bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of lambda gt11 and monoclonal antibodies to map the genes for the six major glycoproteins of equine herpesvirus 1
Authors: Allen G P , Yeargan M R
Published: January 2020

To localize the genes for the major glycoproteins of equine herpesvirus 1 (EHV-1), a library of the EHV-1 genome was constructed in the lambda gt11 expression vector. Recombinant bacteriophage expressing EHV-1 glycoprotein epitopes as fusion products with beta-galactosidase were detected by immunoscreening with monoclonal antibodies specific for each of six EHV-1 glycoproteins. Seventy-four recombinant lambda gt11 clones reactive with EHV-1 monoclonal antibodies were detected among 4 X 10(5) phage screened. Phage expressing determinants on each of the six EHV-1 glycoproteins were represented in the library. Herpesviral DNA sequences contained in lambda gt11 recombinants expressing epitopes of EHV-1 glycoproteins were used as hybridization probes for mapping insert sequences on the viral genome. Genes for five EHV-1 glycoproteins (gp2, gp10, gp13, gp14, and gp21/22a) mapped to the genome L component; only one EHV-1 glycoprotein (gp17/18) was expressed from the unique S region of the genome where genes of several major glycoproteins of other herpesviruses have been located. Two glycoproteins of EHV-1, gp13 and gp14, mapped to positions colinear with genes of major glycoproteins identified in several other alphaherpesviruses (gC- and gB-like glycoproteins, respectively). The genomic locations of other EHV-1 glycoproteins indicated the existence of major glycoproteins of EHV-1 (gp2, gp10, and gp21/22a) for which no genetic homologs have yet been detected in other herpesviruses. The results confirm the general utility of the lambda gt11 expression system for localizing herpesvirus genes and suggest that the genomic positioning of several high-abundance glycoproteins of EHV-1 may be different from that of the prototype alphaherpesvirus, herpes simplex virus.
Bovine leukemia virus transcription is controlled by a virus-encoded trans-acting factor and by cis-acting response elements
Authors: Derse D
Published: January 2020

Bovine leukemia virus (BLV) gene expression is exquisitely regulated at multiple levels, including a transcriptional control effected by virus-encoded trans-acting factors and cis-acting target sequences. Like the human T-cell leukemia viruses type I and type II, but unlike other RNA tumor viruses, BLV contains several open reading frames at the 3' end of its genome. A subgenomic mRNA which encodes two overlapping reading frames from this region could produce proteins of 38 and 18 kilodaltons (kDa). A series of cis-trans experiments using transfected virus gene constructs in different combinations revealed that expression of the 38-kDa protein was both necessary and sufficient to activate, in trans, the BLV promoter. This activation was specific for the BLV long terminal repeat, as a variety of related retroviral promoters were not responsive to the expression of the 38-kDa protein p38(XBL). Deletion analysis and construction of chimeric promoters identified a 75-base-pair long terminal repeat region which functions like a p38(XBL)-dependent enhancer element.
Herpes simplex virus type 1 pathogenicity in footpad and ear skin of mice depends on Langerhans cell density, mouse genetics, and virus strain
Authors: Sprecher E , Becker Y
Published: January 2020

Skin Langerhans cells have been shown to be very efficient in presenting antigens to T-helper cells and stimulating the immune response. The present study demonstrates their essential role in the control of primary herpetic infections in the skin. Two unrelated stimuli (abrasion and steroids) were shown to cause depletion of the Langerhans cells in the murine epidermis, and both caused enhancement of the virulence of herpes simplex type 1 (HSV-1) in the skin. The Langerhans cell density was found to be lower in the skin of the ear than in the footpad. HSV-1 was consistently more virulent when injected into the ear epidermis than in the footpad. Thus, HSV-1 pathogenicity in mouse skin depends on the mouse age and strain, the virus strain, and the state of the epidermal Langerhans cells. These findings are discussed in relation to the antigen-presenting cell function of the Langerhans cells.
Antigenic comparison of feline coronavirus isolates: evidence for markedly different peplomer glycoproteins
Authors: Fiscus S A , Teramoto Y A
Published: January 2020

The antigenic relationships among seven feline coronavirus isolates were investigated by using a panel of 26 monoclonal antibodies (MAbs). The MAbs were categorized into five immunoreactive groups which were used to delineate two antigenic types of feline coronaviruses. One antigenic type included the more virulent feline infectious peritonitis virus (FIPV) isolates (FIPV-UCD-1, FIPV-UCD-4, FIPV-TN406, FIPV-DF2, and FIPV-79-1146), whereas the second antigenic type was composed of the avirulent isolate FIPV-UCD-2. The feline enteric coronavirus isolate FECV-79-1683 shared some characteristics of both of the major antigenic groups. Epitopes on the nucleocapsid and envelope polypeptides were in general highly conserved among both antigenic types, although a few type-specific antigenic sites were discriminated. The most striking finding was the marked antigenic difference in the peplomer (E2) glycoproteins between the two antigenic types. Seven anti-E2 MAbs reacted with one antigenic type of E2, whereas seven other anti-E2 MAbs recognized a different antigenic form of E2. None of the 14 anti-E2 MAbs reacted with all of the isolates.
Virus attenuation and identification of structural proteins of vaccinia virus that are selectively modified during virus persistence
Authors: Paez E , Dallo S , Esteban M
Published: January 2020

