bluuebunny/crossref_metadata_2025 huggingface dataset:
Ocular avirulence of a herpes simplex virus type 1 strain is associated with heightened sensitivity to alpha/beta interferon
Authors: Su Y H , Oakes J E , Lausch R N
Published: January 2020

BALB/c mice infected on the scarified cornea with herpes simplex virus type 1 strain 35 [HSV-1(35)] rarely developed ocular disease even at challenge doses as high as 10(7) PFU per eye. In contrast, HSV-1(RE) consistently induced stromal keratitis at an inoculum of 2 x 10(4) PFU. The goal of this study was to determine the reason for the difference in virulence between the two HSV strains. Both HSV-1 strains replicated to similar titers in excised corneal "buttons." However, after in vivo infection of the cornea, the growth of strain 35 was evident only during the first 24 h postinfection, whereas the replication of strain RE persisted for at least 4 days. In vitro tests revealed that HSV-1(35) was greater than 10 times more sensitive to alpha/beta interferon (IFN-alpha/beta) than HSV-1(RE). Both strains induced comparable serum levels of IFN after intraperitoneal inoculation. The kinetics of HSV-1(35) clearance from the eye was markedly altered by treatment with rabbit anti-IFN-alpha/beta. Virus titers exceeding 10(4) PFU per eye could be demonstrated 4 to 5 days postinfection in mice given a single inoculation of antiserum 1 h after infection. Furthermore, anti-IFN treatment in 3-week-old mice infected with HSV-1(35) led to the development of clinically apparent corneal disease which subsequently progressed to stromal keratitis in the majority of recipients. These results indicate that the striking difference in the capacity of HSV-1(35) and HSV-1(RE) to induce corneal disease was related to the inherently greater sensitivity of strain 35 to IFN-alpha/beta produced by the host in response to infection.
Isolation and characterization of herpes simplex virus mutants containing engineered mutations at the DNA polymerase locus
Authors: Marcy A I , Yager D R , Coen D M
Published: January 2020

We have derived Vero cell lines containing the herpes simplex virus DNA polymerase (pol) gene that complement temperature-sensitive pol mutants. These cell lines were used to recover viruses containing new mutations at the pol locus. Two spontaneously arising host-range mutants, 6C4 and 7E4, were isolated. These mutants did not grow efficiently on Vero cells or synthesize late polypeptides but formed plaques on a cell line containing the pol gene (DP6 cells). Whereas mutant 6C4 specified a wild-type-size Pol protein, we detected no full-length Pol protein in 7E4-infected cell extracts. Complementation studies demonstrated that 6C4 and 7E4 contain different mutations and indicated that 6C4 is in a complementation group different from that of pol temperature-sensitive mutant tsC7 or tsD9. A mutant in which 2.2 kilobases of pol sequences were replaced with the Escherichia coli lacZ gene under the control of the herpes simplex virus thymidine kinase promoter was constructed. This mutant formed blue plaques on DP6 cells in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. Using this virus in marker rescue experiments, we engineered three mutants containing deletions in the pol coding region which grew efficiently on DP6 cells but not on Vero cells and which differed in their synthesis of Pol polypeptides. The lacZ insertion virus was also used to introduce a deletion in the region upstream of the pol long open reading frame, which removes a short open reading frame that could encode a 10-amino-acid peptide. This mutant grew to similar titers on Vero and DP6 cells, indicating that these sequences are not essential for growth of the virus in tissue culture.
Mechanism of chemical activation of expression of the endogenous ecotropic murine leukemia provirus Emv-3
Authors: Mercer J A , Lee K H , Nexø B A , Jenkins N A , Copeland N G
Published: January 2020

