bluuebunny/crossref_metadata_2025 huggingface dataset:
Deletion mutants in the gene encoding the herpes simplex virus type 1 immediate-early protein ICP0 exhibit impaired growth in cell culture
Authors: Sacks W R , Schaffer P A
Published: January 2020

We report the construction and characterization of deletion mutants in the herpes simplex virus type 1 gene encoding the immediate-early protein ICP0. In the event that ICP0 proved to play an essential role in virus replication, ICP0-transformed Vero cells were generated to serve as permissive hosts for such mutants. Two mutants, dlX0.7 and dlX3.1, were isolated in these cells by a marker rescue-transfer procedure involving the rescue of an ICP4 deletion mutant and the simultaneous insertion of a linked deletion in the ICP0 gene. Mutant dlX0.7 contained a 700-base-pair deletion in both copies of ICP0. The deletion lay entirely within the transcript specified by the gene. dlX0.7 induced the synthesis of an ICP0-specific mRNA that was approximately 0.7 kilobases smaller than the corresponding mRNA specified by wild-type virus. The 3.1-kilobase deletion in both copies of the ICP0 gene in mutant dlX3.1 removed the majority of the transcriptional-regulatory signals and coding sequences, retaining only sequences at the 3' end of the gene. As expected, no ICP0-specific mRNA was detected in dlX3.1-infected Nero cells (G418-resistant Vero cells). Both mutants grew in all cells tested, although their burst sizes were 10- to 100-fold lower than that of wild-type virus. Although the plaque sizes of dlX0.7 and dlX3.1 were equally small on Nero and ICP0-transformed cells, the plating efficiency of the mutants was 15- to 50-fold greater on ICP0-transformed cells than on Nero cells. The mutants exhibited modest interference with the growth of wild-type virus in mixed infections, an effect that was abolished by UV irradiation of the mutants, implying that interference required viral gene expression. Polypeptide profiles generated by the mutants in Nero cells were qualitatively similar to that of wild-type virus. Quantitatively, only slight reductions in the levels of certain late viral polypeptides were observed, a phenomenon also borne out by analysis of viral glycoproteins. Both mutants induced the synthesis of significant, although reduced, levels of viral DNA relative to wild-type virus. Taken together, the results demonstrate that ICP0 is not essential for productive infection in cell culture but that this protein plays a significant role in viral growth, as indicated by the impaired abilities of the mutants to replicate.
Myristylation is required for intracellular transport but not for assembly of D-type retrovirus capsids
Authors: Rhee S S , Hunter E
Published: January 2020

The role of myristylation, a fatty acid modification of nascent polypeptides, in the assembly and intracellular transport of D-type retroviral capsids was investigated through the use of oligonucleotide-directed mutagenesis. Myristic acid is normally esterified through an amide linkage to a glycine residue at the amino terminus of the Mason-Pfizer monkey virus gag gene products. Mutant pA-1, which has a codon for valine substituted for that of the normally myristylated glycine, is completely noninfectious. While the mutant gag polyprotein precursors are synthesized at normal levels, they are not myristylated and are not cleaved to the mature virion proteins. No extracellular virus particles are released from mutant pA-1-infected cells, but intracytoplasmic A-type particles (capsids) accumulate in the cytoplasm. Since none of the intracellular capsids can be found associated with the plasma membrane, these results strongly suggest that myristylation is a critical signal for intracytoplasmic transport of completed viral capsids to their normal site of budding and release.
Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA
Authors: Pirisi L , Yasumoto S , Feller M , Doniger J , DiPaolo J A
Published: January 2020

Human keratinocytes and fibroblasts isolated from foreskin were transformed by transfection with recombinant human papillomavirus type 16 (HPV16) DNA. The transformed cells exhibited an extended (fibroblasts) or indefinite (keratinocytes) life-span compared with that of normal controls. In addition, HS27, a human fibroblast cell line previously transfected with origin-defective simian virus 40, was successfully transfected. HPV16 sequences were stably maintained in the cells, and extensive amplification and rearrangements occurred with continuous culturing. Moreover, both fibroblasts and keratinocytes expressed several specific HPV16 mRNAs. Because HPV16-transfected cells had viral transcripts and because transfection with the vector alone did not extend the life-span of the cells, it is likely that the virus was responsible for the indefinite life-span. Transfected fibroblast and keratinocyte lines will be useful for investigating the molecular biology of HPV16 and the interactions between the viral DNA and the human genome. Moreover, transfected keratinocytes provide a model for analyzing the effects of HPV16 on the differentiation properties of human epithelial cells.
Genetic determinants of neoplastic diseases induced by a subgroup F avian leukosis virus
Authors: Simon M C , Neckameyer W S , Hayward W S , Smith R E
Published: January 2020

