bluuebunny/crossref_metadata_2025 huggingface dataset:
Cationic liposomes (Lipofectin) mediate retroviral infection in the absence of specific receptors
Authors: Innes C L , Smith P B , Langenbach R , Tindall K R , Boone L R
Published: January 2020

We have used cationic liposomes (Lipofectin) to facilitate retrovirus infection of cells lacking the homologous viral receptor. Ecotropic murine leukemia virus and packaged retroviral vectors were shown to infect mink cells, and amphotropic packaged retroviral vectors were shown to infect hamster cells in the presence of Lipofectin but not in the presence of Polybrene. Lipofectin-mediated infection of cells lacking the homologous receptor results in a titer approximately 0.1% of the titer in cells with the homologous receptor, using the standard Polybrene protocol. The use of Lipofectin may provide a simple means to experimentally infect a wide variety of cells with viruses not normally infectious for the species, tissue, or cell type of interest.
Characterization of murine monoclonal antibodies to the tat protein from human immunodeficiency virus type 1
Authors: Brake D A , Goudsmit J , Krone W J , Schammel P , Appleby N , Meloen R H , Debouck C
Published: January 2020

A panel of murine monoclonal antibodies (MAbs) to the human immunodeficiency virus type 1 trans-activator tat protein were characterized. The anti-tat MAbs were mapped to the different domains of the tat protein by Western blot (immunoblot) and Pepscan analyses. One-half of the MAbs tested mapped to the amino-terminal proline-rich region, and one-third of the MAbs tested mapped to the lysine-arginine-rich region of tat. The individual MAbs were tested for inhibition of tat-mediated trans activation, using a cell-based in vitro assay system. MAbs which mapped to the amino-terminal region of the tat protein demonstrated the highest degree of inhibition, whereas MAbs reactive to other portions of the molecule exhibited a less pronounced effect on tat function.
A new avian leukosis virus-based packaging cell line that uses two separate transcomplementing helper genomes
Authors: Cosset F L , Legras C , Chebloune Y , Savatier P , Thoraval P , Thomas J L , Samarut J , Nigon V M , Verdier G
Published: January 2020

An avian leukosis virus-based packaging cell line was constructed from the genome of the Rous-associated virus type 1. The gag, pol, and env genes were separated on two different plasmids; the packaging signal and the 3' long terminal repeat were removed. On a plasmid expressing the gag and pol genes, the env gene was replaced by the hygromycin resistance gene. The phleomycin resistance gene was inserted in the place of the gag-pol genes on a plasmid expressing the env gene. The plasmid containing the gag, pol, and Hygror genes was transfected into QT6 cells. Clones that produced high levels of p27gag were transfected with the plasmid containing the Phleor and env genes. Clones that produced high levels of env protein (as measured by an interference assay) were tested for their ability to package NeoR-expressing replication-defective vectors (TXN3'). One of the clones (Isolde) was able to transfer the Neo+ phenotype to recipient cells at a titer of 10(5) resistance focus-forming units per ml. Titers of supernatants of cells infected with Rous-associated virus type 1 prior to transfection by Neor vectors were similar. Tests for recombination events that might result in intact helper virus showed no evidence for the generation of replication-competent virus. The use of selectable genes inserted next to the viral genes to generate high-producer packaging cell lines is discussed.
Two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity
Authors: Andries K , Dewindt B , Snoeks J , Wouters L , Moereels H , Lewi P J , Janssen P A
Published: January 2020

A variety of chemically different compounds inhibit the replication of several serotypes of rhinoviruses (common-cold viruses). We noticed that one of these antiviral compounds, WIN 51711, had an antiviral spectrum clearly distinctive from a consensus spectrum or other capsid-binding compounds, although all of them were shown to share the same binding site. A systematic evaluation of all known rhinovirus capsid-binding compounds against all serotyped rhinoviruses was therefore initiated. Multivariate analysis of the results revealed the existence of two groups of rhinoviruses, which we will call antiviral groups A and B. The differential sensitivity of members of these groups to antiviral compounds suggests the existence of a dimorphic binding site. The antiviral groups turned out to be a reflection of a divergence of rhinovirus serotypes on a much broader level. Similarities in antiviral spectra were highly correlated with sequence similarities, not only of amino acids lining the antiviral compound-binding-site, but also of amino acids of the whole VP1 protein. Furthermore, analysis of epidemiological data indicated that group B rhinoviruses produced more than twice as many clinical infections per serotype than group A rhinoviruses did. Rhinoviruses belonging to the minor receptor group were without exception all computed to lie in the same region of antiviral group B.
Hepatitis B viruses with precore region defects prevail in persistently infected hosts along with seroconversion to the antibody against e antigen
Authors: Okamoto H , Yotsumoto S , Akahane Y , Yamanaka T , Miyazaki Y , Sugai Y , Tsuda F , Tanaka T , Miyakawa Y , Mayumi M
Published: January 2020