To investigate the genetic stability of vaccinia virus DNA, we have tested whether alterations occurred in the polypeptide composition of this complex virus during persistent infections. We found that variants isolated at various passages in Friend erythroleukemia cells persistently infected with vaccinia virus contained, in addition to an 8-megadalton (MDa) deletion on the left terminus of the viral genome, major alterations in the sizes of three structural proteins with molecular masses of about 39, 21, and 14 kDa. Alterations in isoelectric points were also observed in proteins of 48, 27, and 14 kDa. The 14-kDa protein is part of the virus envelope, and the variants increased the size of this protein from 0.5 to 3 kDa with increasing passage number. Alteration in size of the 14-kDa protein is a dominant trait since it appeared in the whole virus population by passage 48. With more passages, some variants were found to increase or decrease the size of a 39-kDa core protein by about 2 kDa and to decrease the size of an envelope protein of 21 kDa by about 2 kDa. These three proteins were immunogenic in mice and elicited a strong host immune response. Major alterations in the sizes of these proteins were prevented by continuous treatment of the persistently infected cultures with interferon. However, after interferon was removed, protein modifications appeared with increasing passage number. Generation of the 8-MDa deletion and alterations in the size of the 14-kDa protein correlated with a marked decrease in virulence of these variants. Our findings suggest that during virus persistence, specific mutations are introduced in the vaccinia virus genome that lead to protein alterations and to highly attenuated viruses.
Functional differences in the peplomer glycoproteins of feline coronavirus isolates
Authors: Fiscus S A , Teramoto Y A
Published: January 2020

The feline coronaviruses can be divided into two distinct antigenic groups on the basis of antigenic differences found on the peplomer (E2) glycoprotein of the virus. Because the E2 glycoprotein is responsible for many of the biological functions of coronaviruses, experiments were done to determine whether there were any E2 functional differences between these two antigenic groups. The avirulent feline infectious peritonitis virus (FIPV) isolate FIPV-UCD-2, which has one antigenic type of E2, was less rapidly internalized and could not spread from cell to cell in the presence of neutralizing antibody. Two virulent isolates, FIPV-DF2 and FIPV-79-1146, as well as the non-FIP-causing feline enteric coronavirus (FECV) isolate FECV-79-1683, all of which have the second antigenic type of E2, were very rapidly internalized and were able to spread from cell to cell despite the presence of neutralizing antibody. The avirulent FIPV-UCD-2 and FECV-79-1683 isolates were more labile at 37 degrees C at pHs of 6.5 and above than were the virulent FIPV-DF2 and FIPV-79-1146 isolates.
Monoclonal antibody-aided characterization of cellular p220 in uninfected and poliovirus-infected HeLa cells: subcellular distribution and identification of conformers
Authors: Etchison D , Etchison J R
Published: January 2020

A monoclonal antibody directed against the Mr-220,000 subunit (p220) of the mRNA cap-binding complex has been prepared and used to analyze the sucrose gradient sedimentation and subcellular location of p220 and its poliovirus-induced cleavage products. The antibody reacted with p220 on immunoblots of cell lysates from uninfected cells, but only with several smaller polypeptides, the p220 cleavage products, in cell lysates from poliovirus-infected cells. The sedimentation of p220 antigens from uninfected or infected cells was analyzed by immunoblot and by enzyme-linked immunosorbent assay (ELISA) of sucrose gradient fractions. The results indicate that antibody reactivity was partially influenced by antigen conformation. Major forms of intact p220 and cleaved p220 were identified by immunoblot, and these had similar sedimentation properties. ELISA analysis of the same gradient fractions detected only uncleaved p220; p220 cleavage products were not recognized. Furthermore, the antibody recognized two forms of native uncleaved p220, one of which appeared to bind antibody with greater affinity. This result suggested the existence of conformational variants of p220. The differential reactivity of the antibody for cleaved versus uncleaved p220 served as a useful control during indirect immunofluorescence analysis to determine the subcellular distribution of p220 antigens. The distribution of p220 in uninfected cells was mainly cytoplasmic, but some nuclear antigens were also apparent. After poliovirus infection only the nuclear pattern remained. Disappearance of the cytoplasmic pattern confirmed the inability of the antibody to react with native p220 cleavage products. The cytoplasmic pattern also disappeared after human rhinovirus 14 infection, but not after mengovirus infection, results which correlated with the ability of human rhinovirus 14 and the inability of mengovirus to induce the cleavage of p220. The results demonstrate that p220 is not likely to be associated with the cytoskeleton and hint at the possibility of a partially nuclear location.
A mouse model for poliovirus neurovirulence identifies mutations that attenuate the virus for humans
Authors: La Monica N , Almond J W , Racaniello V R
Published: January 2020

A mutation in the genome of poliovirus type 3 that is known to reduce neurovirulence in humans similarly reduces neurovirulence in mice when incorporated into a mouse-adapted-human poliovirus recombinant. Viral recombinants with a uracil at nucleotide position 472 in the 5'-noncoding regions of their genomes are unable to replicate in the mouse brain. Viral recombinants with a cytosine at this position are neurovirulent in mice. Neurovirulence of poliovirus in mice may therefore prove to be a useful indicator of the genetic stability of new attenuating mutations created by site-directed mutagenesis.
Levels of bovine papillomavirus RNA and protein expression correlate with variations in the tumorigenic phenotype of hamster cells
Authors: Zhang Y L , Lewis A , Wade-Glass M , Schlegel R
Published: January 2020