Two subgroup F avian leukosis viruses, ring-necked pheasant virus (RPV) and RAV-61, were previously shown to induce a high incidence of a fatal proliferative disorder in the lungs of infected chickens. These lung lesions, termed angiosarcomas, appear rapidly (4 to 5 weeks after infection), show no evidence of proto-oncogene activation by proviral integration, and are not induced by avian leukosis viruses belonging to other subgroups. To identify the viral sequences responsible for induction of these tumors, we constructed recombinant viruses by exchanging genomic segments of molecularly cloned RPV with those of a subgroup A leukosis virus, UR2AV. The ability to induce rapid lung tumors segregated only with the env sequences of RPV; the long terminal repeat of RPV was not required. However, recombinants carrying both env and long terminal repeat sequences of RPV induced lung tumors with a shorter latency. In several cases, recombinant viruses exhibited pathogenic properties differing from those of either parental virus. Recombinants carrying the gag-pol region of RPV and the env gene of UR2AV induced a high incidence of a muscle lesion termed infiltrative intramuscular fibromatosis. One recombinant, EU-8, which carries the gag-pol and LTR sequences of RPV, and the env gene of UR2AV, induced lymphoid leukosis after an unusually short latent period. The median time of death from lymphoid leukosis was 6 to 7 weeks after infection with EU-8 compared with approximately 5 months for UR2AV.
Methylation as a modulator of expression of human immunodeficiency virus
Authors: Bednarik D P , Mosca J D , Raj N B
Published: January 2020

When Vero or murine cells were stably transfected with the human immunodeficiency virus (HIV) long terminal repeat (LTR) that directs the chloramphenicol acetyltransferase (CAT) gene (pU3R-III-CAT), expression was suppressed. Treatment with the nucleoside analog 5-azacytidine (5-azaC) restored CAT expression. S1 nuclease analysis and a nuclear run-on assay demonstrated that activation of the latent HIV LTR by 5-azacytidine occurred at the transcriptional level. Southern blot analysis demonstrated that this activation was due to the demethylation of cytosine residues in the LTR enhancer. Thus, the HIV LTR appears to be susceptible to transcriptional inactivation by methylation, a process that is proposed to play a modulatory role in viral latency.
Isolation and characterization of phosphonoacetic acid-resistant mutants of human cytomegalovirus
Authors: D'Aquila R T , Summers W C
Published: January 2020

Mutants of the human cytomegalovirus (HCMV) that were 6- to 13-fold more resistant to phosphonoacetic acid than the wild-type HCMV (Towne) were isolated. Extracts from mycoplasma-free, mutant-infected cells had phosphonoacetate-resistant DNA polymerase activity in vitro. This strongly suggests that the selected mutations are in the HCMV DNA polymerase genes of these viruses.
Proteolysis of the p220 component of the cap-binding protein complex is not sufficient for complete inhibition of host cell protein synthesis after poliovirus infection
Authors: Bonneau A M , Sonenberg N
Published: January 2020

Infection of cells with poliovirus results in the complete shutoff of host protein synthesis. It is presumed that proteolysis of the p220 component of the cap-binding protein complex that is required for the translation of host mRNAs is responsible for the shutoff phenomenon. In this paper, we show that when cells are infected with poliovirus in the presence of guanidine or 3-methylquercetin, both inhibitors of poliovirus replication, complete cleavage of p220 occurs by 3.5 h postinfection. However, under these conditions only 55 to 77% of host protein synthesis is suppressed. Results obtained with extracts prepared from poliovirus-infected cells were similar to those obtained in vivo. These results suggest that complete inhibition of host protein synthesis after poliovirus infection requires at least one event in addition to proteolysis of p220. Thus, proteolysis of p220 is probably necessary but not sufficient for total suppression of host protein synthesis after poliovirus infection.
Lymphoid cells in the spleens of woodchuck hepatitis virus-infected woodchucks are a site of active viral replication
Authors: Korba B E , Wells F , Tennant B C , Cote P J , Gerin J L
Published: January 2020

Lymphoid cells were purified from the spleens of 15 woodchucks and examined for the presence of woodchuck hepatitis virus (WHV). Lymphoid cells from the spleens of eight of eight chronically infected animals contained high levels of WHV RNA and DNA. A 100-fold lower level of WHV DNA was found in the spleen from one of five animals that had recovered from acute WHV infections 2 years before this analysis. No WHV nucleic acids were observed in either of two uninfected animals. WHV DNA patterns in the lymphoid cells from the spleens of the chronically infected animals, which included the presence of single-stranded DNA and RNA-DNA hybrid molecules, were identical to those observed in WHV-infected liver. WHV DNA in these cells was present in intact, 27-nm core particles which also contained the endogenous DNA polymerase activity. These results indicate that the spleen is a site of active WHV DNA replication and is most likely a major source of WHV-infected cells in the circulating lymphoid cell population.
Inhibition of reovirus type 3 binding to host cells by sialylated glycoproteins is mediated through the viral attachment protein
Authors: Pacitti A F , Gentsch J R
Published: January 2020

