that whereas the D variant of encephalomyocarditis virus caused diabetes only in SJL/J mice, Mengo- 2T caused diabetes in strains of mice resistant to encephalomyocarditis-induced diabetes (i.e., CBA/J, C3H/HeJ, CE/J, AKR/J, C57BL/6J). The ability of Mengo- 2T to induce diabetes in encephalomyocarditis-resistant mice was found to be due to the greater capacity of Mengo- 2T as compared to the D variant of encephalomyocarditis virus to replicate in and destroy the islets of these animals. Although Mengo- 2T and the D variant of encephalomyocarditis virus are antigenically indistinguishable by hyperimmune sera, our studies show that these viruses have different host ranges and tissue tropisms .
Replication of endogenous avian retrovirus in permissive and nonpermissive chicken embryo fibroblasts
Authors: Humphries E H , Allen R
Published: January 2020

Clones of chicken embryo fibroblasts exogenously infected with the endogenous avian retrovirus were analyzed to examine the replication of this virus in permissive (Gr+) and nonpermissive (Gr-) cells. The results demonstrate that the endogenous virus was capable of infecting both Gr+ and Gr- cells with equal efficiency. Infected clones of Gr+ and Gr- cells differed, however, in two significant ways. At the time of their initial characterization, the Gr+ clones produced 100- to 1,000-fold more virus than the Gr- clones. Further, the amount of virus produced by Gr+ clones did not change significantly during serial passage of the cells. In contrast, continued passage of the infected Gr- clones resulted in a gradual increase in the amount of virus produced. Individual clones of infected Gr- cells produced infectious virus at rates that, initially, differed by a factor of more than 10(4). The large differences in the production of virus by these clones could not be explained by equally large differences in the number of infected cells within the clonal populations. Greater than 80% of the clonal populations examined ultimately produced virus at rates that were not significantly different from the rates observed in infected Gr+ cells. Virus produced by these infected Gr- cells exhibited the same restricted replication upon establishing a new infection in nonpermissive cells. Analysis of the appearance of free and integrated viral DNA sequences during endogenous virus infection of Gr+ and Gr- cells demonstrated that, after an initial delay in the synthesis of free viral DNA in Gr- cells, the nonpermissive cells ultimately acquired as many integrated viral DNA sequences as were found in infected Gr+ cells. These results indicate that a majority of the infectious particles of the endogenous virus are capable of establishing infection in a Gr- cell and, ultimately, of producing virus at a rate that is not significantly different from that produced by infected Gr+ cells. The virus produced from the Gr- cells is not a stable genetic variant of the original endogenous virus that is capable of unrestricted replication in nonpermissive cells. The reduced efficiency with which the endogenous virus initially replicates in nonpermissive cells and the increased length of time required for infected Gr- cells to produce maximal virus titers suggest that the endogenous virus may utilize a different mechanism of replication in Gr+ and Gr- fibroblasts.
Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from a chronically infected liver
Authors: Rogler C E , Summers J
Published: January 2020

We have isolated and determined the structure of a recombinant clone in lambda phage Charon 30 which contains woodchuck hepatitis virus sequences integrated in woodchuck genomic DNA sequences. This clone, in contrast to previously reported clones (Ogston et al., Cell 29:385-394, 1982), was isolated from a chronically infected liver which never developed hepatocellular carcinoma. Southern blot analysis of viral sequences in the clone in conjunction with electron microscope heteroduplex analysis showed that the integrated viral sequences did not contain internal rearrangements, as have those from hepatomas, but were colinear with the cloned viral genome except for the deletion of approximately 500 base pairs of viral sequences (between positions 1,000 and 1,550 on the viral map). Therefore, the integration was probably a defective genome incapable of supporting viral replication. However, the complete open reading frames coding for the viral X, core, presurface , and surface antigen genes were present, indicating that the viral sequences could code for viral antigens. Southern blot analysis of the normal cellular flanking sequences, using flanking sequence probes from the clone, showed that no detectable rearrangements of cellular DNA (less than 50 base pairs) had occurred at the site of viral integration.
Cooperative effects between two acyclovir resistance loci in herpes simplex virus
Authors: Darby G , Churcher M J , Larder B A
Published: January 2020