Three independent cell lines were established from primary cultures of LSH hamster embryo cells infected with bovine papillomavirus type 1 (BPV-1). Although these cell lines differed in their in vitro saturation densities, none was capable of colony formation in soft agar. Interestingly, two cell lines (BPV-HE1 and BPV-HE3) were tumorigenic in nude mice, syngeneic hamsters, and allogeneic hamsters, whereas BPV-HE2 was not. All three cell lines contained similar numbers of the BPV-1 genome (approximately 50 to 200 copies per cell). However, the nontumorigenic BPV-HE2 cell line contained very low levels of BPV-specific RNA and only small amounts of the BPV-1 E5 transforming protein. The efficiency and rate of tumor formation by BPV-HE1 and BPV-HE3 correlated directly with the apparent amount of viral E5 protein. This analysis suggests that there is a threshold level of BPV protein synthesis required for tumorigenicity, there is a continuum of tumorigenic phenotypes which may depend upon the level of BPV protein expression, and BPV-transformed hamster cells can withstand allogeneic transplantation.
Characterization of a recombinant vaccinia virus expressing human melanoma-associated antigen p97
Authors: Hu S L , Plowman G D , Sridhar P , Stevenson U S , Brown J P , Estin C D
Published: January 2020

p97 is a cell surface glycoprotein expressed at high levels in most human melanomas but present only in trace amounts in normal adult tissues. We are interested in exploring the possibility of using recombinant vaccinia virus to express a specific tumor-associated antigen as a vaccine against human cancer. To this end, we constructed a recombinant virus, v-p97NY, which contains the entire coding sequence for p97 under the control of the vaccinia virus 7.5K promoter. Upon infection of tissue culture cells, v-p97NY expressed high levels of a membrane-bound glycoprotein immunoreactive with a p97-specific monoclonal antibody. Immunization of mice with this recombinant elicited high-titered antibodies against p97. Spleen cells isolated from these mice proliferated in vitro when stimulated either with purified p97 protein or with syngeneic cells expressing p97 antigen. Delayed-type hypersensitivity was also observed in immunized mice after challenge with p97-expressing cells. These findings indicate the potential usefulness of v-p97NY and similar recombinants in tumor immunotherapy.
Cloning and characterization of a papillomavirus associated with papillomas and carcinomas in the European harvest mouse (Micromys minutus)
Authors: O'Banion M K , Reichmann M E , Sundberg J P
Published: January 2020

Individuals in a colony of European harvest mice (Micromys minutus) were diagnosed with a variety of skin tumors including papillomas, trichoepitheliomas, and sebaceous carcinomas. Papillomavirus group-specific antigens and viruslike particles were detected in the papillomas. A 7.6-kilobase supercoiled circular DNA, which was cleaved once by EcoRI, was visualized in papilloma extracts by low-stringency Southern blot hybridization with a bovine papillomavirus type 2 probe. The molecule was cloned in pUC18, and a restriction map was generated. The molecule was shown to be colinear with the genome of human papillomavirus type 1a by partial sequence analysis. The DNA hybridized to human papillomavirus type 1a, rabbit oral papillomavirus, and the genome of Mastomys natalensis papillomavirus at Tm - 33 degrees C but not to the DNAs of 13 other papillomaviruses. Transformation of NIH 3T3 or C127I cells by tail papilloma extracts or transfected viral DNA was not observed. All 17 tumors examined contained large amounts of viral DNA in a supercoiled, unintegrated form as revealed by Southern blot hybridization. Furthermore, many extracts (25 of 35) from normal organs and skin of individuals with lesions elsewhere on their bodies contained viral DNA. This represents the first reported molecular cloning of a papillomavirus genome from a mouse species.
Nucleotide sequence and genome organization of canine parvovirus
Authors: Reed A P , Jones E V , Miller T J
Published: January 2020

The genome of a canine parvovirus isolate strain (CPV-N) was cloned, and the DNA sequence was determined. The entire genome, including ends, was 5,323 nucleotides in length. The terminal repeat at the 3' end of the genome shared similar structural characteristics but limited homology with the rodent parvoviruses. The 5' terminal repeat was not detected in any of the clones. Instead, a region of DNA starting near the capsid gene stop codon and extending 248 base pairs into the coding region had been duplicated and inserted 75 base pairs downstream from the poly(A) addition site. Consensus sequences for the 5' donor and 3' acceptor sites as well as promotors and poly(A) addition sites were identified and compared with the available information on related parvoviruses. The genomic organization of CPV-N is similar to that of feline parvovirus (FPV) in that there are two major open reading frames (668 and 722 amino acids) in the plus strand (mRNA polarity). Both coding domains are in the same frame, and no significant open reading frames were apparent in any of the other frames of both minus and plus DNA strands. The nucleotide and amino acid homologies of the capsid genes between CPV-N and FPV were 98 and 99%, respectively. In contrast, the nucleotide and amino acid homologies of the capsid genes for CPV-N and CPV-b (S. Rhode III, J. Virol. 54:630-633, 1985) were 95 and 98%, respectively. These results indicate that very few nucleotide or amino acid changes differentiate the antigenic and host range specificity of FPV and CPV.
La Crosse virus infection of mammalian cells induces mRNA instability
Authors: Raju R , Kolakofsky D
Published: January 2020