The interaction of mammalian reoviruses with sialylated glycoproteins was studied and found to be highly serotype specific in that attachment of type 3 Dearing reovirus to murine L cell receptors could be strongly inhibited by bovine submaxillary mucin (BSM), fetuin, and alpha 1 acid glycoprotein, albeit at different efficiencies, whereas attachment of type 1 Lang reovirus was inhibited only by fetuin. We subsequently demonstrated, by using reassortants between type 3 and 1 reoviruses, that inhibition of reovirus attachment to cell receptors was specified by the viral attachment protein gene S1. Using a solid-phase binding assay, we further demonstrated that the ability of reovirus type 3 or reassortant 1HA3 and the inability of reovirus type 1 or reassortant 3HA1 to bind avidly to BSM was a property of the viral S1 genome segment and required the presence of sialic acid residues on BSM oligosaccharides. Taken together, these results demonstrated that there is a serotype-specific difference in the ability of the reovirus attachment protein, sigma 1, to interact with sialylated oligosaccharides of glycoproteins. Interaction of reovirus type 3 with sialylated oligosaccharides of BSM is dramatically affected by the degree of O-acetylation of their sialic acid residues, as indicated by the findings that chemical removal of O-acetyl groups stimulated reovirus type 3 attachment to BSM, whereas preferential removal of residues lacking or possessing reduced amounts of O-acetyl groups per sialic acid molecule with Vibrio cholerae sialidase abolished binding. We also demonstrated that BSM was 10 times more potent in inhibiting attachment of infectious reovirus to L cells than was V. cholerae-treated BSM. The results are consistent with the hypothesis that sialylated oligosaccharides on host cells or erythrocytes may act as binding sites or components of binding sites for type 3 reovirus through a specific interaction with the virus attachment protein.
Herpes simplex virus infection generates large tandemly reiterated simian virus 40 DNA molecules in a transformed hamster cell line
Authors: Matz B
Published: January 2020

When the simian virus 40 (SV40)-transformed Syrian hamster cell line Elona is infected with herpes simplex virus type 1, an excessive amplification of SV40-specific DNA sequences occurs. Analysis of total DNA from herpes simplex virus-infected cells revealed that amplified DNA sequences were present predominantly in a high-molecular-weight form, consisting of a tandem array of many unit-length SV40 DNA molecules. Repeat units of amplified DNA were found to be very similar to standard SV40 DNA as was shown by restriction analyses, except for a small deletion close to the origin of replication, which could also be detected in the chromosomal DNA of uninfected cells. A procedure, devised for selective enrichment of amplified SV40 DNA molecules from the bulk of cellular and herpesviral DNA, allowed molecular cloning of single repeat units and nucleotide sequence analysis of the relative genomic region.
A standardized nomenclature for endogenous mouse mammary tumor viruses
Authors: Kozak C , Peters G , Pauley R , Morris V , Michalides R , Dudley J , Green M , Davisson M , Prakash O , Vaidya A
Published: January 2020

We propose a revised standardized nomenclature for endogenous mouse mammary tumor viruses based on characterization by molecular cloning techniques and genetic segregation data.
Replication of latent Epstein-Barr virus genomes in Raji cells
Authors: Adams A
Published: January 2020

The replication of the 50 to 60 latent, predominantly extrachromosomal, Epstein-Barr virus genomes maintained by the Burkitt-lymphoma-derived Raji cell line was investigated by using a Meselson-Stahl density transfer approach. Samples of DNA isolated from cells cultivated for different periods in bromodeoxyuridine-supplemented medium were fractionated according to density, and the distribution of viral and cellular DNAs among the heavy-, hybrid-, and light-density species was quantitated. The results indicate that the majority of latent Epstein-Barr virus DNA plasmids each replicate once during the cell cycle.
DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome
Authors: Jones E V , Puckett C , Moss B
Published: January 2020

Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A.
Evolution of pseudorabies virions containing genomes with an invertible long component after repeated passage in chicken embryo fibroblasts
Authors: Lomniczi B , Gielkens A , Csobai I , Ben-Porat T
Published: January 2020

The genome of pseudorabies virus consists of two components, short (S) and long (L). Only the S component is bracketed by inverted repeats, and only the S component inverts itself relative to the L component, giving rise to two isomeric forms of the genome. An attenuated vaccine strain of pseudorabies virus (Norden), however, has a genome which is found in four isomeric forms (B. Lomniczi, M. L. Blankenship, and T. Ben-Porat, J. Virol. 49:970-979, 1984). To determine the basis for the atypical structure of the genome of the Norden strain, we examined more than 40 field isolates of pseudorabies virus; all contained genomes in which the L component was fixed in only one orientation relative to the S component. Several independently generated vaccine strains which have been passaged extensively in chicken embryos and chicken embryo fibroblast (CEF) cell cultures were also analyzed; they possessed an invertible L component. Furthermore, emergence of pseudorabies virus variants with an invertible L component was observed after passage of the virus in CEF, but not in rabbit kidney or pig kidney, cells. The invertibility of the L component was associated consistently with a translocation of sequences from the left end of the genome to a position next to the inverted repeat sequence of the S component. Three observations indicate that genomes with an invertible L component (and the translocation) have a selective growth advantage over standard pseudorabies virus when grown in CEF. The proportion of virions with such genomes does not increase linearly as would be expected if the translocation events occurred repeatedly, most genomes eventually experiencing the translocation. Instead, after a lag, the proportion of such virions in the population increases relatively rapidly. The genome structures that are generated upon independent passage in CEF of each virion population were relatively homogeneous. Some heterogeneity was observed at relatively early stages of the emergence of the genomes carrying the translocation; at later stages, virions with genomes with a specific size translocation predominated in the virus population. Parallel passages in CEF of the same pseudorabies virus strain resulted in the emergence of populations of virions with genomes with different size translocations. However, in each of the passaged populations of virions the majority of virions had genomes with the same size translocation. The most likely interpretation of these results is that virions with genomes carrying the translocations that emerge upon passage of the virus in CEF have a selective advantage when grown in these cells.
Stimulation of expression of the human endogenous retrovirus genome by female steroid hormones in human breast cancer cell line T47D
Authors: Ono M , Kawakami M , Ushikubo H
Published: January 2020