DBA/2 mice carry a single endogenous ecotropic murine leukemia provirus, Emv-3. This provirus is defective; it is very poorly expressed in young DBA/2 mice. The defect in Emv-3 is caused by a single base substitution in codon 3 of p15gag. The resulting amino acid substitution inhibits myristylation of the gag precursor and subsequent virus assembly. Despite this defect, percutaneous treatment of DBA/2 mice with the carcinogen and mutagen 7,12-dimethylbenz[a]anthracene (DMBA) induces ecotropic murine leukemia virus replication in virtually all treated mice. We hypothesized that this induction is the result of a DMBA-induced reverse mutation in codon 3 of p15gag which allows for efficient myristylation. We tested this hypothesis by isolating ecotropic viruses from DMBA-treated mice and determining the DNA sequences of selected regions of p15gag, including codon 3. In support of the above-described model, all of the viruses examined contained single nucleotide substitutions in codon 3. In addition, most of the replication-competent viruses that were sequenced appeared to result from simple mutation of Emv-3 rather than recombination with other endogenous murine leukemia viruses. These studies may provide a basis for development of a sensitive assay for the mutagenic activity of a variety of chemical carcinogens in vivo.
Assembly of gag-beta-galactosidase proteins into retrovirus particles
Authors: Jones T A , Blaug G , Hansen M , Barklis E
Published: January 2020

We studied the expression of beta-galactosidase (beta-gal) and 15 gag-beta-gal fusion proteins in the presence of Moloney murine leukemia virus wild-type core (gag) proteins. Analysis indicated that proteins retaining the amino-terminal portion of gag through the capsid protein-coding region were incorporated into retrovirus particles. Proteins which deleted portions of the capsid protein were assembled into virions at low efficiency, indicating the importance of capsid protein interactions in retrovirus assembly. Fusion proteins which retained the amino-terminal matrix protein of the gag polyprotein but which lacked the capsid protein were released efficiently from cells in a nonviral form. The nonviral form was characterized by a high sedimentation coefficient and a low density, suggestive of membrane vesicles. While beta-gal was present in the cytoplasm of expressing cells, all fusion constructs were associated with cellular membranes. gag-beta-gal proteins which were capable of release from cells demonstrated a two-component immunofluorescence staining pattern consisting of a circle of fluorescence around the nucleus and a punctate pattern of staining throughout the remainder of the cell. Interestingly, fusions within the matrix protein were trapped intracellularly and yielded distinct perinuclear staining patterns, possibly localizing to the rough endoplasmic reticulum and/or Golgi. This observation suggests that Moloney murine leukemia virus gag proteins travel to the plasma membrane by vesicular transport associated with the cytoplasmic face of intracellular vesicles.
Phytohemagglutinin-induced activity of cyclic AMP (cAMP) response elements from cytomegalovirus is reduced by cyclosporine and synergistically enhanced by cAMP
Authors: Niller H H , Hennighausen L
Published: January 2020

The 19-base-pair enhancer repeat of the human cytomegalovirus immediate-early 1 gene mediates cyclic AMP- and phytohemagglutinin-induced expression in Jurkat T cells. Synergistic activity was observed in the presence of both drugs, suggesting a convergence of the protein kinase A and C pathways on this transcription element. In addition, the immunosuppressive drug cyclosporine strongly reduced the ability of the 19-base-pair repeat to activate gene expression in phytohemagglutinin-stimulated T cells.
Myristoylation of the poliovirus polyprotein is required for proteolytic processing of the capsid and for viral infectivity
Authors: Kräusslich H G , Hölscher C , Reuer Q , Harber J , Wimmer E
Published: January 2020

The poliovirus polyprotein is cotranslationally linked to myristic acid at its amino-terminal glycine residue. We investigated the role of myristoylation in the viral replication cycle by site-directed mutagenesis of this glycine codon. Synthetic full-length RNA transcripts carrying a Gly-to-Ala mutation (G4002A) gave no infectious virus on transfection into permissive cells (HeLa). However, mutant viral RNA was replicated in the transfected cells, albeit at a reduced level. The virus-specific polypeptide P1, the precursor for the capsid proteins, was found in HeLa cells transfected with wild-type or mutant RNA, but only the wild-type P1 was myristoylated; the G4002A mutant P1 was not myristoylated. We also introduced the G4002A mutation into an in vitro transcription-translation vector encoding poliovirus P1 precursor. Processing of the mutant precursor by poliovirus-infected cell lysate (providing 3Cpro and 3CDpro activities) was severely inhibited, whereas the normally inefficient cleavage by purified 3Cpro was not affected. These results suggest that the myristic acid moiety of the P1 precursor may be required for efficient processing by 3CDpro.
The level of c-fgr RNA is increased by EBNA-2, an Epstein-Barr virus gene required for B-cell immortalization
Authors: Knutson J C
Published: January 2020