La Crosse virus infection of BHK cells leads to a dramatic shutoff of not only host protein synthesis but also viral protein synthesis later in infection. This shutoff can be accounted for by the loss of the cytoplasmic cellular and viral mRNAs. The induction of mRNA instability requires extensive virus replication, since when cycloheximide is added early in infection the preexisting viral and cellular mRNAs do not decrease upon incubation of the cultures. Pretreatment of the cultures with actinomycin D does not affect the ability of La Crosse virus infection to induce mRNA instability, and examination of the rRNAs shows no evidence of specific degradation due to activation of the interferon-associated latent RNase. The induction of mRNA instability therefore does not appear to operate through an interferon pathway. Viral mRNA synthesis, on the other hand, is not turned off during infection, and the cap-dependent endonuclease involved in viral mRNA initiation may be responsible for the mRNA instability.
A heat-sensitive inhibitor in poliovirus-infected cells which selectively blocks phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the double-stranded RNA-activated protein kinase
Authors: Ransone L J , Dasgupta A
Published: January 2020

We partially purified an inhibitor from poliovirus-infected HeLa cells which specifically blocked phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the double-stranded RNA-activated protein kinase. The inhibitory activity eluted from a sizing column with an approximate molecular weight of 80,000 to 100,000 and was sensitive to heat, suggesting a protein nature for the inhibitor. No specific virus-encoded protein purified with the inhibitor. The inhibition of phosphorylation of the alpha subunit of eucaryotic initiation factor 2 was not due to a protein phosphatase associated with the inhibitor. The inhibitor did not seem to prevent phosphorylation of the double-stranded RNA-activated protein kinase but inhibited the phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the activated kinase. Double-stranded RNA-induced inhibition of in vitro protein synthesis in reticulocyte lysates could be prevented by the addition of the partially purified inhibitor during preincubation of lysate with double-stranded RNA.
Site-specific mutations in vectors that express antigenic and temperature-sensitive phenotypes of the M gene of vesicular stomatitis virus
Authors: Li Y , Luo L Z , Snyder R M , Wagner R R
Published: January 2020

Full-length cDNA copies of mRNAs coding for the matrix (M) proteins of vesicular stomatitis virus and its mutant tsO23(III) were cloned in pBSM13- (BlueScribe). The authenticity of these clones was demonstrated by restriction enzyme mapping, DNA sequencing, and in vitro transcription and translation to identify the two M proteins by Western immunoblotting with epitope-specific monoclonal antibodies. Site-directed mutants were constructed by primer extension of synthetic oligodeoxynucleotides with one or two nucleotide changes to alter the glycine at amino acid 21 of the wild-type (wt) M gene to glutamic acid, alanine, or proline. Similarly, a revertant was created in the M gene of mutant tsO23 by a Glu-21----Gly substitution. A series of wt- and mutant-M-gene chimeras was also constructed to create mutant and revertant clones with Leu----Phe and His----Tyr alterations at amino acids 111 and 227, respectively. We then moved the wt and tsO23 M genes and their site-specific mutants and chimeras cloned in pBSM13- into the eucaryotic expression vector pTF7 directed by the T7 bacteriophage RNA polymerase of the vaccinia virus recombinant vTF1-6,2. Western blot analysis of the M proteins transiently expressed in CV-1 cells by plasmids carrying M genes altered at amino acid 21 revealed that the critical antigenic determinant (epitope 1) is expressed only by the Gly-21 M protein and not by Glu-21, Ala-21, or Pro-21 M proteins. Of particular interest is an apparent conformational change, evidenced by slightly but significantly retarded electrophoretic migration, in plasmid-expressed M proteins with amino acids substituted for glycine at position 21. The glutamic acid at position 21 of tsO23 is not responsible for its temperature-sensitive phenotype, because a tsO23 revertant plasmid with glycine substituted at position 21 fails to rescue tsO23 virus in cells infected at the restrictive temperature; conversely, plasmids expressing wt M protein with substitutions of glutamic acid, alanine, or proline at position 21 are just as effective in marker rescue of tsO23 as is the Gly-21 wt M protein. Marker rescue experiments with wt- and mutant-M-gene chimeras support the hypothesis of K. Morita, R. Vanderoef, and J. Lenard (J. Virol. 61:256-263, 1987) that the temperature-sensitive phenotype of tsO23 is due to a phenylalanine substituted for leucine at amino acid 111, rather than the His-227----Tyr substitution or the Gly-21----Glu substitution, which independently accounts for the loss of epitope 1 in the mutant M protein of tsO23.(ABSTRACT TRUNCATED AT 400 WORDS)
Structural state of newly replicated closed circular simian virus 40 DNA
Authors: Rao B S , Martin R G
Published: January 2020

We have studied the early transition of newly replicated, segregated daughter molecules of simian virus 40 (SV40) into their mature, fully supercoiled state. The DNA of SV40 replicating in African green monkey kidney CV1 cells was chronically labeled with [14C]thymidine and pulse-labeled with [3H]thymidine. The cells were lysed and the viral DNA was isolated. Density gradient centrifugation of viral DNA in cesium chloride revealed that the pulse-labeled, newly synthesized, closed circular supercoiled DNA molecules banded at a slightly higher density (delta sigma = 0.0025) than the chronically labeled DNA, suggesting that the newly completed molecules were in a different structural state. Electrophoresis of DNA in agarose gels at appropriate chloroquine concentrations demonstrated that the mobility of the pulse-labeled closed, superhelical DNA was retarded relative to that of the chronically labeled DNA. These observations indicated that the newly completed SV40 DNA molecules existed in a structural state more relaxed than that of mature DNA by one or two linking numbers.
Molecular basis for cytolytic T-lymphocyte recognition of the murine cytomegalovirus immediate-early protein pp89
Authors: Del Val M , Volkmer H , Rothbard J B , Jonjić S , Messerle M , Schickedanz J , Reddehase M J , Koszinowski U H
Published: January 2020