The acyclovir-resistant mutant SC16 R9C2 (H.J. Field, G. Darby, and P. Wildy , J. Gen. Virol. 49:115-124, 1980) has been shown to contain two resistance loci which segregate independently on recombination with wild-type virus. One locus is in thymidine kinase, and the other is in DNA polymerase. Both induced enzymes have altered properties, thymidine kinase showing a low affinity for acyclovir and low activity, and DNA polymerase showing a low affinity for acyclovir triphosphate. Other properties of both enzymes are described which distinguish them from their wild-type counterparts. Recombinants containing either mutant thymidine kinase ( RSC -11) or mutant DNA polymerase ( RSC -26), but not both, have been used to investigate the relative contribution of each lesion to resistance and pathogenicity. Although SC16 R9C2 and both recombinants grow as well as does wild-type virus in tissue culture, they are considerably attenuated in vivo, the greatest attenuation of virulence being seen with SC16 R9C2 and RSC -26. With respect to both acyclovir resistance and in vivo growth, the lesions appear to behave synergistically. Cross resistance studies have shown the recombinant RSC -26, which contains mutant DNA polymerase but which evidently expresses wild-type thymidine kinase, to be cross resistant to both 5-iodo-2'-deoxyuridine and 5-trifluoromethyl-2'-deoxyuridine but not to (E)-5-(2-bromovinyl)-2'-deoxyuridine or 9-beta-D-arabinofuranosyladenine.
Molecular cloning and characterization of avian sarcoma virus UR2 and comparison of its transforming sequence with those of other avian sarcoma viruses
Authors: Neckameyer W S , Wang L H
Published: January 2020

Avian sarcoma virus UR2 and its associated helper virus, UR2AV , were molecularly cloned into lambda gtWES X lambda B by using unintegrated viral DNAs. One UR2 and several UR2AV clones were obtained. The UR2 DNA was subsequently cloned into pBR322. Both UR2 and UR2AV DNAs were tested for their biological activity by transfection onto chicken embryo fibroblasts. When cotransfected with UR2AV DNA, UR2 DNA was able to induce transformation of chicken embryo fibroblasts with a morphology similar to that of parental UR2 . UR2 -specific protein with kinase activity and UR2 -specific RNA were detected in the transfected cells. Transforming virus, UR2 ( UR2AV ), was produced from the doubly transfected cells. Five of the six UR2AV clones tested were also shown to be biologically active. The insert of the UR2 DNA clone is 3.4 kilobases in length and contains two copies of the long terminal repeat. Detailed restriction mapping showed that UR2 DNA shared with UR2AV DNA 0.8 kilobases of 5' sequence, including a portion of 5' gag, and 1.4 kilobases of 3' sequence, including a portion of 3' env. The UR2 transforming sequence, ros, is ca. 1.2 kilobases. No significant homology was found between v-ros and the conserved regions of v-src, v-yes, or v- abl . By contrast, a significant homology was found between v-ros and v-fps. The v-fps-related sequence was mapped within a 300-base-pair sequence in the middle of ros.
Expression of xenotropic-like env RNA sequences in normal DBA/2 and NZB mouse tissues
Authors: Lee E J , Kaminchik J , Hankins W D
Published: January 2020

Using a DNA probe prepared from cloned env gene sequences of Friend spleen focus-forming viruses, we detected the differential expression of multiple RNA species in uninfected DBA/2 fibroblasts and in various tissues from adult DBA/2 and NZB mice. The size of the major RNA species detected was estimated to be 24S . The 24S RNA species was enriched in polyadenylate-selected preparations and thus may represent a message for endogenous viral envelope glycoproteins. The viral origin of the 24S RNA was further characterized by its hybridization to DNA probes containing the long terminal repeats of Harvey murine sarcoma virus, mouse mammary tumor virus, or the U3 region of an endogenous xenotropic virus. Although the env-related 24S RNA failed to react with either Harvey murine sarcoma virus or mouse mammary tumor virus long terminal repeat probes, it hybridized well with the xenotropic virus long terminal repeat probe. Therefore, it is likely that the RNA detected with the Friend spleen focus-forming virus env probe reflects transcription of xenotropic envelope sequences in uninfected tissues. Our finding that the level of 24S RNA varied in different organs indicated some tissue specificity in the expression of these xenotropic-like env proteins.
Localization of a type-specific antigenic site on herpes simplex virus type 2 glycoprotein D
Authors: Rawls W E , Balachandran N , Sisson G , Watson R J
Published: January 2020