The C gene of hepatitis B virus (HBV) codes for a nucleocapsid protein made of 183 amino acid residues and is preceded in phase by the precore (pre-C) region, encoding 29 residues. The pre-C-region product is required for the synthesis and secretion of hepatitis B e antigen (HBeAg), which is made of the C-terminal 10 amino acid residues of the pre-C-region product and the N-terminal 149 residues of the C-gene product. HBV mutants with pre-C-region defects prevailed in the circulation of three asymptomatic carriers as they seroconverted from HBeAg to the corresponding antibody (anti-HBe), and these mutants finally replaced nondefective HBV. HBV DNA clones were propagated from sera of an additional 15 carriers with anti-HBe and sequenced for the pre-C region. Essentially all HBV DNA clones (56 of 57 [98%]) revealed mutations that prohibited the translation of a functional pre-C-region product. A point mutation from G to A at nucleotide 83, converting Trp-28 (TGG) to a stop codon (TAG), was by far the commonest and was observed in HBV DNA clones from 16 (89%) of 18 carriers seropositive for anti-HBe. In addition, there were point mutations involving ATG codon to abort the translation initiation of the pre-C region, as well as deletion and insertion to induce frameshifts. Such mutations leading to pre-C-region defects were rarely observed in persistently infected individuals positive for HBeAg or in patients with type B acute hepatitis after they had seroconverted to anti-HBe. These results would indicate a selection of pre-C-defective mutants in persistently infected hosts, along with seroconversion to anti-HBe, by immune elimination of hepatocytes harboring nondefective HBV with the expression of HBeAg.
Naturally occurring human parainfluenza type 3 viruses exhibit divergence in amino acid sequence of their fusion protein neutralization epitopes and cleavage sites
Authors: Coelingh K V , Winter C C
Published: January 2020

Many human parainfluenza type 3 virus (PIV3) strains isolated from children with respiratory illness are resistant to neutralization by monoclonal antibodies (MAbs) which recognize epitopes in antigenic site A or B of the fusion (F) protein of the prototype 1957 PIV3 strain. The F protein genes of seven PIV3 clinical isolates were sequenced to determine whether their neutralization-resistant phenotypes were associated with specific differences in amino acids which are recognized by neutralizing MAbs. Several clinical strains which were resistant to neutralization by site A or B MAbs had amino acid differences at residues 398 or 73, respectively. These specific changes undoubtedly account for the neutralization-resistant phenotype of these isolates, since identical substitutions at residues 398 or 73 in MAb-selected escape mutants confer resistance to neutralization by site A or B MAbs. The existence of identical changes in naturally occurring and MAb-selected neutralization-resistant PIV3 strains raises the possibility that antigenically different strains may arise by immune selection during replication in partially immune children. Three of the seven clinical strains examined had differences in their F protein cleavage site sequence. Whereas the prototype PIV3 strain has the cleavage site sequence Arg-Thr-Lys-Arg, one clinical isolate had the sequence Arg-Thr-Arg-Arg and two isolates had the sequence Arg-Thr-Glu-Arg. The different cleavage site sequences of these viruses did not affect their level of replication in either continuous simian or bovine kidney cell monolayers (in the presence or absence of exogenous trypsin or plasmin) or in the upper or lower respiratory tract of rhesus monkeys. We conclude that two nonconsecutive basic residues within the F protein cleavage site are sufficient for efficient replication of human PIV3 in primates.
In vitro synthesis and processing of herpes simplex virus type 2 gG-2, using cell-free transcription and translation systems
Authors: Weldon S K , Su H K , Fetherston J D , Courtney R J
Published: January 2020

Translation of in vitro-synthesized herpes simplex virus type 2 (HSV-2) gG-2 mRNA in a reticulocyte lysate system was used to study the processing of HSV-2 gG-2. In the presence of canine pancreatic microsomal membranes, a single species that is protected from trypsin digestion was detected. This product comigrates with the 104,000-Mr (104K) high mannose intermediate seen in HSV-2-infected-cell lysates. Endo-beta-N-acetylglucosaminidase H treatment of the in vitro-synthesized 104K protein yielded a single product migrating at 100 K. The 72K and 31K cleavage products of gG-2 were not observed in the in vitro system. These data show that the molecular weight of the nonglycosylated form of the gG-2 protein is 100,000 and that the cotranslational processing of this protein in the endoplasmic reticulum yields the 104K high-mannose intermediate.
Vaccination with inactivated influenza A virus during pregnancy protects neonatal mice against lethal challenge by influenza A viruses representing three subtypes
Authors: Mbawuike I N , Six H R , Cate T R , Couch R B
Published: January 2020

A single intraperitoneal injection of pregnant mice with a monovalent Formalin-inactivated influenza A virus vaccine protected their offspring against a lethal challenge dose of the same influenza A virus H3N2, H2N2, and H1N1 subtypes, as well as against challenge with the other two subtypes. Degree of protection was vaccine dose related. Cross-fostering of neonates indicated that protection was conferred by breast milk antibodies. Serum virus-specific neutralizing antibodies in the mothers and neonates correlated with resistance to vaccine virus, but were detected against other subtypes only in a complement enhancement test or when high doses of vaccine were given.
Replication-dependent transactivation of the polyomavirus late promoter
Authors: Cahill K B , Roome A J , Carmichael G G
Published: January 2020