The murine cytomegalovirus protein pp89, which is encoded by gene ieI, is a nonstructural regulatory protein expressed in the immediate-early phase of the viral replication cycle and located mainly in the nucleus of infected cells. Protection of BALB/c (H-2d) mice against a lethal murine cytomegalovirus challenge infection is achieved by vaccination with a recombinant vaccinia virus, MCMV-ieI-VAC, expressing pp89 as the only murine cytomegalovirus gene product. The protection is entirely mediated by T lymphocytes of the CD8+ subset. In the present report, we analyzed the molecular basis of the recognition of pp89 by BALB/c CD8+ cytolytic T lymphocytes. A series of internal and terminal deletion mutants of gene ieI was constructed and cloned in vaccinia virus, and the antigenicity and immunogenicity of the fragments of pp89 expressed by the recombinants were studied. A region of only one-sixth of the protein, from amino acids 154 to 249 and encoded by the fourth exon of gene ieI, was sufficient for both the recognition in vitro of the protein by pp89-specific cytotoxic T lymphocytes and the induction in vivo of pp89-specific cytotoxic T lymphocytes. By using synthetic peptides, the sequence between residues 161 and 179, which is located within the defined domain, was identified as an epitope presented to BALB/C cytotoxic T lymphocytes by the class I major histocompatibility antigen Ld.
Activation of the human immunodeficiency virus long terminal repeat by herpes simplex virus type 1 is associated with induction of a nuclear factor that binds to the NF-kappa B/core enhancer sequence
Authors: Gimble J M , Duh E , Ostrove J M , Gendelman H E , Max E E , Rabson A B
Published: January 2020

It has been previously shown that herpes simplex virus type 1 (HSV-1) infection of HeLa cells results in augmentation of gene expression directed by the human immunodeficiency virus (HIV) long terminal repeat (LTR). This effect is presumably mediated by protein interactions with the LTR. We have used two different assays of DNA-protein interactions to study the HSV-induced activation of the HIV LTR. Activation of the HIV LTR is associated with increased protein binding to LTR sequences in a region including the NF-kappa B/core enhancer and the Sp1 binding sequences as monitored by an exonuclease protection assay. Gel retardation assays demonstrated that HSV-1 infection resulted in the induction of a nuclear factor(s) that binds to the NF-kappa B/core enhancer sequence. In addition to the activation of the HIV LTR, HSV induction of NF-kappa B activity may be important for the regulation of HSV gene expression during a herpesvirus infection.
A tagged helper-free Friend virus causes clonal erythroblast immortality by specific proviral integration in the cellular genome
Authors: Spiro C , Gliniak B , Kabat D
Published: January 2020

A colinear molecular clone of the Lilly-Steeves polycythemia strain of Friend spleen focus-forming virus (SFFV) was modified by inserting a 215-base-pair tag of simian virus 40 DNA into its nonfunctional pol gene region. The DNA was then transfected into psi-2 packaging cells, and helper-free tagged SFFV was recovered in the culture medium. Injection of this helper-free virus into NIH/Swiss mice caused transient mild splenomegaly and formation of spleen foci at 9 to 10 days. Although the vast majority of infected erythroblast clones then differentiated and died out, rare cell clones that were present in only 20 to 30% of the mice grew extensively by 26 to 33 days to form transplantable leukemias. The clonality of these leukemias was established by Southern blot analysis of their DNAs by using several restriction endonucleases and the simian virus 40 tag as a hybridization probe. All transplantable leukemias lacked helper virus contamination and contained a single tagged SFFV provirus that expressed the mitogenic env gene product gp55. The SFFV proviruses in these leukemias also appeared to be integrated into a few tightly clustered sites in the cellular genome. Although the tagged SFFV caused polycythemia during the polyclonal early stage of erythroblastosis, growth of the helper-free clonal erythroleukemias caused severe anemia. These results suggest that a single SFFV can cause mitosis of erythroblasts, and that cell immortalization also occurs when the provirus integrates into a critical site in the host genome. We propose that mice with clonal-stage leukemia become anemic because the immortalizing proviral integrations interfere with the cellular commitment to differentiate.
Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide
Authors: Gripon P , Diot C , Thézé N , Fourel I , Loreal O , Brechot C , Guguen-Guillouzo C
Published: January 2020