The efficient immortalization of primary resting human B lymphocytes by Epstein-Barr virus (EBV) requires several viral genes and presumably the altered expression of an unknown number of cellular genes as well. In this paper, I show that infection of primary human B cells with EBV increased the transcript level of the proto-oncogene, c-fgr, 10-fold. This effect on the level of c-fgr transcripts in B cells was not secondary to blast formation, because levels of c-fgr RNA were also increased 10-fold in two proliferating EBV-negative Burkitt's lymphoma-derived cell lines, Ramos and BJAB, 2 days after infection with EBV. Two lines of evidence indicated that EBV nuclear antigen 2 (EBNA-2) mediates this increase in c-fgr RNA levels: acute infection of BJAB and Ramos cells by a mutant strain of EBV that lacked the EBNA-2 open reading frame, P3HR1, did not affect c-fgr RNA levels; and cell lines constitutively expressing only the EBNA-2 gene of EBV had increased levels of c-fgr RNA relative to those in the parental cell lines. Since P3HR1, a nonimmortalizing strain of EBV, failed to affect c-fgr RNA levels and since a viral gene required for B-cell immortalization was responsible for the induction of c-fgr, the data indicate a possible role of c-fgr expression in B-lymphocyte immortalization by EBV and a mechanism by which EBNA-2 contributes to the immortalizing activity of EBV.
Antibodies that block rhinovirus attachment map to domain 1 of the major group receptor
Authors: Lineberger D W , Graham D J , Tomassini J E , Colonno R J
Published: January 2020

The vast majority of human rhinovirus serotypes utilize the intercellular adhesion molecule 1 (ICAM-1) as the attachment site on susceptible cells. Twelve murine monoclonal antibodies were isolated and shown by competition binding studies to recognize three distinct, nonoverlapping epitopes on the ICAM-1 receptor. Titration of three antibodies representing each of the binding sites demonstrated that they were equally effective at blocking viral attachment. By using in vitro transcription and translation systems, a series of progressive C-terminal truncations of ICAM-1 molecules was generated. Immunoprecipitation of these fragments with each of the three antibodies indicated that all three epitopes reside within the first 82 amino acids of the receptor. Attempts to demonstrate specific binding of these in vitro-synthesized receptor fragments to virions were unsuccessful. The inability to show virion binding was most likely due to a failure of the lysates to properly glycosylate the receptor molecule, since native, unglycosylated receptor molecules isolated from cell membranes were also inactive in virus binding assays.
Specific effect of interferon on the herpes simplex virus type 1 transactivation event
Authors: De Stasio P R , Taylor M W
Published: January 2020

Human recombinant gamma interferon and to a lesser extent alpha interferon were shown to inhibit herpes simplex virus type 1 replication in the human cell lines WISH and HEp-2. Dot blot analysis of viral DNA synthesis and viral RNA transcription indicated that the inhibition occurred at an early step in infection. A study of the early events after herpes simplex virus type 1 infection indicated that adsorption, penetration, uncoating, and transport of viral DNA were not affected by interferon. Northern (RNA) blot analysis revealed that both immediate-early and delayed-early gene transcription was inhibited by interferon. Transactivation of the immediate-early responsive element linked to a reporter gene (CAT or tk) was specifically inhibited by both classes of interferon. Our data would indicate that either the transactivating protein VP16 or the complex formed between VP16 and a host protein(s) is attenuated by interferon.
Spleen necrosis virus gag polyprotein is necessary for particle assembly and release but not for proteolytic processing
Authors: Weaver T A , Talbot K J , Panganiban A T
Published: January 2020