When a plasmid containing the wild-type polyomavirus intergenic regulatory region fused to the bacterial cat gene was introduced into mouse NIH 3T3 cells along with a plasmid coding for the early viral proteins (T antigens), chloramphenicol transacetylase enzyme activity and mRNA levels were increased about 10-fold over levels observed in the absence of early proteins. To investigate this transactivation phenomenon further, 11 specific deletion mutant derivatives of the wild-type parent plasmid were constructed and studied. One mutant (NAL) with a minimal level of chloramphenicol transacetylase expression in the absence of T antigens was capable of being transactivated more than 40-fold. A number of other mutants, however, had little capacity for transactivation. Each of these mutants had in common a defect in large T-antigen-mediated DNA replication. Interestingly, one of the transactivation-defective mutants showed a basal late promoter activity fivefold higher than that of wild type and replicated in mouse cells in the absence of large T antigen. Subsequently, a small deletion abolishing viral DNA replication was introduced into those mutants capable of transactivation. The effect of the second deletion was to eliminate both replication and transactivation. Finally, wild-type and mutant constructs were transfected into Fisher rat F-111 cells in the presence or absence of early proteins. No transactivation or replication was ever observed in these cells. We concluded from these studies that the observed transactivation of the polyomavirus late promoter by one or more of the viral early proteins was due to either higher template concentration resulting from DNA replication or replication-associated changes in template conformation.
Transactivation of a human cytomegalovirus early promoter by gene products from the immediate-early gene IE2 and augmentation by IE1: mutational analysis of the viral proteins
Authors: Malone C L , Vesole D H , Stinski M F
Published: January 2020

Expression from a human cytomegalovirus early promoter (E1.7) has been shown to be activated in trans by the IE2 gene products (C.-P. Chang, C. L. Malone, and M. F. Stinski, J. Virol. 63:281-290, 1989). Using wild-type and mutant viral proteins, we have defined the protein regions required for transactivation of the E1.7 promoter in IE2 and for augmentation of transactivation in the IE1 protein. Two regions of the IE2 proteins were found to be essential for transactivation. One near the amino terminus is within 52 amino acids encoded by exon 3. The second comprises the carboxyl-terminal 85 amino acids encoded by exon 5. The IE2 protein encoded by an mRNA which lacks the intron within exon 5 and the IE2 protein encoded by exon 5 had no activity for transactivation of the E1.7 promoter. Although the IE1 gene product alone had no effect on this early viral promoter, maximal early promoter activity was detected when both IE1 and IE2 gene products were present. The IE1 protein positively regulated its enhancer-containing promoter-regulatory region. The IE1 protein alone increased the steady-state level of IE2 mRNA; therefore, IE1 and IE2 are synergistic for expression from the E1.7 promoter. Like the IE2 proteins, the IE1 protein requires for activity 52 amino acids encoded by exon 3. IE1 also requires amino acids encoded by exon 4. Since the IE1 and IE2 proteins have 85 amino acids in common at the amino-terminal end encoded by exons 2 and 3, the difference between these specific transactivators resides in their carboxyl-terminal amino acids encoded by exons 4 and 5, respectively.
Complete nucleotide sequence of infectious Coxsackievirus B3 cDNA: two initial 5' uridine residues are regained during plus-strand RNA synthesis
Authors: Klump W M , Bergmann I , Müller B C , Ameis D , Kandolf R
Published: January 2020

A full-length reverse-transcribed, infectious cDNA copy of coxsackievirus B3 (CVB3) was used to determine the nucleotide sequence of this cardiotropic enterovirus. Comparison of the nucleotide sequence and the deduced amino acid sequence of the viral precursor polyprotein with the sequences of other group B coxsackieviruses (CVB1 and CVB4) demonstrates a high degree of genetic identity. They share about 80% homology at the nucleotide level and about 90% when the amino acid sequences of the polyproteins are compared. The potential processing sites of the coxsackievirus polyproteins, as deduced from alignment with the poliovirus sequence, are conserved among these enteroviruses with the exception of the cleavage sites between VP1 and 2Apro and between polypeptides 2B and 2C. Comparison of the 5' termini of the enteroviral genomes reveals a high degree of identity, including the initial 5' consensus UUAAAACAGC, suggesting essential functions in virus replication. An important finding concerning the molecular basis of infectivity was that both recombinant CVB3 cDNA and in vitro-synthesized CVB3 RNA transcripts are infectious, although two initial 5' uridine residues found on the authentic CVB3 RNA were missing. Here, we report that cDNA-generated CVB3, as well as CVB3 generated by in vitro-synthesized RNA transcripts, regains the authentic initial 5' uridine residues during replication in transfected cells, indicating that the picornaviral primer molecule VPg-pUpU may be uridylylated in a template-independent fashion. The generation of virus or virus mutants with infectious recombinant CVB3 cDNA and in vitro-synthesized infectious CVB3 transcripts should provide a valuable means for studying the molecular basis of the pathogenicity of this cardiotropic enterovirus.
Transcription of the Epstein-Barr virus genome during latency in growth-transformed lymphocytes
Authors: Sample J , Kieff E
Published: January 2020