Expression of the human endogenous retrovirus genome HERV-K, homologous to mouse mammary tumor virus, was investigated in cultured human tumor cells. In several cell lines, the HERV-K genome was expressed as an 8.8-kilobase poly(A)+ RNA which appeared to be a full-size transcript of this genome. In the human breast cancer cell line T47D, stimulation of HERV-K genome expression by progesterone was observed after estradiol treatment.
Oligomerization and origin DNA-binding activity of simian virus 40 large T antigen
Authors: Runzler R , Thompson S , Fanning E
Published: January 2020

Simian virus 40 (SV40) large tumor antigen (T antigen) exists in multiple molecular forms, some of which are separable by zone velocity sedimentation of soluble extracts from infected monkey cells. Three subclasses of this antigen from SV40-infected monkey cells have been separated and characterized: the 5S, 7S, and 14S forms. Newly synthesized T antigen occurs primarily in the 5S form. Chemical cross-linking provided evidence that the 14S form is primarily a tetramer, whereas the 5S and 7S forms could not be cross-linked into oligomers. The DNA-binding properties of each subclass were investigated after immunopurification. The affinities of the three forms for SV40 DNA and for a synthetic 19-base-pair sequence from binding site I are very similar (equilibrium dissociation constant [KD], 0.3 to 0.4 nM). The specific activity of DNA binding was greatest for the 5S and 7S subclasses and least for the 14S subclass. Moreover, the specific activity of the 5S and 7S subclasses increased sharply at about 40 h after infection, whereas the activity of the 14S subclass was maintained at a constant low level throughout infection. A model relating oligomerization and DNA binding of T antigen in infected cells is presented.
Posttranslational processing of the Epstein-Barr virus-encoded p63/LMP protein
Authors: Mann K P , Thorley-Lawson D
Published: January 2020

In this paper we describe the posttranslational processing of the p63/LMP (latent membrane protein) encoded by Epstein-Barr virus in transformed B cells. Specifically, we show that after synthesis, free LMP disappeared with a half-life of about 0.5 h. This was caused by the association of LMP with an insoluble complex. All detectable LMP in the plasma membrane was insoluble. This interaction was resistant to nondenaturing detergents but readily dissociated with 8 M urea or by boiling in 0.5% sodium dodecyl sulfate, suggesting that LMP may be associated with cytoskeletal elements. Most of the Nonidet P-40-insoluble LMP was phosphorylated (ppLMP) primarily on serine but also on threonine residues. No phosphotyrosine was detected. Furthermore, greater than 90% of the ppLMP resided in the Nonidet P-40-insoluble fraction, suggesting a strong correlation between complexing and phosphorylation. Additionally, ppLMP was found to be associated with a 53,000-molecular-weight phosphoprotein (pp53) of unknown origin. Finally, LMP turned over extremely rapidly, with a half-life of about 2 h. Taken together, these properties suggest that although LMP falls broadly within the category of phosphorylated, cytoskeleton-associated oncoproteins, it is nevertheless clearly different from any previously described member of this family.
Nerve growth factor deprivation results in the reactivation of latent herpes simplex virus in vitro
Authors: Wilcox C L , Johnson E M
Published: January 2020

Primary sympathetic neuronal cultures were maintained for up to 5 weeks after inoculation with herpes simplex virus (HSV) without evidence of viral infection. Upon deprivation of nerve growth factor, the cultures produced infectious HSV, indicating that the cultures harbored latent HSV. This study demonstrates a function of nerve growth factor in the maintenance of HSV latency.
Herpes simplex virus virion stimulatory protein mRNA leader contains sequence elements which increase both virus-induced transcription and mRNA stability
Authors: Blair E D , Blair C C , Wagner E K
Published: January 2020

To investigate the role of 5' noncoding leader sequence of herpes simplex virus type 1 (HSV-1) mRNA in infected cells, the promoter for the 65,000-dalton virion stimulatory protein (VSP), a beta-gamma polypeptide, was introduced into plasmids bearing the chloramphenicol acetyltransferase (cat) gene together with various lengths of adjacent viral leader sequences. Plasmids containing longer lengths of leader sequence gave rise to significantly higher levels of CAT enzyme in transfected cells superinfected with HSV-1. RNase T2 protection assays of CAT mRNA showed that transcription was initiated from an authentic viral cap site in all VSP-CAT constructs and that CAT mRNA levels corresponded to CAT enzyme levels. Use of cis-linked simian virus 40 enhancer sequences demonstrated that the effect was virus specific. Constructs containing 12 and 48 base pairs of the VSP mRNA leader gave HSV infection-induced CAT activities intermediate between those of the leaderless construct and the VSP-(+77)-CAT construct. Actinomycin D chase experiments demonstrated that the longest leader sequences increased hybrid CAT mRNA stability at least twofold in infected cells. Cotransfection experiments with a cosmid bearing four virus-specified transcription factors (ICP4, ICP0, ICP27, and VSP-65K) showed that sequences from -3 to +77, with respect to the viral mRNA cap site, also contained signals responsive to transcriptional activation.
Human papillomavirus types 6 and 11 mRNAs from genital condylomata acuminata
Authors: Chow L T , Nasseri M , Wolinsky S M , Broker T R
Published: January 2020