We investigated the possibility of infecting normal adult human hepatocytes maintained in pure cultures or in cocultures with hepatitis B virus (HBV). Several assays with different infectious sera and hepatocyte populations from various donors identified only limited HBV replication, with significant variations from one cell preparation to another. The addition of 1.5% dimethyl sulfoxide to the culture medium markedly enhanced the infection process. Indeed, hepatitis B e antigen secretion, the appearance of both HBV DNA replicative forms and major HBV transcripts, and the release of complete HBV particles into the medium were demonstrated. It is possible that the significant increase in intracellular HBV DNA in dimethyl sulfoxide-treated cells was related to enhanced adsorption of the virus. When viral particles produced by a transfected HepG2 cell line were used to infect normal hepatocytes, the same results were obtained. In addition, comparative assays with hepatocytes from three different donors showed that although high amounts of intracellular viral DNA were found in all cases, viral replicative intermediates were visualized in only one case. These findings suggest that this HBV-producing cell line could serve as a reproducible source of infectious virus and that primary culturing of human hepatocytes represents a unique tool for analyzing intracellular regulating factors which, in addition to the penetration step, modulate HBV replication.
Translocation of rubella virus glycoprotein E1 into the endoplasmic reticulum
Authors: Hobman T C , Shukin R , Gillam S
Published: January 2020

Rubella virus (RV) contains four structural proteins, C (capsid), E2a, E2b, and E1, which are derived from posttranslational processing of a single polyprotein precursor, p110. C protein is nonglycosylated and is thought to interact with RV RNA to form a nucleocapsid. E1 and E2 are membrane glycoproteins that form the spike complexes located on the virion exterior. Two different E1 cDNAs were used to analyze the requirements for translocation of E1 into the endoplasmic reticulum. Analysis of expression of these cDNAs both in vivo and in vitro showed that RV E1 was stably expressed and glycosylated in COS cells and correctly targeted into microsomes in the absence of E2 glycoprotein. The results provide experimental evidence that translocation of RV E1 glycoprotein into the endoplasmic reticulum is mediated by a signal peptide contained within the 69 carboxyl-terminal residues of E2.
Retinoic acid-induced differentiation of retrovirus-infected HL-60 cells is associated with enhanced transcription from the viral long terminal repeat
Authors: Collins S J
Published: January 2020

I infected different human leukemic cell lines with an amphotropic retrovirus vector (designated PA317/N2) which confers G418 resistance and contains the Moloney murine leukemia virus long terminal repeat. In retrovirus-infected G418-resistant HL-60 cells, induction of granulocyte differentiation by retinoic acid was invariably accompanied by a marked increase (5- to 10-fold) in the transcriptional activity of the integrated retroviral long terminal repeat.
Evidence for two points of restriction in the expression of adenovirus type 2 in cultured epidermal keratinocytes
Authors: Aneskievich B J , Taichman L B
Published: January 2020

Cultures of epidermal keratinocytes contain two populations of cells, a basal undifferentiated population and a suprabasal terminally differentiated population. When exposed to wild-type adenovirus type 2 (wtAd2), the suprabasal cells are positive by immunofluorescence for capsid antigen and exhibit cytopathic effects (CPE) (R.F. LaPorta, and L.B. Taichman, Virology 110:137-146, 1981). The basal cells, although infected, are not positive for capsid antigen and do not display CPE. Despite CPE and capsid antigens in suprabasal cells, yields of virus from the entire culture are very low (10 PFU per cell). These observations suggest that Ad2 expression is restricted at different times in the viral life cycle in basal and suprabasal cells. To test this hypothesis, we isolated host range (hr) mutants of Ad2 on two lines of squamous cell carcinoma (SCC) keratinocytes which were shown to be restrictive for wtAd2 replication. The hrAd2 mutants produced high yields of progeny virus in epidermal cell cultures (500 to 600 PFU per cell). However, the pattern of CPE induction in these cultures was like that produced by wtAd2, i.e., basal cells were CPE negative and suprabasal cells were CPE positive. The high yield of hrAd2 progeny indicated that the restriction present in suprabasal cells was overcome. However, the failure of hrAd2 mutants to induce CPE in basal cells indicated that the hrAd2 mutants remain restricted in the basal population and supported our hypothesis that a second and distinct restriction exists in basal keratinocytes.
A single-base change in gene 10 of bacteriophage T7 permits growth on Shigella sonnei
Authors: Pierce J C , Masker W E
Published: January 2020

Bacteriophage T7 can extend its host range to include Shigella sonnei D2 371-48 by a mutation called ss found in the T7 major capsid protein, the gene 10 product. We show that a single A-to-C transversion at position 23150 in the T7 genome is responsible for the T7 ss mutant phenotype that allows the phage to avoid DNA degradation and undergo productive infection. The ss mutation causes an amino acid substitution of proline for glutamine at position 61 of the 344-amino-acid T7 major capsid protein.
Relationships among genes and gene products of bacteriophage BF23
Authors: Nagasu T , Yoshinari K , Kikuchi S , Mizobuchi K
Published: January 2020

Twenty-five gene products of bacteriophage BF23 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functions were studied in relation to type I and II genes classified by means of genetic complementation tests. All the type I mutants were defective in the synthesis of a tail protein, L3. In addition, 4 type I gene products, L5 (gp21), L7 (gp20), L8 (gp29), and L9 (gp25), were identified as constituents of tails (gp21 denotes that a protein is a product of gene 21). Three type IIb mutants in genes 10, 14, and 19 diminished substantially the production of late proteins, including tail and head proteins, and the two other type IIb mutants in genes 1 and 2 were defective in the synthesis of both early and late proteins. Of 14 type IIa mutants, at least 6 were defective in phage DNA synthesis and 2 were defective in the synthesis of head proteins. The defect in the head donor activities of type IIa mutants in extract complementation tests was due to the failure of the formation of mature heads containing DNA. The above results support directly the results of the genetic characterization of BF23 genes.
Poliovirus proteinase 3C: large-scale expression, purification, and specific cleavage activity on natural and synthetic substrates in vitro
Authors: Nicklin M J , Harris K S , Pallai P V , Wimmer E
Published: January 2020