Nuclear run-on assays revealed extensive transcription of the Epstein-Barr virus genome during latent infection in in vitro-infected human fetal lymphoblastoid cells (IB-4). The EBER genes were the most heavily transcribed viral genes in these cells. Their transcription was partially inhibited in the presence of 1 microgram of alpha-amanitin per ml and fully inhibited at 100 micrograms/ml, consistent with RNA polymerase III transcription. All other transcription was inhibited at 1 microgram of alpha-amanitin per ml, consistent with RNA polymerase II sensitivity to alpha-amanitin. Other than EBER transcription, almost no transcription occurred from the U1 region. Specifically, no transcription was detected from the U1 latent promoter. RNA polymerase II transcription was highest in IR1, extending rightward through U2 and IR2 into the U3 domain and gradually decreased, but was measurable throughout the rest of the genome. This is consistent with EBNA gene transcription initiation within IR1. The higher level of transcription of the IR1 and U2 domains, which encode EBNA-LP and EBNA-2, as opposed to the domains which encode EBNA-3A, EBNA-3B, or EBNA-3C or EBNA-1, correlated with a higher level of EBNA-LP/EBNA-2 mRNA. Transcription extended through U4 into U5, even though no known latent-gene mRNAs are expressed from U4 downstream of the EBNA-1 open reading frame. This may result from inefficient termination of EBNA gene transcription. Leftward transcription from the latent membrane protein promoter was lower than EBNA transcription, although the latent membrane protein mRNA was the most abundant of the latent-gene mRNAs, indicating that this mRNA is more efficiently processed or has a longer half-life. Although transcription was detected from the DL strong early promoters and to a lesser extent from other early promoters, early mRNAs were less abundant than EBNA mRNAs or undetectable, suggesting that there may be posttranscriptional as well as transcriptional control over early mRNA expression in these latently infected cells.
Identification of a cis-acting element in human immunodeficiency virus type 2 (HIV-2) that is responsive to the HIV-1 rev and human T-cell leukemia virus types I and II rex proteins
Authors: Lewis N , Williams J , Rekosh D , Hammarskjöld M L
Published: January 2020

A simian virus 40 late replacement vector encoding human immunodeficiency virus type 1 (HIV-1) gp120 (pGP120) was used to define a region within the HIV-2 genome that could work as a rev-responsive element (RRE). Our previous work showed that gp120 expression in this system required a functional RRE in cis and required the rev protein in trans (M.-L. Hammarskjöld, J. Heimer, B. Hammarskjöld, I. Sangwan, L. Albert, and D. Rekosh, J. Virol. 63:1959-1966, 1989). Using pGP120, we first mapped an RRE to a 1,042-base-pair (bp) Sau3a fragment in the env region of HIV-2. Both HIV-1 rev (rev1) and HIV-2 rev (rev2) could work in conjunction with this fragment. Further mapping showed that a 272-bp subfragment within the 1,042-bp region was sufficient as an RRE. Surprisingly, the smaller fragment worked only with the rev1 protein and not with its homologous rev2 protein. In addition, the rev2 protein failed to function together with the RRE from HIV-1. We also utilized this system to examine the ability of the rex genes of human T-cell leukemia virus types I and II to functionally substitute for rev. These experiments showed that complementation by both the rexI and rexII proteins required the presence of an RRE. The rex proteins worked well in conjunction with either the HIV-1 or the HIV-2 RRE (the 1,042-bp as well as the 272-bp fragment).
The Epstein-Barr virus BRLF1 immediate-early gene product transactivates the human immunodeficiency virus type 1 long terminal repeat by a mechanism which is enhancer independent
Authors: Quinlivan E B , Holley-Guthrie E , Mar E C , Smith M S , Kenney S
Published: January 2020

The Epstein-Barr virus (EBV) immediate-early gene product, BRLF1, transactivates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat. BRLF1-induced transactivation of HIV-1 promoter constructs is accompanied by an increase in plasmid mRNA and is reporter gene independent. Previously, BRLF1 transactivation of EBV promoters has been mapped to regions which function as enhancer elements. Deletional analysis demonstrates that BRLF1 transactivation of the HIV-1 promoter does not require the HIV-1 enhancer. Thus, the EBV BRLF1 gene product may transactivate by at least two different mechanisms, one mechanism involving certain enhancer elements and another mechanism which is enhancer independent.
Pattern of transcription of the genome of equine infectious anemia virus
Authors: Noiman S , Yaniv A , Sherman L , Tronick S R , Gazit A
Published: January 2020

The pattern of expression of the equine infectious anemia virus (EIAV) genome in a persistently infected canine cell line was determined. Five EIAV-specific transcripts (8.2, 5.0, 4.0, 2, and 1.8 kilobases [kb]) were detected by using subgenomic restriction enzyme fragments of EIAV DNA and EIAV-specific oligonucleotides as probes. The 8.2-kb mRNA could be shown to represent viral genomic RNA, whereas the smaller transcripts were generated by splicing events. Evidence was obtained that indicated that each subgenomic RNA species shared a common 5'-splice donor. The 5.0-kb mRNA was found to be expressed at relatively low levels, was difficult to detect consistently, and appeared to be generated by a single splicing event which linked the 5' exon to the 3' region of pol. The 4.0-kb transcript was concluded to be the env mRNA on the basis of its hybridization pattern with the various probes and its abundance. The 2-kb species was found to be multiply spliced and was encoded by sequences derived from orf2 but was not detected by probes representing 3'-env/3'-orf sequences. The 1.8-kb species was shown to consist of sequences representing orf1, part of orf2, and the 3'-orf/env and may represent the message for the EIAV trans-activator gene.
cis-acting regulatory elements near the Epstein-Barr virus latent-infection membrane protein transcriptional start site
Authors: Ghosh D , Kieff E
Published: January 2020