We have identified and mapped a number of RNA species of human papillomavirus types 6 and 11 from condylomata acuminata by the electron microscopic R-loop technique. Each of the early (E)- and late (L)-region open reading frames (ORFs) deduced from the DNA sequences was represented in one or more transcripts. In addition, RNA species that could encode the modulator of DNA replication and the repressor of transcription, functions recently identified in the genetically similar bovine papillomavirus type 1, were also detected. Some ORFs were 5' proximal in one or more transcripts, whereas others were not 5' proximal in any species, suggesting that internal initiation of translation might be required to gain access to these latter ORFs. Virtually all transcripts had their 5' ends located in the E region and were polyadenylated at one of two sites, i.e., at the end of the E region or at the end of the L region. The great majority of the RNAs were derived from the E region of the genome, with one species approximately 50 to 100 times more abundant than the others. For most of the RNAs, the 5' end mapped near nucleotide 700; minor populations had 5' ends near nucleotide 100 or 1200. By correlating our mapping data with the genomic DNA sequences as well as available RNA structures and cDNA sequences of several papillomaviruses, we predict a number of mRNA splice donor and acceptor sites and suggest that the papillomaviruses have sophisticated usage of ORFs through alternative promoters, mRNA splice sites, and polyadenylation sites.
Physical Analysis of the
Campoletis sonorensis
Virus Multipartite Genome and Identification of a Family of Tandemly Repeated Elements
Authors: Theilmann David A. , Summers Max D.
Published: January 2020

This report is an analysis of cross-hybridizing sequences found within the 28 superhelical (SH) DNAs of the multipartite genome of the polydnavirus
Campoletis sonorensis
virus (CsV). A Southern cross-blot hybridization analysis showed that the majority of CsV
Eco
RI restriction fragments cross-hybridize to multiple
Eco
RI fragments. These sequence homologies were analyzed by hybridizing recombinant clones of the CsV SH DNAs B, H, M, and O
1
to Southern blots of undigested CsV DNA, using different hybridization stringencies. The results indicated that homologous regions among the SH DNAs include closely related sequences that are detectable under stringent conditions and related but more diverged sequences which are only detectable under reduced stringencies. A sequence that hybridized to the majority of the CsV SH DNAs was identified and subcloned from the SH DNAs O
1
, H, and B. Nucleotide sequence data revealed that these homologous regions contained a family of imperfectly conserved repeated elements. These repeat elements were arranged singly or in direct tandem arrays and had an average length of 540 base pairs. Within the sequenced regions that contained the repeated elements six putative open reading frames were identified. These results show that the CsV genome consists of SH DNAs with complex sequence interrelationships that may have arisen due to multiple recombinational events.
The dodecahedral framework of the bacteriophage phi 6 nucleocapsid is composed of protein P1
Authors: Ktistakis N T , Lang D
Published: January 2020

The outer layer of the bacteriophage phi 6 nucleocapsid (NC) was removed by EDTA and reassociated with the core in the presence of Ca2+ or Mg2+. The core was relatively inaccessible to trypsin digestion, was composed of protein P1, and was in the dodecahedral framework reported previously. (H.T. Steely, Jr., and D. Lang, J. Virol. 51:479-483, 1984; Y. Yang and D. Lang, J. Virol. 51:484-488, 1984). The double-stranded RNA genome became RNase sensitive after EDTA treatment of the nucleocapsid.
Genome segment reassortment between two serotypes of bluetongue virus in a natural host
Authors: Stott J L , Oberst R D , Channell M B , Osburn B I
Published: January 2020

The occurrence of genome segment reassortment between two antigenically related orbiviruses was demonstrated in cattle. Individual virus clones were isolated by cell culture plaque assays directly from the blood of a calf infected with two serotypes of bluetongue virus. The majority (89%) of progeny viruses isolated from the calf represented reassortant viruses. A minimum of six genome segments participated in reassortment, with 16 unique reassortant constellations being identified. Such genome segment reassortment between unique, though antigenically related, orbiviruses has undoubtedly played a major role in generating the extensive phenotypic and genotypic diversity that is characteristic of this serogroup.
The pseudorabies virus gII gene is closely related to the gB glycoprotein gene of herpes simplex virus
Authors: Robbins A K , Dorney D J , Wathen M W , Whealy M E , Gold C , Watson R J , Holland L E , Weed S D , Levine M , Glorioso J C
Published: January 2020