The nature of spleen necrosis virus pol gene expression and the role of gag and gag-pol polyproteins in virion assembly was investigated. The DNA sequence of the gag-pol junction revealed that the two genes occupy the same open reading frame but are separated by an in-frame amber stop codon. Biochemical analysis of gag-pol translational readthrough in vitro and in Escherichia coli suggests that, in a manner similar to that in other mammalian type C retroviruses, amber stop codon suppression is required for pol gene expression. Removal of the gag stop codon had little or no effect on synthesis or cleavage of the polyprotein but interrupted particle assembly. This block could be overcome by complementation with wild-type gag protein.
Cytomegalovirus activates transcription directed by the long terminal repeat of human immunodeficiency virus type 1
Authors: Barry P A , Pratt-Lowe E , Peterlin B M , Luciw P A
Published: January 2020

Proteins encoded by a variety of DNA viruses activate gene expression from the promoter within the long terminal repeat (LTR) of the human immunodeficiency virus type 1 (HIV-1). The mechanism by which immediate-early (IE) gene products of human cytomegalovirus (CMV) activate expression from the HIV-1 LTR was examined in transient expression assays in cultures of human cells by using plasmids containing the LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and a plasmid expressing the CMV IE gene. Analysis of clustered site mutations within the HIV-1 LTR revealed that sequences from nucleotides -6 to +20 (relative to the start site of transcription) are critical for responsiveness to transactivation by CMV IE gene products. This region partially overlaps the trans-acting response element (+19 to +42) required for function of the HIV-1 transactivator. The CMV IE gene was shown to increase the steady-state levels of both prematurely terminated and full-length transcripts initiated within the LTR. These results support a model in which CMV IE gene products act through a specific regulatory element in the HIV-1 LTR to increase viral transcription.
Hemagglutinin mutations related to attenuation and altered cell tropism of a virulent avian influenza A virus
Authors: Philpott M , Hioe C , Sheerar M , Hinshaw V S
Published: January 2020

The H5 hemagglutinin (HA) of a highly virulent avian influenza virus, A/Turkey Ontario/7732/66 (H5N9), was previously shown to have five neutralizing epitopes, and escape mutants within one epitope (group 1) were markedly attenuated (M. Philpott, B. C. Easterday, and V. S. Hinshaw, J. Virol. 63:3453-3458, 1989). To define the genetic changes related to these antigenic and biologic properties, the HA genes of mutants within each of the epitope groups were sequenced by using the polymerase chain reaction. The mutations in the attenuated group 1 mutants were located near the distal tip of the HA molecule in close proximity to the receptor-binding site, on the basis of alignment with the three-dimensional structure of the H3 HA. All group 1 mutations involved charged amino acids. The group 1 mutants, similar to the wild-type virus, spread systemically and were recovered from the spleens of infected chickens but, unlike the wild-type virus, failed to produce severe necrosis in the spleens. Viral replication in the spleens was investigated by in situ hybridization of spleen sections from chickens infected with the wild-type or attenuated mutants. Wild-type virus replication was demonstrated in large, mononuclear, macrophagelike cells; however, group 1 mutant virus was detected attached only to erythrocytes within the red pulp. These results suggest that the attenuated mutants differ in their cell tropism within the spleen.
The adeno-associated virus rep gene suppresses herpes simplex virus-induced DNA amplification
Authors: Heilbronn R , Bürkle A , Stephan S , zur Hausen H
Published: January 2020

Herpes simplex virus (HSV) induces within the host cell genome DNA amplification which can be suppressed by coinfection with adeno-associated virus (AAV). To characterize the AAV functions mediating this effect, cloned AAV type 2 wild-type or mutant genomes were transfected into simian virus 40 (SV40)-transformed hamster cells together with the six HSV replication genes (encoding UL5, UL8, major DNA-binding protein, DNA polymerase, UL42, and UL52) which together are necessary and sufficient for the induction of SV40 DNA amplification (R. Heilbronn and H. zur Hausen, J. Virol. 63:3683-3692, 1989). The AAV rep gene was identified as being responsible for the complete inhibition of HSV-induced SV40 DNA amplification. Likewise, rep inhibited origin-dependent HSV replication. rep neither killed the transfected host cells nor interfered with gene expression from the cotransfected amplification genes. This points to a specific interference with HSV-induced DNA amplification.
Genetic mapping of a cloned sequence responsible for susceptibility to ecotropic murine leukemia viruses
Authors: Kozak C A , Albritton L M , Cunningham J
Published: January 2020