A herpes simplex virus type 1 strain isolated from a recurrent lesion of the nose reacted with monoclonal antibodies recognizing a type 2-specific site on glycoprotein D but not with monoclonal antibodies recognizing other type 2-specific sites. DNA sequence analysis of the glycoprotein D gene of the isolate revealed a single nucleotide alteration which changed the codon for asparagine to one encoding histidine at amino acid 97 in the protein. Histidine is located at this position in glycoprotein of herpes simplex virus type 2; thus, the monoclonal antibody 17 beta A3 recognizes an epitope located at this region of the protein.
Comparative sequence analysis of rotavirus genomic segment 6--the gene specifying viral subgroups 1 and 2
Authors: Both G W , Siegman L J , Bellamy A R , Ikegami N , Shatkin A J , Furuichi Y
Published: January 2020

Cloned DNA copies of rotavirus genomic segment 6 from simian 11 (subgroup 1) and human strain Wa (subgroup 2) rotaviruses have been used to determine the nucleotide sequences of the gene that determines viral subgroup specificity. Both genomic segments are 1,356 nucleotides in length and possess 5'- and 3'-terminal untranslated regions of 23 and 142 nucleotides, respectively. The inferred amino acid sequence reveals VP6 to be a polypeptide of 397 amino acids in which more than 90% of the amino acid sequence is conserved between the two viruses. There are 34 amino acid changes between the subgroup 1 and 2 polypeptides, most clustered in three regions of the molecule at residues 39 through 62, 80 through 122, and 281 through 315.
Epitopes on foot-and-mouth disease virus outer capsid protein VP1 involved in neutralization and cell attachment
Authors: Baxt B , Morgan D O , Robertson B H , Timpone C A
Published: January 2020

Foot-and-mouth disease virus structural protein VP1 elicits neutralizing and protective antibody and is probably the viral attachment protein which interacts with cellular receptor sites on cultured cells. To study the relationships between epitopes on the molecule related to neutralization and cell attachment, we tested monoclonal antibodies prepared against type A12 virus, isolated A12 VP1, and a CNBr-generated A12 VP1 fragment for neutralization and effect on viral absorption. The antibodies selected for analysis neutralized viral infectivity with varying efficiencies. One group of antibodies caused a high degree of viral aggregation and inhibited the adsorption of virus to cells by 50 to 70%. A second group of antibodies caused little or no viral aggregation but inhibited the adsorption of virus to cells by 80 to 90%. One antibody, which is specific for the intact virion, caused little viral aggregation and had no effect on the binding of virus to specific cellular receptor sites. Thus, at least three antigenic areas on the surface of foot-and-mouth disease virus which were involved in neutralization were demonstrated. One of the antigenic sites appears to have been responsible for interaction with the cellular receptor sites on the surface of susceptible cells.
Heteroduplex analysis of molecular clones of the pathogenic Friend virus complex: Friend murine leukemia virus, Friend mink cell focus-forming virus, and the polycythemia- and anemia-inducing strains of Friend spleen focus-forming virus
Authors: Gonda M A , Kaminchick J , Oliff A , Menke J , Nagashima K , Scolnick E M
Published: January 2020

The pathogenic Friend virus complex is of considerable interest in that, although members of this group are genetically related, they differ markedly in biochemical and biological properties. Heteroduplex mapping of molecular clones of the Friend virus complex, which includes the replication-competent ecotropic Friend murine leukemia virus (F-MuLV) and mink cell focus-forming virus (F-MCF) and replication-defective polycythemia- and anemia-inducing strains of spleen focus-forming virus (SFFVp and SFFVa, respectively), was employed to provide insight into the molecular basis of their relationships. In heteroduplexes of F-MuLV X F-MCF, a major substitution of 0.89 kilobases in the env gene of F-MCF was discerned. Heteroduplexes of SFFVp X F-MuLV or F-MCF and SFFVa X F-MuLV or F-MCF showed several major deletions in the pol gene region and a single major deletion in the 3' half of the env gene region of SFFVp and SFFVa. A major substitution of 0.89 kilobases was mapped to the 5' end of the env deletion of SFFVp and SFFVa in heteroduplexes with F-MuLV, similar to that seen in F-MuLV X F-MCF heteroduplexes. In contrast, this env gene region was totally homologous in F-MCF X SFFVp or SFFVa and SFFVp X SFFVa heteroduplexes. Our results suggest that (i) both SFFVp and SFFVa lack part of the env gene at its 3' end, corresponding to the p15(E) coding region, (ii) major deletions occur in the pol and env genes which account for the replication defectiveness of SFFVp and SFFVa, (iii) minor substitutions occur in the gag gene region of SFFVa that are not present in SFFVp, F-MuLV, or F-MCF, (iv) a major substitution exists in the gp70 region of the env gene between F-MuLV and F-MCF that probably accounts for the differences in their host range specificities, (v) this substitution in F-MCF is identical to the gp70 part of the gp52 coding region of SFFVp and SFFVa, and (vi) heteroduplexes to F-MCF show unambiguously that no additional large substitutions are present in SFFVp or SFFVa that could account for differences in their leukemogenicity.
The gene order for rubella virus structural proteins is NH2-C-E2-E1-COOH
Authors: Oker-Blom C
Published: January 2020