We have looked for conserved DNA sequences between four herpes simplex virus type 1 (HSV-1) glycoprotein genes encoding gB, gC, gD, and gE and pseudorabies virus (PRV) DNA, HSV-1 DNA fragments representing these four glycoprotein-coding sequences were hybridized to restriction enzyme fragments of PRV DNA by the Southern blot procedure. Specific hybridization was observed only when HSV-1 gB DNA was used as probe. This region of hybridization was localized to a 5.2-kilobase (kb) region mapping at approximately 0.15 map units on the PRV genome. Northern blot (RNA blot) analysis, with a 1.2-kb probe derived from this segment, revealed a predominant hybridizing RNA species of approximately 3 kb in PRV-infected PK15 cells. DNA sequence analysis of the region corresponding to this RNA revealed a single large open reading frame with significant nucleotide homology with the gB gene of HSV-1 KOS 321. In addition, the beginning of the sequenced PRV region also contained the end of an open reading frame with amino acid homology to HSV-1 ICP 18.5, a protein that may be involved in viral glycoprotein transport. This sequence partially overlaps the PRV gB homolog coding sequence. We have shown that the PRV gene with homology to HSV-1 gB encoded the gII glycoprotein gene by expressing a 765-base-pair segment of the PRV open reading frame in Escherichia coli as a protein fused to beta-galactosidase. Antiserum, raised in rabbits, against this fusion protein immunoprecipitated a specific family of PRV glycoproteins of apparent molecular mass 110, 68, and 55 kilodaltons that have been identified as the gII family of glycoproteins. Analysis of the predicted amino acid sequence indicated that the PRV gII protein shares 50% amino acid homology with the aligned HSV-1 gB protein. All 10 cysteine residues located outside of the signal sequence, as well as 4 of 6 potential N-linked glycosylation sites, were conserved between the two proteins. The primary protein sequence for HSV-1 gB regions known to be involved in the rate of virus entry into the cells and cell-cell fusion, as well as regions known to be associated with monoclonal antibody resistance, were highly homologous with the PRV protein sequence. Furthermore, monospecific antibody made against PRV gII immunoprecipitated HSV-1 gB from infected cells. Taken together, these findings suggest significant conservation of structure and function between the two proteins and may indicate a common evolutionary history.
Poliovirus proteinase 2A induces cleavage of eucaryotic initiation factor 4F polypeptide p220
Authors: Kräusslich H G , Nicklin M J , Toyoda H , Etchison D , Wimmer E
Published: January 2020

Poliovirus infection of HeLa cells induces rapid shutoff of host protein synthesis, whereas translation of poliovirus RNA is not inhibited. It is presumed that shutoff is the result of proteolytic cleavage of component p220 of eucaryotic initiation factor 4F. To study whether poliovirus proteinase 2A is involved in this cleavage, we translated synthetic RNAs that contained the coding region for poliovirus-specific polypeptides P1 and 2A in vitro and assayed for cleavage of p220. We report here that cleavage of p220 occurred in all cases when active proteinase 2A was translated and that disruption of the coding sequence of 2A by linker insertion or deletion prevented processing of p220 in vitro. Activity of 2A was determined by its ability to cleave at the P1-P2 site of a segment of the poliovirus polyprotein. We also constructed a plasmid in which the 3'-most 500 nucleotides of the nontranslated region of encephalomyocarditis virus were linked to the coding sequence for poliovirus polypeptide 2A. Translation of the RNA transcript of this clone was very efficient and yielded a fusion protein that included 2A; this polypeptide also induced cleavage of p220. In vitro translation in the presence of antibodies against 2A specifically inhibited processing of p220, whereas incubation of in vitro translation products with antibodies against 2A after translation was completed did not prevent proteolysis of p220.
Isolation and characterization of cDNA clones corresponding to transcripts from the BamHI H and F regions of the Epstein-Barr virus genome
Authors: Pfitzner A J , Tsai E C , Strominger J L , Speck S H
Published: January 2020

The Epstein-Barr virus (EBV) mutant P3HR1 is incapable of immortalizing B lymphocytes because of a 6.8-kilobase deletion in the BamHI W, Y, and H regions of the viral genome (M. Rabson, L. Gradoville, L. Heston, and G. Miller, J. Virol. 44:834-844, 1982). To characterize transcripts that are encoded in this region, poly(A)+ RNA from the EBV-transformed lymphoblastoid cell line JY was isolated, and this RNA was used to generate a cDNA library in lambda gt10. By screening 500,000 recombinant bacteriophages with the BamHI H fragment, we isolated 10 cDNA clones and characterized them in detail. One group of six cDNA clones was derived from a 2.9-kilobase early transcript encoded by the IR2 repeat element and showed restriction site polymorphism for the enzyme SmaI. The second group consisted of four cDNA clones, all of which contained the BamHI-H right reading frame (BHRF1), and used the polyadenylation signal at base pair 662 in the BamHI F fragment. Computer analysis of the hydrophobicity of the BHRF1 protein revealed that it is likely to be a membrane protein. Northern blotting experiments with RNA from an EBV producer line, B95-8, and a tightly latent lymphoblastoid B-cell line, IB4, revealed that BHRF1 is contained in at least two different mRNA species which can be detected during the latent cycle of EBV. These data and the recent characterization of a spliced transcript (containing five exons in common with other known latent messages [M. Bodescot and M. Perricaudet, Nucleic Acids Res. 14:7103-7113, 1986]) suggest that alternative splicing is used to generate transcripts containing BHRF1, as for the EBV nuclear antigen 1 transcripts. Furthermore, the observation that a potential oncogene activated in human follicular lymphomas is homologous to the BHRF1-encoded polypeptide (M. L. Cleary, S.D. Smith, and J. Sklar, Cell 47:19-28, 1986) suggests a possible role for this putative viral protein in EBV-induced growth transformation of B lymphocytes.
Antigenic site II of the rabies virus glycoprotein: structure and role in viral virulence
Authors: Prehaud C , Coulon P , LaFay F , Thiers C , Flamand A
Published: January 2020