Proteinase 3C of poliovirus type 2 (Sabin) was expressed at 4% total protein in Escherichia coli. The protein was soluble and could be purified by a simple scheme. It was weakly active on the capsid precursor P1 (expressed in vitro), which contains two cleavage sites. The products of processing P1 were 1ABC and 1D (VP1). The activity was insensitive to Triton X-100. Crude extracts of cells infected with poliovirus type 1 (Mahoney) gave strong processing and yielded 1AB (VP0), 1C (VP3), and 1D in the same assay system but were sensitive to detergent. 3C from cell extracts that was separated from its precursors resembled the recombinant proteinase in its activity. Recombinant 3C cleaved the peptide dansyl-Glu-Glu-Glu-Ala-Met-Glu-Gln-Gly-Ile-Thr-Asn-Lys-NH2 at the Gln-Gly bond. We conclude that 3C is merely the core of the Gln-Gly-cleaving activity which processes P1 in vivo and that there is probably a hydrophobic contact between a larger 3C precursor and its P1 substrate which allows the second processing reaction: 1ABC, 1D----1AB, 1C, 1D.
Mouse mammary tumor virus proviruses in T-cell lymphomas lack a negative regulatory element in the long terminal repeat
Authors: Hsu C L , Fabritius C , Dudley J
Published: January 2020

The nucleotide sequences of long terminal repeats (LTRs) from several mouse mammary tumor virus (MMTV) proviruses acquired in mouse T-cell lymphomas were determined. All MMTV proviruses cloned from a C57BL/6 lymphoma contained an identical LTR deletion of 491 base pairs (approximately -655 to -165), whereas an MMTV provirus from a BALB/c T-cell lymphoma had a 430-base-pair deletion in the same U3 region. MMTV proviruses with LTR deletions were acquired in these tumors 10 times more frequently than proviruses with intact LTRs. Because the deletions removed a portion of the glucocorticoid response element or "regulated" enhancer, the transcriptional activity of the deleted MMTV LTRs was assessed in both transient expression and stable transfection experiments. Plasmids were constructed in which the deleted or full-length MMTV LTRs were placed upstream of the chloramphenicol acetyltransferase gene. Results from transfection experiments with these constructs showed that the basal expression of the deleted MMTV LTR in the absence of glucocorticoids was higher than that of the full-length Mtv-17 or C3H MMTV LTRs under the same conditions. Moreover, the C3H LTR with a similar deletion (-637 to -255) also promoted high basal levels of chloramphenicol acetyltransferase activity. These results, coupled with the observation in lymphomas of high basal levels of transcription from MMTV proviruses with deleted LTRs, suggested that these proviruses lack negative regulatory elements in their LTRs. Loss of the negative regulatory element may contribute to the selective propagation of proviruses with deleted LTRs.
Epitope mapping of the human immunodeficiency virus type 1 gp120 with monoclonal antibodies
Authors: Dowbenko D , Nakamura G , Fennie C , Shimasaki C , Riddle L , Harris R , Gregory T , Lasky L
Published: January 2020

A soluble form of recombinant gp120 of human immunodeficiency virus type 1 was used as an immunogen for production of murine monoclonal antibodies. These monoclonal antibodies were characterized for their ability to block the interaction between gp120 and the acquired immunodeficiency syndrome virus receptor, CD4. Three of the monoclonal antibodies were found to inhibit this interaction, whereas the other antibodies were found to be ineffective at blocking binding. The gp120 epitopes which are recognized by these monoclonal antibodies were mapped by using a combination of Western blot (immunoblot) analysis of gp120 proteolytic fragments, immunoaffinity purification of fragments of gp120, and antibody screening of a random gp120 gene fragment expression library produced in the lambda gt11 expression system. Two monoclonal antibodies which blocked gp120-CD4 interaction were found to map to adjacent sites in the carboxy-terminal region of the glycoprotein, suggesting that this area is important in the interaction between gp120 and CD4. One nonblocking antibody was found to map to a position that was C terminal to this CD4 blocking region. Interestingly, the other nonblocking monoclonal antibodies were found to map either to a highly conserved region in the central part of the gp120 polypeptide or to a highly conserved region near the N terminus of the glycoprotein. N-terminal deletion mutants of the soluble envelope glycoprotein which lack these highly conserved domains but maintain the C-terminal CD4 interaction sites were unable to bind tightly to the CD4 receptor. These results suggest that although the N-terminal and central conserved domains of intact gp120 do not appear to be directly required for CD4 binding, they may contain information that allows other parts of the molecule to form the appropriate structure for CD4 interaction.
Genetic structure and function of an early transcript of visna virus
Authors: Mazarin V , Gourdou I , Quérat G , Sauze N , Vigne R
Published: January 2020