The order of translation in vivo of the genes coding for rubella virus structural proteins was studied in infected B-Vero cells. The proteins were sequentially pulse-chase labeled with [35S]methionine after synchronization of translation initiation with hypertonic salt treatment. A sequential labeling procedure ("window-labeling") to specifically label defined segments of the structural proteins was also used. The labeled proteins were identified by sodium dodecyl sulfate-gel electrophoresis after immunoprecipitation with specific antisera directed against the two virion glycoproteins (E1 and E2a/E2b) and the nucleocapsid (C) protein. The order of translation was found to be NH2-C-E2-E1-COOH. We have previously shown that the structural proteins are synthesized in vitro from a cytoplasmic 24S subgenomic mRNA as a 110,000-dalton (p110) precursor (Oker-Blom et al., J. Virol. 49:403-408, 1984). Here, it is shown that p110 is precipitated with anti-C, anti-E2, and anti-E1 sera, indicating that p110 is the precursor of all three structural proteins. Two major in vitro translation products (Mrs, 66,000 and 62,000) that could represent preterminated polypeptide chains or proteolytic cleavage products were precipitated with anti-C and anti-E2 sera, but not with anti-E1 serum, indicating, in conformity with the in vivo results, that the genes for the C and E2 proteins are adjacent to each other. Using these specific antisera, we have also confirmed the identity of the unglycosylated forms of E1 (Mr, 53,000) and E2 (Mr, 30,000) immunoprecipitated from tunicamycin-treated infected cells.
Nucleotide sequence of an infectious molecularly cloned genome of ground squirrel hepatitis virus
Authors: Seeger C , Ganem D , Varmus H E
Published: January 2020

We have determined the complete nucleotide sequence of an infectious cloned genome of ground squirrel hepatitis virus (GSHV), a nonpathogenic member of the hepadnavirus group. The genome is 3,311 base pairs long and contains the major open reading frames described for the related human and woodchuck hepatitis B viruses (HBV and WHV, respectively). These reading frames include genes for the major structural proteins (the surface and core antigens), unassigned open reading frames (A and B), the longer of which is presumed to encode the viral DNA polymerase, and an open reading frame preceding and continuous with the surface antigen gene. The arrangement of these open reading frames is similar to that encountered in the genomes of HBV and WHV: all of the reading frames are encoded on the same strand, they are positioned in the same fashion with respect to each other, and a large portion (at least 51%) of the genome can be translated in two reading frames. Comparisons of the predicted translational products of the three mammalian hepadnaviruses reveal 78% amino acid homology between the proteins of GSHV and WHV and 43% homology between those of GSHV and HBV. In addition, a perfect direct repeat of 10 to 11 base pairs, separated by ca. 46 to 223 base pairs, is present in the three mammalian viruses and in duck hepatitis B virus; the position of the repeats near the 5' termini of the two strands of virion DNA suggests a role in viral replication.
Physical state and transcription of the cottontail rabbit papillomavirus genome in warts and transplantable VX2 and VX7 carcinomas of domestic rabbits
Authors: Georges E , Croissant O , Bonneaud N , Orth G
Published: January 2020