Twelve monoclonal antibodies neutralizing the CVS strain of rabies virus were used to characterize antigenic site II of the viral glycoprotein. Nineteen antigenic mutants resistant to neutralization by some of these antibodies were selected; some continued to normally or partially bind the antibody, whereas others did not. Mutations conferring resistance to neutralization by site II-specific monoclonal antibodies were localized into two clusters, the first between amino acids 34 and 42 (seven groups of mutants) and the second at amino acids 198 and 200 (three groups of mutants). Two intermediate mutations were identified at positions 147 and 184. Four mutations resulted in reduced pathogenicity after intramuscular inoculation of the virus in adult mice. One of the mutants, M23, was 300 times and the others were 10 to 30 times less pathogenic than CVS. In three cases the attenuated phenotype was related to an important modification of antigenic site II, whereas the other known antigenic sites were unchanged.
Entry of herpes simplex virus 1 in BJ cells that constitutively express viral glycoprotein D is by endocytosis and results in degradation of the virus
Authors: Campadelli-Fiume G , Arsenakis M , Farabegoli F , Roizman B
Published: January 2020

The BJ cell line which constitutively expresses herpes simplex virus 1 glycoprotein D is resistant to infection with herpes simplex viruses. Analysis of clonal lines indicated that resistance to superinfecting virus correlates with the expression of glycoprotein D. Resistance was not due to a failure of attachment to cells, since the superinfecting virus absorbed to the BJ cells. Electron microscopic studies showed that the virions are juxtaposed to coated pits and are then taken up into endocytic vesicles. The virus particles contained in the vesicles were in various stages of degradation. Viral DNA that reached the nucleus was present in fewer copies per BJ cell than that in the parental BHKtk- cells infected at the same multiplicity. Moreover, unlike the viral DNA in BHKtk- cells which was amplified, that in BJ cells decreased in copy number. The results suggest that the glycoprotein D expressed in the BJ cell line interfered with fusion of the virion envelope with the plasma membrane but not with the adsorption of the virus to cells and that the viral proteins that mediate adsorption to and fusion of membranes appear to be distinct.
Herpes simplex virus type 1-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 lacZ insertion mutant
Authors: Goldstein D J , Weller S K
Published: January 2020

Herpes simplex virus (HSV) encodes a ribonucleotide reductase consisting of two subunits (140 and 38 kilodaltons) whose genes map to coordinates 0.56 to 0.60 on the viral genome. Host cell lines containing the HpaI F fragment which includes the reductase subunit genes of HSV type 1 strain KOS (coordinates 0.535 to 0.620) were generated. Transfection of these cells with a plasmid containing the immediate-early ICP0 gene resulted in the expression of ICP6; interestingly, ICP4 plasmids failed to induce expression, indicating an unusual pattern of ICP6 regulation. One such cell line (D14) was used to isolate a mutant with the structural gene of lacZ inserted into the ICP6 gene such that the lacZ gene is read in frame with the N-terminal region of ICP6. This mutant generated a protein containing 434 amino acids (38%) of the N terminus of ICP6 fused to beta-galactosidase under control of the endogenous ICP6 promoter. Screening for virus recombinants was greatly facilitated by staining virus plaques with 5-bromo-4-chloro-3-indoyl-beta-D-galactoside (X-gal). Enzyme assays of infected BHK cells indicated that the mutant is incapable of inducing viral ribonucleotide reductase activity. Surprisingly, although plaque size was greatly reduced, mutant virus yield was reduced only four- to fivefold compared with that of the wild type grown in exponentially growing Vero cells. Mutant virus plaque size, yields, and ability to synthesize viral DNA were more severely compromised in serum-starved cells as compared with the wild type grown under the same condition. Although our evidence suggests that the HSV type 1 ribonucleotide reductase is not required for virus growth and DNA replication in dividing cells, it may be required for growth in nondividing cells.
Antibody-mediated growth of influenza A NWS virus in macrophagelike cell line P388D1
Authors: Ochiai H , Kurokawa M , Hayashi K , Niwayama S
Published: January 2020