During the early step of the lytic cycle, visna provirus is first transcribed into two small multispliced mRNAs of 1.6 and 1.2 kilobases which may encode factors regulating the replication of visna virus (R. Vigne, V. Barban, G. Quérat, V. Mazarin, I. Gourdou, and N. Sauze, Virology 161:218-227, 1987). By cDNA cloning and nucleotide sequencing, we determined that the 1.2-kilobase mRNA is 1,174 nucleotides long without the 3'-polyadenylated tail and is composed of four exons, two of which originated from the 5' and 3' ends, respectively, of the env gene region. Two overlapping open reading frames are present in each of these two exons. They were translated in vitro and gave rise to three proteins, two of 19 and 17 kilodaltons, termed VEP1, and one of 16.5 kilodaltons, termed STM. Only the VEP1 proteins were recognized by a hyperimmune anti-visna virus serum of infected sheep. Transient-expression assays performed in eucaryotic cells demonstrated that the cDNA clone described here has a trans-acting effect on transcription of the visna virus genes.
Poorly expressed endogenous ecotropic provirus of DBA/2 mice encodes a mutant Pr65gag protein that is not myristylated
Authors: Copeland N G , Jenkins N A , Nexø B , Schultz A M , Rein A , Mikkelsen T , Jørgensen P
Published: January 2020

DBA/2 mice carry a single endogenous ecotropic murine leukemia provirus designated Emv-3. Although this provirus appears to be nondefective by genomic restriction enzyme mapping, weanling mice do not produce virus and only about one-third of adult mice ever express virus. 5-Iododeoxyuridine and 5-azacytidine, two potent inducers of ecotropic virus expression, are relatively ineffective at inducing Emv-3 expression. However, the chemical carcinogen 7,12-dimethylbenz(a)anthracene can induce ecotropic virus expression in approximately 95% of treated DBA/2 mice. Previous experiments involving DNA transfection and marker rescue analysis of molecularly cloned Emv-3 DNA suggested that Emv-3 carries a small defect(s) in the gag gene, not detectable by restriction enzyme mapping, that inhibits virus expression in vivo and in vitro. Using a combination of approaches, including DNA sequencing, peptide mapping, and metabolic labeling of cells with [3H]myristate, we have demonstrated that the defect in Emv-3 most likely results from a single nucleotide substitution within the gene for p15gag that inhibits myristylation of the Pr65gag N terminus. Myristylation of Pr65gag is thought to be required for this protein to associate with the plasma membrane and is essential for virus particle formation. These results provide a conceptual framework for understanding how Emv-3 expression is regulated during development and after chemical induction.
Four viral genes independently contribute to attenuation of live influenza A/Ann Arbor/6/60 (H2N2) cold-adapted reassortant virus vaccines
Authors: Snyder M H , Betts R F , DeBorde D , Tierney E L , Clements M L , Herrington D , Sears S D , Dolin R , Maassab H F , Murphy B R
Published: January 2020

Clinical studies previously demonstrated that live influenza A virus vaccines derived by genetic reassortment from the mating of influenza A/Ann Arbor/6/60 (H2N2) cold-adapted (ca) donor virus with epidemic wild-type influenza A viruses are reproducibly safe, infectious, immunogenic, and efficacious in the prevention of illness caused by challenge with virulent wild-type virus. These influenza A reassortant virus vaccines also express the ca and temperature sensitivity (ts) phenotypes in vitro, but the genes of the ca virus parent which specify the ca, ts, and attenuation (att) phenotypes have not adequately been defined. To identify the genes associated with each of these phenotypes, we isolated six single-gene substitution reassortant viruses, each of which inherited only one RNA segment from the ca parent virus and the remaining seven RNA segments from the A/Korea/1/82 (H3N2) wild-type virus parent. These were evaluated in vitro for their ca and ts phenotypes and in ferrets, hamsters, and seronegative adult volunteers for the att phenotype. We found that the polymerase PA gene of the ca parent specifies the ca phenotype and that the PB2 and PB1 genes independently specify the ts phenotype. The PA, M, PB2, and PB1 genes of the ca donor virus each contribute to the att phenotype. The finding that four genes of the ca donor virus contribute to the att phenotype provides a partial explanation for the observed phenotypic stability of ca reassortant viruses following replication in humans.
Uptake of reovirus serotype 1 by the lungs from the bloodstream is mediated by the viral hemagglutinin
Authors: Verdin E M , Lynn S P , Fields B N , Maratos-Flier E
Published: January 2020

We used the mammalian reoviruses to determine the molecular basis of the clearance of a virus from the bloodstream by specific organs. Reovirus serotypes 1 (T1) and 3 (T3) were radiolabeled with [35S]methionine or 125I, and the viruses were injected intravenously into weanling rats. The distribution of radioactivity within the animals was determined at various times after the injection. Both viruses were cleared rapidly from the bloodstream and concentrated in different organs. Reovirus T1 was found predominantly in the lungs and liver, whereas T3 was found predominantly in the liver, with very little virus in the lungs. Using intertypic reassortants, we determined that the T1 S1 gene, which encodes the viral hemagglutinin (sigma 1 protein), is responsible for the difference in uptake of T1 and T3 by the lungs. The genetic mapping was extended by using several approaches. (i) T1 subjected to limited proteolytic digestion with chymotrypsin was cleared efficiently by the lungs despite the removal of sigma 3 and digestion of mu 1C to delta. (ii) Uptake of T1 by the lungs was totally inhibited by incubation of T1 with an anti-sigma 1 monoclonal antibody or its Fab fragment before injection. (iii) A reovirus T1 variant in the sigma 1 protein was poorly taken up by the lungs. These data indicate that clearance of reovirus from the bloodstream by the lungs is dependent on the presence of the T1 sigma 1 protein.