A mouse cDNA that confers susceptibility to ecotropic murine leukemia viruses following transfection into human EJ cells has been cloned and sequenced. We show that this sequence is likely to be Rec-1, the chromosome 5 locus originally defined by studies with somatic cell hybrids as responsible for virus susceptibility, and provide a specific chromosomal map position for this locus by analysis of an interspecies backcross. This locus maps in the distal region of chromosome 5 and is thus not within the cluster of retrovirus-related genes near the centromere.
The site of an immune-selected point mutation in the transmembrane protein of human immunodeficiency virus type 1 does not constitute the neutralization epitope
Authors: Wilson C , Reitz M S , Aldrich K , Klasse P J , Blomberg J , Gallo R C , Robert-Guroff M
Published: January 2020

We previously reported the in vitro generation of a neutralization-resistant variant of the molecularly cloned isolate of human immunodeficiency virus type 1 (HIV-1), HXB2D. The molecular basis for the resistance was shown to be a point mutation in the env gene, causing the substitution of threonine for alanine at position 582 of gp41. Here, we show the variant to be resistant to syncytium inhibition as well as to neutralization by the immune-selecting serum. Moreover, 30% of HIV-positive human sera able to neutralize the parental virus have significantly decreased ability to neutralize the variant. As the A-to-T substitution thus has general relevance to the interaction of HIV-1 with the host immune system, we investigated further the biologic and immunologic bases for the altered properties. Synthetic peptides corresponding to the 582 region failed to compete in infectivity, neutralization, or syncytium inhibition assays and did not elicit neutralizing antibodies. Furthermore, human antibodies, affinity purified on synthetic peptide resins, bound to gp41 and peptides from the 582 region but did not possess neutralizing antibody activity. Some viral constructs in which the AVERY sequence in the 582 region was altered by site-directed mutagenesis were not infectious, indicating that the primary structure in this region is crucial for viral infectivity. Constructs predicted to possess a local secondary structure similar to that of the variant nevertheless behaved like the parental virus and remained neutralization sensitive. These results suggest that the requirements for neutralization resistance in this region are very precise. Our results with synthetic peptides show that the 582 region does not by itself constitute a neutralization epitope. Moreover, the degree of flexibility in amino acid substitution which allows maintenance of neutralization sensitivity suggests that position 582 does not form part of a noncontiguous neutralization epitope. The basis for neutralization resistance of the immune-selected variant is more likely a conformational change altering a neutralization epitope at a distant site.
Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1
Authors: Wang F , Tsang S F , Kurilla M G , Cohen J I , Kieff E
Published: January 2020

Several lines of evidence are compatible with the hypothesis that Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) or leader protein (EBNA-LP) affects expression of the EBV latent infection membrane protein LMP1. We now demonstrate the following. (i) Acute transfection and expression of EBNA-2 under control of simian virus 40 or Moloney murine leukemia virus promoters resulted in increased LMP1 expression in P3HR-1-infected Burkitt's lymphoma cells and the P3HR-1 or Daudi cell line. (ii) Transfection and expression of EBNA-LP alone had no effect on LMP1 expression and did not act synergistically with EBNA-2 to affect LMP1 expression. (iii) LMP1 expression in Daudi and P3HR-1-infected cells was controlled at the mRNA level, and EBNA-2 expression in Daudi cells increased LMP1 mRNA. (iv) No other EBV genes were required for EBNA-2 transactivation of LMP1 since cotransfection of recombinant EBNA-2 expression vectors and genomic LMP1 DNA fragments enhanced LMP1 expression in the EBV-negative B-lymphoma cell lines BJAB, Louckes, and BL30. (v) An EBNA-2-responsive element was found within the -512 to +40 LMP1 DNA since this DNA linked to a chloramphenicol acetyltransferase reporter gene was transactivated by cotransfection with an EBNA-2 expression vector. (vi) The EBV type 2 EBNA-2 transactivated LMP1 as well as the EBV type 1 EBNA-2. (vii) Two deletions within the EBNA-2 gene which rendered EBV transformation incompetent did not transactivate LMP1, whereas a transformation-competent EBNA-2 deletion mutant did transactivate LMP1. LMP1 is a potent effector of B-lymphocyte activation and can act synergistically with EBNA-2 to induce cellular CD23 gene expression. Thus, EBNA-2 transactivation of LMP1 amplifies the biological impact of EBNA-2 and underscores its central role in EBV-induced growth transformation.
Human cytomegalovirus immediate-early two protein region involved in negative regulation of the major immediate-early promoter
Authors: Hermiston T W , Malone C L , Stinski M F
Published: January 2020