The physical state and the transcription of the genome of cottontail rabbit papillomavirus (CRPV) in non-virus-producing warts and in the VX2 and VX7 transplantable carcinomas of domestic rabbits were compared. The CRPV DNA present in VX2 and VX7 carcinomas (10 to 20 and 100 to 200 genome equivalents per diploid cell, respectively) was found to be entirely integrated into the cellular DNA, most probably as head-to-tail tandem repeats, in contrast to warts, in which viral DNA (10 to 100 copies per diploid cell) was found only as free, mainly monomeric, molecules. In the VX7 tumor, ca. 50% of the viral DNA molecules were found to be longer than one genome length, indicating that viral DNA rearrangements had occurred. A major viral transcript of 1,250 bases was detected in warts and in VX2 and VX7 carcinomas. Complementary sequences were localized within the E region, the putative transforming region inferred from the nucleotide sequence of the CRPV genome (I. Giri, O. Danos, and M. Yaniv, manuscript in preparation). Analysis of heteroduplexes formed between single-stranded CRPV DNA and polyadenylated RNAs from the VX2 tumor showed that the 1,250-base RNA resulted from the splicing of the sequences corresponding to the open reading frame E6 to those corresponding to the 3' third of E2. A second viral transcript, measuring 2,000 bases, was detected in warts and, in lesser amounts than the 1,250-base species, in VX2 carcinoma, and a 2,100-base RNA was found in VX7 carcinoma. Complementary sequences to these messengers were localized to the same part of the genome as the 1,250-base species and to a contiguous fragment situated upstream. Heteroduplex analysis showed that the 2,000-base species from VX2 carcinoma resulted from the splicing of the sequences corresponding to E6 and E7 to those corresponding to the 3' third of E2. The sequences spliced out upon the maturation of the two messengers of VX2 carcinoma correspond to E1, the two-thirds of E2, and most of E4. Additional transcripts were found in VX7 carcinoma, a major 3,100-base species transcribed from the E region, and several minor species, measuring from 2,400 bases, which all hybridize with a subgenomic fragment contained in the L region encoding the viral capsid polypeptides. This could account for the antiviral antibodies found in animals bearing the VX7 carcinoma.
Molecular basis of the glycoprotein-C-negative phenotype of herpes simplex virus type 1 macroplaque strain
Authors: Draper K G , Costa R H , Lee G T , Spear P G , Wagner E K
Published: January 2020

The basis for the inability of the macroplaque (MP) strain of herpes simplex virus type 1 to express mature glycoprotein C (gC) was examined. RNA transfer (Northern) blot analysis with hybridization probes from the region of the herpes simplex virus type 1 DNA known to encode the gC gene indicated that gC mRNA was produced in MP-infected HeLa cells at levels relative to other mRNAs comparable with that seen in KOS-infected cells. Comparative nucleotide sequence analysis of the gC gene from the MP and KOS strains, coupled with the results of recently reported marker rescue experiments, indicates that the inability of MP to produce gC is due to a frameshift mutation in the gC-coding sequence. Because two different (out-of-phase) open reading frames overlap the gC-coding sequence in the region of the mutation, MP mRNA can encode two gC-related polypeptides. Two polypeptides of the predicted size and precipitable by anti-gC antibodies were produced by in vitro translation of MP mRNA. These polypeptides have not been detected in extracts from infected cells with the same antibodies. Comparative nucleotide sequence analyses led to several corrections in the published sequence for the gC gene and the 17,800-molecular-weight polypeptide gene just to the right in KOS DNA. These relatively minor effects on the predicted amino code sequence of gC are tabulated.
Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus in vivo
Authors: Byrne J A , Oldstone M B
Published: January 2020

Our data show that 1 X 10(7) to 1.5 X 10(7) lymphocytic choriomeningitis virus-specific, H-2-restricted cloned cytotoxic T lymphocytes (CTL) administered intravenously into acutely infected mice totally cleared virus from the spleens (10(4) to 10(5) PFU per spleen reduced to less than 50 PFU per spleen) by 24 h. This activity was genetically restricted in that cloned CTL could reduce titers of infectious virus in syngeneic C57BL/6 mice but not allogeneic BALB/c mice. Dose-response analysis indicated that at least 3 X 10(6) to 5 X 10(6) cloned CTL injected intravenously were needed to reduce significant amounts of infectious virus in the spleens. No infectious virus could be recovered from the spleens for at least 4 days after injection of cloned CTL. Hence, CTL play a major role in elimination of infectious virus from spleens during lymphocytic choriomeningitis virus infection. Our results also indicate that cloned CTL propagated in vitro for long periods of time can mediate a biologically relevant effect in vivo. These cells should be of considerable value in defining the precise manner in which CTL bring about control of viral infection, analyzing lymphocyte trafficking, and the potential use of cloned CTL in immunotherapy against viral disease.
Entry of adenovirus 2 into HeLa cells
Authors: Svensson U , Persson R
Published: January 2020