Epstein-Barr virus (EBV) latent-infection membrane protein (LMP) gene cis-acting regulatory sequences were assayed in human B lymphocytes by using chloramphenicol acetyltransferase (cat) gene expression as a reporter. The activities of progressively longer upstream elements from bases -55 to -2350 were compared. At least two positive cis-activating regulatory components (-155 to -147 and -234 to -205) upstream of the LMP promoter were defined. LMP promoter cat gene constructs were more active in a Burkitt's lymphoma cell line latently infected with the B95 EBV strain than in the same cells latently infected with the P3HR1 EBV strain. Since the P3HR1- and B95-infected cells differ in EBNA-2 and EBNA-LP expression, EBNA-2 or EBNA-LP is a likely transactivator of the LMP promoter. Probable cognate sequences for known transcription factors in the LMP promoter are discussed.
The amino terminus of polyomavirus middle T antigen is required for transformation
Authors: Cook D N , Hassell J A
Published: January 2020

In polyomavirus-transformed cells, pp60c-src is activated by association with polyomavirus middle T antigen. These complexes have a higher tyrosine kinase activity compared with that of unassociated pp60c-src. Genetic analyses have revealed that the carboxy-terminal 15 amino acids of pp60c-src and the amino-terminal half of middle T antigen are required for this association and consequent activation of the tyrosine kinase. To define in greater detail the borders of the domain in middle T antigen required for activation of pp60c-src, we constructed a set of unidirectional amino-terminal deletion mutants of middle T antigen. Analysis of these mutants revealed that the first six amino acids of middle T antigen are required for it to activate the kinase activity of pp60c-src and to transform Rat-1 fibroblasts. Analysis of a series of insertion and substitution mutants confirmed these observations and further revealed that mutations affecting the first four amino acids of middle T antigen reduced or abolished its capacity to activate the kinase activity of pp60c-src and to transform Rat-1 cells in culture. Our results suggest that the first four amino acids of middle T antigen constitute part of a domain required for activation of the pp60c-src tyrosyl kinase activity and for consequent cellular transformation.
Characterization of the murine cytomegalovirus early transcription unit e1 that is induced by immediate-early proteins
Authors: Bühler B , Keil G M , Weiland F , Koszinowski U H
Published: January 2020

The regulation of murine cytomegalovirus early (E) gene expression was studied in the cell line B25, which is stably transfected with the immediate-early ie1/ie3 gene complex. Infection of B25 cells in the presence of the protein synthesis inhibitor cycloheximide resulted in the expression of some E genes, whereas for the expression of other E genes prior protein synthesis was still mandatory, thus showing differences in the expression requirements of individual E genes. Transcription unit e1, a member of the E genes induced by immediate-early products of the ie1/ie3 gene complex, was characterized. It is located between map units 0.709 and 0.721 of the genome of murine cytomegalovirus strain Smith. A 2.6-kilobase RNA specified in this region is spliced from three exons of 912, 177, and 1,007 or 1,020 nucleotides, which are separated by introns of 93 and 326 nucleotides. The second AUG located in the first exon 119 nucleotides downstream of the 5' cap site is followed by an open reading frame of 990 nucleotides. The predicted polypeptide of 330 amino acids has a calculated molecular mass of 36.4 kilodaltons. Transfection with e1 revealed three antigenically related proteins of 36, 37, and 38 kilodaltons; these proteins probably represent differently modified forms of the predicted protein. These three proteins are phosphorylated and are associated with intranuclear inclusion bodies. A 33-kilodalton protein also derived from e1 was identified as a product of nonspliced transcripts. Comparison of amino acid sequences revealed homology between the murine cytomegalovirus transcription unit e1 and a human cytomegalovirus E transcription unit.
Positive and negative regulation of the minute virus of mice P38 promoter
Authors: Gavin B J , Ward D C
Published: January 2020

5' deletion mutants of the minute virus of mice P38 promoter were constructed and analyzed for transcriptional activity in vitro and in vivo. In uninfected mouse A9 cell extracts, 107 base pairs upstream of the RNA start sites are required for optimal activity. Within this region, the only readily recognizable cis-acting control elements are a GC box and a TATA box. However, in infected cell extracts, deletion of a sequence between -167 and -121, which shares homology with the 30-base-pair trans-activation region (TAR) of H-1 virus (S. L. Rhode and S. M. Richard, J. Virol. 61:2807-2815, 1987), results in a three- to fourfold decrease in transcriptional activity. Interestingly, in vivo transfection experiments demonstrate a three- to eightfold increase in transcription relative to the wild-type promoter when the TAR element homology region is deleted and reveal a functional role for a CCAAT motif which lies immediately downstream of the TAR element. These results indicate both positive and negative regulation of the P38 promoter.
Multigene families in African swine fever virus: family 110
Authors: Almendral J M , Almazán F , Blasco R , Viñuela E
Published: January 2020