The immediate-early two (IE2) gene products of human cytomegalovirus negatively regulate gene expression from the major immediate-early promoter in permissive human fibroblasts. A mutational analysis of the IE2 proteins indicated that the carboxyl-terminal region is required for negative regulation. The IE2 proteins that lack amino acid residues 365 to 519, or the carboxyl-terminal amino acids failed to negatively regulate. Most of the amino-terminal portion of the IE2 protein was not required for negative regulation. A possible explanation of the negative effect on downstream expression by the IE2 proteins is discussed.
Identification of a site on herpes simplex virus type 1 glycoprotein D that is essential for infectivity
Authors: Muggeridge M I , Wilcox W C , Cohen G H , Eisenberg R J
Published: January 2020

Herpes simplex virus glycoprotein D (gD) plays an essential role during penetration of the virus into cells. There is evidence that it recognizes a specific receptor after initial attachment of virions to cell surface heparan sulfate and also that gD-1, gD-2, and gp50 (the pseudorabies virus gD homolog) bind to the same receptor. Although the antigenic structure of gD has been studied intensively, little is known about functional regions of the protein. Antigenic site I is a major target for neutralizing antibodies and has been partially mapped by using deletion mutants and neutralization-resistant viruses. Working on the assumption that such a site may overlap with a functional region of gD, we showed previously that combining two or more amino acid substitutions within site I prevents gD-1 from functioning and is therefore lethal. We have now used a complementation assay to measure the functional activity of a panel of deletion mutants and compared the results with an antigenic analysis. Several mutations cause gross changes in protein folding and destroy functional activity, whereas deletions at the N and C termini have little or no effect on either. In contrast, deletion of residues 234 to 244 has only localized effects on antigenicity but completely abolishes functional activity. This region, which is part of antigenic site Ib, is therefore essential for gD-1 function. The complementation assay was also used to show that a gD-negative type 1 virus can be rescued by gD-2 and by two gD-1-gD-2 hybrids but not by gp50, providing some support for the existence of a common receptor for herpes simplex virus types 1 and 2 but not pseudorabies virus. Alternatively, gp50 may lack a signal for incorporation into herpes simplex virions.
Cloning and characterization of cDNAs encoding equine infectious anemia virus tat and putative Rev proteins
Authors: Stephens R M , Derse D , Rice N R
Published: January 2020

We isolated and characterized six cDNA clones from an equine infectious anemia virus-infected cell line that displays a Rev-defective phenotype. With the exception of one splice site in one of the clones, all six cDNAs exhibited the same splicing pattern and consisted of four exons. Exon 1 contained the 5' end of the genome; exon 2 contained the tat gene from mid-genome; exon 3 consisted of a small section of env, near the 5' end of the env gene; and exon 4 contained the putative rev open reading frame from the 3' end of the genome. The structures of the cDNAs predict a bicistronic message in which Tat is encoded by exons 1 and 2 and the presumptive Rev protein is encoded by exons 3 and 4. tat translation appears to be initiated at a non-AUG codon within the first 15 codons of exon 1. Equine infectious anemia virus-specific tat activity was expressed in transient transfections with cDNA expression plasmids. The predicted wild-type Rev protein contains 30 env-derived amino acids and 135 rev open reading frame residues. All of the cDNAs had a frameshift in exon 4, leading to a truncated protein and thus providing a plausible explanation for the Rev-defective phenotype of the original cells. We used peptide antisera to detect the faulty protein, thus confirming the cDNA sequence, and to detect the normal protein in productively infected cells.
Feedback regulation of human immunodeficiency virus type 1 expression by the Rev protein
Authors: Felber B K , Drysdale C M , Pavlakis G N
Published: January 2020