Adenovirus 2 (Ad2) uncoating was analyzed as the destabilization of virions which renders the parental genome sensitive to DNase treatment. This event demonstrated a strong temperature dependence, and an Arrhenius plot of initial uncoating rates revealed an inflection point at around 16 degrees C. Activation energies of 331 kJ/mol below and 88 kJ/mol above this temperature were obtained for the uncoating process. Penetration of Ad2 through the plasma membrane was completely inhibited by sodium azide, whereas uncoating was only slightly influenced. This indicated that uncoating had already taken place at the outside of the plasma membrane. Incubations of Ad2 with isolated plasma membranes and cell homogenates showed that intact and metabolizing cells were required for uncoating. We further suggest, based on the inhibitory patterns of EDTA, EGTA, dansylcadaverine, and dithiothreitol, that this destabilization of virions follows upon reorganization in the plasma membrane. In the electron microscope the involvement of coated vesicles was shown for the initial uptake of virions, possibly followed by the engagement of acidic vesicles as judged from the effects of lysosomotropic agents on gene expression. The vectorial transport of virions from the plasma membrane to the nucleus was not affected by reagents interfering with the cytoskeletal system. Consequently, we propose that Ad2 virions are internalized by adsorptive endocytosis.
Cloning and analysis of integrated hepatitis virus sequences from a human hepatoma cell line
Authors: Shaul Y , Ziemer M , Garcia P D , Crawford R , Hsu H , Valenzuela P , Rutter W J
Published: January 2020

We report here the isolation by molecular cloning and the analysis by heteroduplex and restriction enzyme mapping of seven distinct DNA fragments containing hepatitis B virus (HBV) sequences from genomic DNA of the PLC/PRF/5 human liver carcinoma cell line (the Alexander cell). No intact full-length HBV genomes were present. Three different patterns of organization of HBV fragments were detected. These included two linear fragments without detectable rearrangement, three other HBV fragments with internal deletions, and two HBV fragments containing long inverted duplications. HBsAg sequences are preferentially included in the integrated fragment, whereas the core gene is preferentially eliminated. Several of the integrated HBV fragments might act as templates for the synthesis of functional HBsAg mRNA, whereas only one clone could produce a full core antigen transcript.
Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC
Authors: Serafini-Cessi F , Dall'Olio F , Pereira L , Campadelli-Fiume G
Published: January 2020

Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983).
Mapping and expression of a human cytomegalovirus major viral protein
Authors: Davis M G , Mar E C , Wu Y M , Huang E S
Published: January 2020

We constructed a DNA fragment map of low-passage Towne strain cytomegalovirus by analyzing cross-blot hybridization and hybridizations of isolated recombinant clones. The abundant late transcripts were located on this map by hybridization of labeled total RNA of virus-infected cells to blotted DNA fragments. The most abundant late transcript, carried by the 11.7-kilobase EcoRI fragment (EcoRI-G), was precisely mapped. The EcoRI fragment was fragmented and subcloned in a plasmid carrying simian virus 40 sequences (pSV-OH, constructed by Chi-Bom Chae, Department of Biochemistry, University of North Carolina, Chapel Hill). One resulting recombinant plasmid, pHD713SV2, was transferred to simian virus 40-transformed monkey kidney cells (COS-1) by DNA transfection. Synthesis of a cytomegalovirus-specific 67-kilodalton protein was detected in these cells by reaction of blotted proteins with virus-specific monoclonal antibody. The 67-kilodalton protein is a major phosphorylated protein found in virions; it is not glycosylated. The location of the gene for this 67-kilodalton protein is therefore assigned to the center of the L-unique region of human cytomegalovirus, at 0.37 to 0.39 map units.
Molecular genetic characterization of the RD-114 gene family of endogenous feline retroviral sequences
Authors: Reeves R H , O'Brien S J
Published: January 2020

RD-114 is a replication-competent, xenotropic retrovirus which is homologous to a family of moderately repetitive DNA sequences present at ca. 20 copies in the normal cellular genome of domestic cats. To examine the extent and character of genomic divergence of the RD-114 gene family as well as to assess their positional association within the cat genome, we have prepared a series of molecular clones of endogenous RD-114 DNA segments from a genomic library of cat cellular DNA. Their restriction endonuclease maps were compared with each other as well as to that of the prototype-inducible RD-114 which was molecularly cloned from a chronically infected human cell line. The endogenous sequences analyzed were similar to each other in that they were colinear with RD-114 proviral DNA, were bounded by long terminal redundancies, and conserved many restriction sites in the gag and pol regions. However, the env regions of many of the sequences examined were substantially deleted. Several of the endogenous RD-114 genomes contained a novel envelope sequence which was unrelated to the env gene of the prototype RD-114 env gene but which, like RD-114 and endogenous feline leukemia virus provirus, was found only in species of the genus Felis, and not in other closely related Felidae genera. The endogenous RD-114 sequences each had a distinct cellular flank which indicates that these sequences are not tandem but dispersed nonspecifically throughout the genome. Southern analysis of cat cellular DNA confirmed the conclusions about conserved restriction sites in endogenous sequences and indicated that a single locus may be responsible for the production of the major inducible form of RD-114.
La Crosse virions contain a primer-stimulated RNA polymerase and a methylated cap-dependent endonuclease
Authors: Patterson J L , Holloway B , Kolakofsky D
Published: January 2020