The genome of African swine fever virus was screened for the existence of repetitive sequences by hybridization between different cloned restriction fragments covering the viral DNA. Several sets of repeated sequences were detected in fragments located close to the DNA ends. One of these groups of repetitions involved fragments located at both ends of the genome. The remaining groups involved fragments that were located exclusively at the left end. The sequence of a 3.2-kilobase segment spanning from 7.5 to 11 kilobases from the left DNA end, which showed a complex pattern of cross-hybridizations, was determined. Two short and three long blocks of direct repeated sequences were found in this DNA region, which accounted for the hybridization results. The repeated sequences formed a family of five homologous genes with an average length of 116 codons (multigene family 110), one of which had a dimeric structure. Transcripts of the five members of the family were detected both in RNA synthesized in vitro by purified African swine fever virions and in RNA isolated at early times after infection. Comparison of the predicted protein sequences revealed a striking conservation of a cysteine-rich domain in the central part of the proteins. In addition, a highly hydrophobic NH2-terminal sequence present in all the proteins suggests that these proteins are processed through the endoplasmic reticulum.
Deletions in herpes simplex virus glycoprotein D define nonessential and essential domains
Authors: Feenstra V , Hodaie M , Johnson D C
Published: January 2020

Herpes simplex virus glycoprotein D (gD) is a major component of the virion envelope and infected cell membranes and is essential for virus entry into cells. We have recently shown that gD interacts with a limited number of cell surface receptors which are required for virus penetration into cells. To define domains of gD which are required for aspects of virus replication including receptor binding, deletion mutations of 5 to 14 amino acids were constructed by using oligonucleotide-directed mutagenesis. Plasmids containing mutant genes for gD were assayed for the ability to rescue a recombinant virus, F-gD beta, in which beta-galactosidase sequences replace gD-coding sequences. Effects on global folding and posttranslational processing of the molecules were assessed by using a panel of monoclonal antibodies which recognize both continuous and discontinuous epitopes. A region near the amino terminus (residues 7 to 21) of gD which is recognized by monoclonal antibodies able to neutralize herpes simplex virus in the absence of complement was not essential for function. In addition, virtually all of the cytoplasmic domain of gD and an extracellular domain close to the membrane were dispensable. In contrast, deletion mutations in the central region of the molecule, save for one exception, led to alterations in global folding of the molecule and maturation of the protein was inhibited.
Structure and function of the Epstein-Barr virus BZLF1 protein
Authors: Packham G , Economou A , Rooney C M , Rowe D T , Farrell P J
Published: January 2020

Five DNA-binding sites for the Epstein-Barr virus BZLF1 protein have been identified within three of the early viral promoters, and four of these binding sites contain a consensus AP-1 site. The part of the BZLF1 protein required for sequence-specific DNA binding to one of these AP-1-like sites was identified by deletion mapping. Site-directed mutagenesis of this DNA target suggests that BZLF1 may work partly by overcoming a cellular repressor of viral transcription.
Sequences in the U5-gag-pol region influence early and late pathogenic effects of Friend and Moloney murine leukemia viruses
Authors: Sitbon M , Ellerbrok H , Pozo F , Nishio J , Hayes S F , Evans L H , Chesebro B
Published: January 2020

Friend replication-competent murine leukemia virus (F-MuLV), clone 57, induces a severe early hemolytic anemia and a later erythroleukemia after inoculation of newborn IRW or ICFW mice, whereas Moloney MuLV (M-MuLV) induces only lymphoid leukemia. We have shown previously that the attenuated hemolytic and erythroleukemogenic abilities of an F-MuLV variant, clone B3, were due mostly to changes in the env gene and long terminal repeat, respectively. For the present study, we derived two constructs exchanging env fragments of F-MuLV 57 and M-MuLV and compared them with two constructs described by Chatis et al. (J. Virol. 52:248-254, 1984) exchanging the U3 region of the long terminal repeat of the same parental viruses. When comparing the hemolytic effect of these constructs with those of the parent, we found that the U5-gag-pol region of F-MuLV was required for development of severe early hemolytic anemia and that, unlike the env of F-MuLV B3, the env of M-MuLV was fully competent in inducing severe early hemolytic anemia when associated with the F-MuLV U5-gag-pol and U3 regions. As expected, induction of erythroleukemia depended on the presence of the F-MuLV U3 region; however, the presence of both the U3 and U5-gag-pol regions of F-MuLV appeared to be synergistic and was associated with a more rapid appearance of erythroleukemia.
Conservation of function of Drosophila melanogaster abl and murine v-abl proteins in transformation of mammalian cells
Authors: Holland G D , Henkemeyer M J , Kaehler D A , Hoffmann F M , Risser R
Published: January 2020

The Drosophila melanogaster abl and the murine v-abl genes encode tyrosine protein kinases (TPKs) whose amino acid sequences are highly conserved. To assess functional conservation between the two gene products, we constructed Drosophila abl/v-abl-chimeric Abelson murine leukemia viruses. In these chimeric Abelson murine leukemia viruses, the TPK and carboxy-terminal regions of v-abl were replaced with the corresponding regions of D. melanogaster abl. The chimeric Abelson murine leukemia viruses were able to mediate morphological and oncogenic transformation of NIH 3T3 cells and were able to abrogate the interleukin-3 dependence of a lymphoid cell line. We also found that a virus that contained both TPK and carboxy-terminal Drosophila abl regions had no in vitro transforming activity for primary bone marrow cells and lacked the ability to induce tumors in susceptible mice. A virus that replaced only a portion of the v-abl TPK region with that of Drosophila abl had low activity in in vitro bone marrow transformation and tumorigenesis assays. These results indicate that the transforming functions of abl TPKs are only partially conserved through evolution. These results also imply that the TPK region of v-abl is a major determinant of its efficient lymphoid cell-transforming activity.
Definition of the sequence requirements for binding of the EBNA-1 protein to its palindromic target sites in Epstein-Barr virus DNA
Authors: Ambinder R F , Shah W A , Rawlins D R , Hayward G S , Hayward S D
Published: January 2020