Rev is an essential regulatory protein of the human immunodeficiency virus type 1 (HIV-1) that affects the transport and half-life of certain viral mRNAs. Rev exerts its function via a unique element, the Rev-responsive element (RRE), located within the env region of HIV-1. It has been previously demonstrated that Rev affects the relative levels of RRE-containing and RRE-lacking mRNAs. We have studied the effects of Rev on the expression of the three different groups of small, multiply spliced mRNAs that lack the RRE sequence and encode the regulatory proteins Tat, Rev, and Nef. To monitor the tat, rev, and nef mRNAs we generated specific S1 nuclease mapping probes that distinguish among them. Analysis of all the mRNA species producing Tat, Rev, and Nef revealed that their levels are coordinately regulated by Rev. They are increased in the absence of Rev protein and are down regulated in the presence of Rev. The corresponding proteins were measured by immunoprecipitations, and their levels are in agreement with the RNA levels. These results verify the model proposing that Rev is a general regulator indirectly affecting all the multiply spliced mRNAs to a similar extent. Therefore, Rev down regulates its own expression and the expression of Tat and Nef.
The Epstein-Barr virus (EBV) BMRF1 promoter for early antigen (EA-D) is regulated by the EBV transactivators, BRLF1 and BZLF1, in a cell-specific manner
Authors: Holley-Guthrie E A , Quinlivan E B , Mar E C , Kenney S
Published: January 2020

The Epstein-Barr virus early antigen diffuse component (EA-D) is essential for Epstein-Barr virus DNA polymerase activity, and its activity is suppressed during latent infection. We investigated the regulation of the promoter (BMRF1) for this early gene by studying its responsiveness in vitro to two immediate-early viral transactivators, BZLF1 (Z) and BRLF1 (R), focusing on the differences in response in lymphoid cells and epithelial cells. In lymphoid cells, Z or R alone produced only small increases in EA-D promoter activity, whereas both transactivators together produced a large stimulatory effect. In epithelial cells, the Z transactivator alone produced maximal stimulation of the EA-D promoter; the effect of R and Z together was no greater than that of Z alone. Deletional analysis and site-directed mutagenesis of the EA-D promoter demonstrated that in epithelial cells the potential AP-1 binding site plays an essential role in Z responsiveness, although sequences further upstream are also important. In lymphoid cells, only the upstream sequences are required for transactivation by the Z/R combination, and the AP-1 site is dispensable. These data suggest that EA-D (BMRF1) promoter regulation by Z and R is cell type specific and appears to involve different mechanisms in each cell type.
Phosphatidylinositol metabolism in cells transformed by polyomavirus middle T antigen
Authors: Ulug E T , Hawkins P T , Hanley M R , Courtneidge S A
Published: January 2020

Associated with the middle T antigen of polyomavirus is a novel phosphatidylinositol (PtdIns) kinase activity which phosphorylates PtdIns at the D-3 position of the inositol ring. We have undertaken an analysis of myo-[3H]inositol-containing compounds in a panel of NIH 3T3 cell lines stably transfected with transforming and nontransforming middle T antigen mutants. All cell lines from which PtdIns 3-kinase activity coprecipitated with middle T antigen exhibited modestly elevated levels of PtdIns(3)P and compounds with predicted PtdIns(3,4)P2 and PtdIns(3,4,5)P3 structures. Complex formation between middle T antigen and PtdIns 3-kinase correlated not with an increase in total inositol phosphate levels but rather with elevated levels of InsP2 and InsP4. A specific increase in the level of an InsP2 species which comigrated in high-pressure liquid chromatography analysis with Ins(3,4)P2 was observed. These results suggest that association of the polyomavirus middle T antigen with PtdIns 3-kinase activates a distinct inositol metabolic pathway.
An Epstein-Barr virus-specific cytotoxic T-cell epitope present on A- and B-type transformants
Authors: Burrows S R , Misko I S , Sculley T B , Schmidt C , Moss D J
Published: January 2020