Purified La Crosse virions in vitro were found to transcribe their negative polarity (-)RNA genomes. This polymerase activity was stimulated by oligonucleotides such as (A)nG, cap analogs such as m7GpppAm, and natural mRNAs such as alfalfa mosaic virus RNA 4. For (A)nG- and alfalfa mosaic virus RNA 4-stimulated reactions, evidence is presented that these RNAs stimulate activity by acting as primers for viral transcription. The cap analogs appear to stimulate activity via an alternative mechanism. Purified La Crosse virions were also found to contain an endonuclease which specifically cleaves alfalfa mosaic virus RNA 4 when this RNA contains a methylated cap group.
Lysis of dengue virus-infected cells by natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity
Authors: Kurane I , Hebblewaite D , Brandt W E , Ennis F A
Published: January 2020

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.
Mapping in vivo initiation sites of RNA transcription and determining their relative use
Authors: Kessler M , Aloni Y
Published: January 2020

Runoff transcripts were generated on viral transcriptional complexes cleaved with restriction enzymes and incubated in vitro with [alpha-32P]UTP under pulse-chase conditions. As viral transcriptional complexes in vitro elongated the nascent RNA preinitiated in vivo, size analysis by gel electrophoresis of the runoff transcripts allowed identification of the in vivo initiation sites. Moreover, scanning the intensities of the runoff bands as they appeared in the autoradiogram of the gel allowed determination of the relative use of these sites. A model system in which the initiation sites of simian virus 40 late RNA were identified and their relative use determined is presented.
Microinjection of anti-interferon antibodies into cells does not inhibit the induction of an antiviral state by interferon
Authors: Arnheiter H , Zoon K C
Published: January 2020

Affinity-purified polyclonal antibodies directed against human lymphoblastoid interferon (IFN), Escherichia coli-derived human IFN-alpha 2, or two synthetic fragments of human IFN-alpha 1 all neutralized the antiviral activity of human alpha IFNs when added to the culture medium of MDBK cells together with IFNs. However, when these antibodies were microinjected into the cytoplasm or the nucleus of cells, subsequent treatment of the cells with IFNs induced full protection against vesicular stomatitis virus. This suggests that IFNs themselves need not act in the cytoplasmic compartment or the nucleus to induce an antiviral state.
Core antigen and antibody in woodchucks after infection with woodchuck hepatitis virus
Authors: Ponzetto A , Cote P J , Ford E C , Purcell R H , Gerin J L
Published: January 2020

The woodchuck hepatitis virus is a naturally occurring hepatitis B-like virus that infects the eastern woodchuck. Direct immunofluorescence staining for woodchuck hepatitis virus core antigen in liver biopsies demonstrated the presence of this antigen in 14 of 17 chronically infected woodchucks, and in 8 of 10 woodchucks undergoing acute infections. Fluorescent localization of woodchuck hepatitis virus core antigen was typically cytoplasmic, and this was confirmed further by electron microscopy. Experimental infection with woodchuck hepatitis virus was achieved in four of four woodchucks inoculated with serum from chronic carrier woodchucks. All infected animals developed a self-limited disease characterized by seroconversion to antibodies against the major viral antigens (core and surface antigens); naturally acquired acute infection demonstrated a similar course. A chimpanzee seronegative for all markers of hepatitis B virus developed a subclinical infection after inoculation with woodchuck hepatitis virus.
Characterization of mRNAs that map in the BglII N fragment of the herpes simplex virus type 2 genome
Authors: Jenkins F J , Howett M K
Published: January 2020