Interaction between the trans-acting DNA-binding protein EBNA-1 and cis-acting sequences in the ori-P region of Epstein-Barr virus DNA is required for maintenance of the viral plasmid state in latently infected B cells and is involved in the regulation of transcription during latency. In the Epstein-Barr virus genome, a total of 26 EBNA-1-binding sites occur within three clustered loci referred to as the family of repeats and dyad symmetry locus of ori-P and the separate BamHI-Q locus. Incubation of a bacterially expressed carboxy-terminal domain of EBNA-1 (28,000-molecular-weight EBNA-1 [28K EBNA-1]) with synthetic monomer and dimer consensus binding sites gave characteristic DNA-protein complexes in a mobility retardation assay. A similar approach with the naturally occurring Q locus confirmed that it contains two distinct but low-affinity binding sites. We then examined the precise sequence requirements for EBNA-1 binding, using a set of 30-base-pair oligonucleotides designed to contain symmetric point mutations within both halves of the palindromic target site. Analysis of all possible single substitutions between positions 1 and 10 in the consensus half-palindrome sequence revealed that positions 9 and 10 did not contribute to EBNA-1 binding and that considerable flexibility could be tolerated at positions 1 and 2. Positions 3 through 8 of the recognition site had the most stringent requirements, with transversions at these positions either reducing or eliminating binding. The relative spacing of the halves of the palindrome was also critical, since the addition or removal of 2 base pairs at the center of the sequence abolished binding. Similar results were obtained when a partially purified preparation of intact Raji EBNA-1 was substituted for the 28K EBNA-1, and the results were further supported by methylation interference studies which indicated contact points between EBNA-1 and the guanine residues at positions -8, -7, and +3 of the binding site. The three naturally occurring EBNA-1-binding loci have previously been shown to differ in their relative affinities for EBNA-1. The present study indicates that the sequence variations occurring within the family of repeats would not affect binding affinity, whereas certain base substitutions within the Q and dyad symmetry sites would be predicted to contribute to the observed lower affinities of these sites. An apparent Kd of 1.5 x 10(-11) M for binding of 28K EBNA-1 to a consensus recognition site was calculated from Scatchard analysis.
The adenovirus EII early promoter has multiple EIA-sensitive elements, two of which function cooperatively in basal and virus-induced transcription
Authors: Manohar C F , Kratochvil J , Thimmapaya B
Published: January 2020

The mechanism by which the adenovirus-encoded nuclear oncogene EIA activates transcription of several viral and host promoters is an important issue in the regulation of eucaryotic gene expression and virus-host cell interactions. Identification of cis-acting elements of the promoters and the cognate host transcription factors that are targets for EIA action is crucial for our understanding of the EIA-mediated control of coordinately regulated genes. The adenovirus EII early promoter has a complex architecture and contains two overlapping promoters with start sites at +1 (major promoter) and -26 (minor promoter). The major promoter responds strongly to virus-encoded trans activators EIA and EIV and contains four elements: a TAGA motif analogous to the TATA box, two EIIF sites present in an inverted orientation, and an ATF/CREB site. To determine precisely the roles played by these cis-acting elements in both basal and virus-induced transcription when the promoter is situated in its natural context, we investigated the phenotype of a series of linker scan promoter substitution mutants inserted into the viral chromosome. Promoter constructs harboring linker scan mutations in each element were rebuilt into a novel EIA- adenovirus vector, and transcriptional activity was monitored in virus-infected cells. In the absence of virus-encoded trans activators, basal activity in vivo was dependent on all four cis-acting elements. Surprisingly, a promoter mutant with only one of the two EIIF sites intact could not promote transcription in vivo, suggesting that the two EIIF sites function cooperatively even in basal transcription. Promoters harboring mutations in either of these two EIIF sites also failed to bind to an infection-specific form of EIIF in gel shift assays and competed only very weakly for EIIF binding with the wild-type promoter fragment. The dramatic cooperativity shown by the two inverted EIIF sites of the EII promoter both in vivo and in vitro could reflect simultaneous contact of both sites by the transcription factor EIIF. Furthermore, promoter mutants with mutations in the TAGA motif, the two EIIF sites, and the single ATF site all failed to respond to virus-encoded trans activators. Whereas recent results demonstrate that EIIF activity can be modulated independently by EIV, leading to transactivation of this promoter, our results and those published previously strongly indicate that the three different transcription factors that bind to TAGA, EIIF, and ATF motifs of the EII early promoter are all targets for EIA regulation in vivo. Thus, strong transactivation of the EII early promoter through these multiple EIA-sensitive elements and independently by the recently discovered EIV pathway suggests that the EII early promoter is stringently regulated in virus-infected cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1
Authors: Schwartz S , Felber B K , Benko D M , Fenyö E M , Pavlakis G N
Published: January 2020