We investigated the internalization and growth of influenza A NWS virus in macrophagelike P388D1 cells. Flow cytometric analysis using fluorescein isothiocyanate-labeled virus showed that the attachment of normal rabbit serum-exposed virus (NS-V) to neuraminidase (NA)-treated cells was noticeably limited compared with that to untreated cells. However, rabbit antiserum-exposed virus (AS-V) could attach equally well to both cells. Virus coated with Fab prepared from antiviral immunoglobulin G could not attach. These data suggest that the NWS virus can infect P388D1 cells in one of two ways, via viral or via Fc receptors, depending on the presence of antibodies. The NS-V could grow in the untreated cells, but not in the NA-treated cells. The highest growth of AS-V in the NA-treated cells was observed at an antibody concentration showing 50% plaque reduction titer. Growth was exponentially decreased toward the lower and higher dilutions of antibodies. By using three different immunoglobulin G subclasses of monoclonal antibodies against hemagglutinin, it was demonstrated that both Fc receptors I and II could take part in this phenomenon. The presence of 20 mM NH4Cl inhibited the growth of both AS-V and NS-V, suggesting that the intracellular pathways after internalization via Fc or viral receptors are similar. These data indicate that the concentration of antibodies has a critical role on the antibody-mediated growth of influenza virus in macrophages.
Two regions of the adenovirus early region 1A proteins are required for transformation
Authors: Whyte P , Ruley H E , Harlow E
Published: January 2020

Regions of the adenovirus type 5 early region 1A (E1A) proteins that are required for transformation were defined by using a series of deletion mutants. Deletion mutations collectively spanning the entire protein-coding region of E1A were constructed and assayed for their ability to cooperate with an activated ras oncogene to induce transformation in primary baby rat kidney cells. Two regions of E1A (amino acids 1 to 85 and 121 to 127) were found to be essential for transformation. Deletion of all or part of the region from amino acids 121 to 127 resulted in a total loss of transforming ability. An adjacent stretch of amino acids (residues 128 to 139), largely consisting of acidic residues, was found to be dispensable for transformation but appeared to influence the efficiency of transformation. Amino acids 1 to 85 made up a second region of the E1A protein that was essential for transformation. Deletion of all or part of this region resulted in a loss of the transforming activity. Even a mutation resulting in a single amino acid change at position 2 of the polypeptide chain was sufficient to eliminate transformation. Deletion of amino acids 86 to 120 or 128 to 289 did not eliminate transformation, although some mutations in these regions had lowered efficiencies of transformation. Foci induced by transformation-competent mutants could be expanded into cell lines that retained their transformed morphology and constitutively expressed the mutant E1A proteins.
Involvement of human papillomavirus type 8 genes E6 and E7 in transformation and replication
Authors: Iftner T , Bierfelder S , Csapo Z , Pfister H
Published: January 2020

We investigated the transforming activity of human papillomavirus type 8 (HPV8) by expressing all early open reading frames from a heterologous promoter in different rodent fibroblast lines. Morphological transformation was observed only in G418-selected mouse C127 and Rat 1 cells containing an intact E6-coding region. E6 of HPV8 did not transform NIH 3T3 cells as did E6 of bovine papillomavirus type 1. C127 cells transformed by E6 were anchorage independent and had a reduced serum requirement but did not form tumors in nude mice. E7 of HPV8 showed no transforming potential, in contrast to E7 of HPV18 and HPV16. It was, however, able to complement an E7 mutant of bovine papillomavirus type 1 with a defect in high-copy-number DNA maintenance. The data indicate that the expression of the HPV8 E6 open reading frame is sufficient to induce morphological transformation in rodent fibroblasts, whereas E7 is involved in the replication of the viral DNA.
Characterization of human immunodeficiency virus type 1 reverse transcriptase by using monoclonal antibodies: role of the C terminus in antibody reactivity and enzyme function
Authors: Tisdale M , Ertl P , Larder B A , Purifoy D J , Darby G , Powell K L
Published: January 2020

We describe the production of eight monoclonal antibodies reactive with human immunodeficiency virus type 1 reverse transcriptase (RT) by immunization of mice with purified recombinant RT. These antibodies were found to react with one or the other of two regions of the enzyme and were found to be useful in immunodeficiency purification of large amounts of the enzyme. One epitope located at the C terminus of the enzyme was of particular interest, since it was present in only the larger, 66-kilodalton (kDa) RT species and not its smaller, 51-kDa counterpart. To define this epitope, a series of mutants was made which synthesized C-terminally truncated RT. These mutants indicated that the same region of the enzyme, when deleted, both removed the C-terminal epitope and drastically reduced RT activity, indicating the importance of this region in the function of the enzyme; however, even the 51-kDa enzyme component had demonstrable activity.
Behavior of intertypic recombinants between virulent and attenuated aphthovirus strains in tissue culture and cattle
Authors: Giraudo A T , Gomes I , de Mello P A , Beck E , La Torre J L , Scodeller E A , Bergmann I E
Published: January 2020

Two aphthovirus intertypic recombinants between the virulent strain A Venceslau and guanidine-resistant attenuated mutants of either strain C3 Resende or O1 Campos were obtained in an attempt to establish the region(s) of the viral genome responsible for attenuation in cattle. Recombinants that inherited the 3' half of the genome from either attenuated parent and the 5' half from the virulent strain were selected and analyzed with respect to their ability to grow in cells of bovine origin and for their virulence in cattle. The results obtained support our previous conclusion, derived from studies with homotypic recombinants between attenuated aphthovirus type O1 and its original virulent strain, that the host range restriction phenotype for fetal bovine kidney cells of the attenuated strain is inherited from the 3' half of the genome. For the intertypic recombinants, however, this restriction is enhanced, presumably by the presence of a heterologous 5' half of the genomic region. In addition, we demonstrate that the results in vitro correlate with those of virulence tests in cattle.