The BglII N DNA fragment (0.580 to 0.620 map units) of herpes simplex virus type 2 strain (333) is of interest because of its transforming potential. This fragment contains either partial or the complete coding sequences for nine mRNA transcripts that can be detected during a lytic infection. Subclones of the BglII N DNA fragment were generated in plasmid vectors, and the approximate locations of the mRNA transcripts were mapped by RNA blot hybridization technology. Precise 5' or 3' ends (or both) of these mRNA species were determined by S1 nuclease mapping, using the BglII N subclones as DNA probes. At least four mRNA transcripts are fully encoded in the BglII N fragment. The coding regions for all of the mRNA transcripts are densely packaged along the BglII N fragment with less than 150 base pairs between neighboring mRNA ends. Analysis of both neutral and alkaline gels failed to reveal the presence of any detectable introns. This manuscript reports a detailed transcription map for this region.
Adenovirus cyt+ locus, which controls cell transformation and tumorigenicity, is an allele of lp+ locus, which codes for a 19-kilodalton tumor antigen
Authors: Subramanian T , Kuppuswamy M , Mak S , Chinnadurai G
Published: January 2020

The early region E1b of adenovirus type 2 (Ad2) codes for two major tumor antigens of 53 and 19 kilodaltons (kd). The adenovirus lp+ locus maps within the 19-kd tumor antigen-coding region (G. Chinnadurai, Cell 33:759-766, 1983). We have now constructed a large-plaque deletion mutant (dl250) of Ad2 that has a specific lesion in the 19-kd tumor antigen-coding region. In contrast to most other Ad2 lp mutants (G. Chinnadurai, Cell 33:759-766, 1983), mutant dl250 is cytocidal (cyt) on infected KB cells, causing extensive cellular destruction. Cells infected with Ad2 wt or most of these other Ad2 lp mutants are rounded and aggregated without cell lysis (cyt+). The cyt phenotype of dl250 resembles the cyt mutants of highly oncogenic Ad12, isolated by Takemori et al. (Virology 36:575-586, 1968). By intertypic complementation analysis, we showed that the Ad12 cyt mutants indeed map within the 19-kd tumor antigen-coding region. The transforming potential of dl250 was assayed on an established rat embryo fibroblast cell line, CREF, and on primary rat embryo fibroblasts and baby rat kidney cells. On all these cells, dl250 induced transformation at greatly reduced frequency compared with wt. The cells transformed by this mutant are defective in anchorage-independent growth on soft agar. Our results suggest that the 19-kd tumor antigen (in conjunction with E1a tumor antigens) may play an important role in the maintenance of cell transformation. Since we have mapped the low-oncogenic or nononcogenic Ad12 cyt mutants within the 19-kd tumor antigen-coding region, our results further indicate that the 19-kd tumor antigen also directly or indirectly plays an important role in tumorigenesis of Ad12. Our results show that the cyt+ locus is an allele of the lp+ locus and that the cyt phenotype may be the result of mutations in specific domains of the 19-kd tumor antigen.
Effect on transformation of mutations in the early region 1b-encoded 21- and 55-kilodalton proteins of adenovirus 5
Authors: Babiss L E , Fisher P B , Ginsberg H S
Published: January 2020

It is well established that the adenovirus 5 genes responsible for the initiation and maintenance of the transformed cell reside in the early region 1a and 1b genes, but it remains unclear how the polypeptides encoded in these genes mediate their functions. To probe the function of the early region 1b-encoded 55- and 21-kilodalton (kd) polypeptides during this process, a series of viral mutants was engineered so that they contained deletions or insertions at 5.4, 5.7, 7.9, or 9.6 map units. By means of either an overlap recombination procedure involving H5dl314 (delta 3.7 to 4.6 map units) cleaved with ClaI, or a marker rescue procedure involving H5dl312 (delta 1.2 to 3.8 map units), viral mutants were isolated by their ability to produce plaques on KB cell line 18 cells, which constitutively express only viral early region 1b functions. DNA sequence analysis confirmed that the series of mutants generated differed in their abilities to express the 21- or the 55-kd polypeptides, or both. Upon infection of cloned rat embryo fibroblast cells with viruses containing mutations affecting the 55-kd protein, the transformation frequency decreased as the size of the predicted truncated polypeptide decreased. Although all of the foci generated by the 55-kd protein mutants were indistinguishable from the foci induced by wild-type virus, they displayed an inefficient ability to grow in soft agar, again in relation to the size of the truncated polypeptide. In contrast, if cloned rat embryo fibroblast cells were transfected with viral DNA, the defectiveness in transformation observed after infection with virions was not as dramatic. However, all of the viruses containing 21-kd mutations were transformation defective, regardless of the mode by which the viral nucleic acid was introduced into the cell.