We have used the polymerase chain reaction technique to clone the small multiply spliced mRNA species produced after infection of human cells by a molecular clone of human immunodeficiency virus type 1 (HIV-1). We identified six Rev-expressing mRNAs, which were generated by the use of two splice acceptors located immediately upstream of the rev AUG. The class of small mRNAs included 12 mRNAs expressing Tat, Rev, and Nef. In addition, HIV-1 produced other multiply spliced mRNAs that used alternative splice sites identified by cloning and sequencing. All of these mRNAs were found in the cytoplasm and should be able to produce additional proteins. The coding capacity of the tat, rev, and nef mRNAs was analyzed by transfection of the cloned cDNAs into human cells. The tat mRNAs produced high levels of Tat, but very low levels of Rev and Nef. All the rev mRNAs expressed high levels of both Rev and Nef and were essential for the production of sufficient amounts of Rev. Therefore, HIV-1 uses both alternatively spliced and bicistronic mRNAs for the production of Tat, Rev, and Nef proteins.
Human immunodeficiency virus-like, nonreplicating, gag-env particles assemble in a recombinant vaccinia virus expression system
Authors: Haffar O , Garrigues J , Travis B , Moran P , Zarling J , Hu S L
Published: January 2020

We report the assembly of human immunodeficiency virus (HIV)-like particles in African green monkey kidney cells coinfected with two recombinant vaccinia viruses, one carrying the HIV-1 gag and protease genes and the other the env gene. Biochemical analysis of particles sedimented from culture supernatants of doubly infected cells revealed that they were composed of gag proteins, primarily p24, as well as the env proteins gp120 and gp41. Thin-section immunoelectron microscopy showed that these particles were 100 to 120 nm in diameter, were characterized by the presence of cylindrical core structures, and displayed the mature gp120-gp41 complexes on their surfaces. Furthermore, thin-section immunoelectron microscopy analysis of infected cells showed that particle assembly and budding occurred at the plasma membrane. Nucleic acid hybridization suggested that the particles packaged only the gag mRNA but not the env mRNA. Therefore, the system we present is well suited for studies of HIV virion maturation. In addition, the HIV-like particles provide a novel and attractive approach for vaccine development.
Echovirus 22 is an atypical enterovirus
Authors: Coller B A , Chapman N M , Beck M A , Pallansch M A , Gauntt C J , Tracy S M
Published: January 2020

Although echovirus 22 (EV22) is classified as an enterovirus in the family Picornaviridae, it is atypical of the enterovirus paradigm, typified by the polioviruses and the coxsackie B viruses. cDNA reverse transcribed from coxsackievirus B3 (CVB3) RNA does not hybridize to genomic RNA of EV22, and conversely, cDNA made to EV22 does not hybridize to CVB3 genomic RNA or to molecular clones of CVB3 or poliovirus type 1. EV22 cDNA does not hybridize to viral RNA of encephalomyocarditis virus or to a molecular clone of Theiler's murine encephalomyelitis virus, members of the cardiovirus genus. The genomic RNA of EV22 cannot be detected by the polymerase chain reaction using generic enteroviral primers. EV22 does not shut off host cell protein synthesis, and the RNA of EV22 is efficiently translated in vitro in rabbit reticulocyte lysates. Murine enterovirus-immune T cells recognize and proliferate against EV22 as an antigen in vitro, demonstrating that EV22 shares an epitope(s) common to enteroviruses but not found among other picornaviruses.
Herpes simplex virus type 1 encodes two Fc receptors which have different binding characteristics for monomeric immunoglobulin G (IgG) and IgG complexes
Authors: Dubin G , Frank I , Friedman H M
Published: January 2020

Two herpes simplex virus type 1 glycoproteins, gE and gI, have been shown to form a complex that binds the Fc domain of immunoglobulin G (IgG). We demonstrate that this complex is required for the binding of monomeric nonimmune IgG but that gE alone is sufficient for binding polymeric IgG in the form of IgG complexes. Evidence that gE but not gI is required for binding IgG complexes is as follows. IgG complexes bound equally well to cells infected with gI-negative mutants or with wild-type virus, whereas cells infected with gE-negative mutants did not bind IgG complexes. Furthermore, L cells transiently transfected to express gE bound IgG complexes. Additional evidence that gI fails to augment binding of IgG complexes comes from experiments in which the gI gene was inducibly expressed in cells after infection. Inducible gI expression failed to increase binding of IgG complexes to infected cells in comparison with cells not capable of inducible gI expression. In contrast, expression of both gE and gI was necessary for binding of monomeric IgG, as demonstrated by flow cytometry using cells infected with gE-negative and gI-negative mutants. These observations demonstrate that herpes simplex virus type 1 Fc receptors (FcRs) have different binding characteristics for monomeric IgG and IgG complexes. Furthermore, it appears that gE is the FcR for IgG complexes and that gE and gI form the FcR for monomeric IgG.
Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification
Authors: Summers J , Smith P M , Horwich A L
Published: January 2020

Primary duck hepatocytes were infected with a mutant duck hepatitis B virus defective in envelope protein but competent for viral DNA synthesis. Cells infected by this mutant accumulated higher levels of viral covalently closed, circular DNA (cccDNA) than those infected by wild-type virus. The accumulation of high levels of cccDNA was due to a failure of the mutant-infected cells to suppress de novo cccDNA synthesis compared with suppression by cells infected by the wild type. The envelope-defective virus failed to establish a persistent infection in vitro, possibly because of a virus-mediated cell death. Therefore, one or both viral envelope proteins are required for regulation of cccDNA synthesis and for maintenance of persistent infection in vitro.