In this report we describe a cytotoxic T-cell epitope in the Epstein-Barr virus nuclear antigen EBNA 6. This epitope is present on both A- and B-type transformants.
N myristoylation of the spleen necrosis virus matrix protein is required for correct association of the Gag polyprotein with intracellular membranes and for particle formation
Authors: Weaver T A , Panganiban A T
Published: January 2020

To determine whether myristoylation is required for spleen necrosis virus replication, we constructed a substitution mutation in the gag gene that alters the putative myristate acceptor glycine residue. This single amino acid change was lethal for virus replication, resulted in aberrant proteolytic processing, and interrupted virion assembly and the release of virus from cells. Immunofluorescence analysis indicated that the amount of Gag polyprotein at the cell periphery and in Golgi-associated vesicles is severely reduced in the myristoylation mutant, indicating that correct intracellular targeting is affected by a lack of myristoylation. Coexpression of wild-type Gag polyprotein did not complement and rescue the replication-defective phenotype of the myristoylation mutant. Thus, it appears that the nonmyristoylated polyproteins are incapable of interacting with their myristoylated counterparts to form biologically active particles.
O glycosylation of glycoprotein G of human respiratory syncytial virus is specified within the divergent ectodomain
Authors: Collins P L
Published: January 2020

cDNAs encoding the G glycoprotein of respiratory syncytial virus and the hemagglutinin-neuraminidase (HN) glycoprotein of parainfluenza virus type 3 were modified by site-specific mutagenesis and restriction fragment replacement to encode chimeric proteins consisting of the cytoplasmic and transmembrane domains of one protein fused to the ectodomain of the other. In the case of the HN ectodomain attached to the G transmembrane and cytoplasmic domains, cell surface expression of the chimera was reduced. Otherwise, the presence of the heterologous transmembrane and cytoplasmic domains had little effect on the processing of the HN or G ectodomain, as assayed by the acquisition of N-linked and O-linked carbohydrates, transport to the cell surface and, in the case of HN, folding, oligomerization, and hemadsorption activity. These results showed that the synthesis and processing of each ectodomain did not require the homologous transmembrane and cytoplasmic domains. In particular, O glycosylation of the G protein was specified fully by its ectodomain, even though this domain is highly divergent among the respiratory syncytial virus antigenic subgroups. In addition, whereas the cytoplasmic and transmembrane domains of the G protein were relatively highly conserved, they were nonetheless fully replaceable without significantly affecting processing.
Temperature-sensitive poliovirus mutant fails to cleave VP0 and accumulates provirions
Authors: Compton S R , Nelsen B , Kirkegaard K
Published: January 2020

A temperature-sensitive mutant of poliovirus, VP2-103, was isolated and characterized. A single nucleotide change, resulting in the substitution of glutamine for arginine at amino acid 76 of the capsid protein VP2, prevented the maturation of virions at the nonpermissive temperature. Particles indistinguishable from the previously elusive provirions were observed; these particles have been proposed to be penultimate in virion morphogenesis. Cleavage of VP0 into VP2 and VP4, the products found in mature virions, was not observed in VP2-103-infected cells at the nonpermissive temperature. The cleavage of VP0 in wild-type poliovirus-infected cells is dependent on RNA packaging; this reaction has been postulated to be autocatalytic. The existence of RNA-containing provirionlike particles in VP2-103-infected cells shows that RNA packaging can be uncoupled from VP0 cleavage.
Inhibition of antigen-induced and interleukin-2-induced proliferation of bovine peripheral blood leukocytes by inactivated bovine herpes virus 1
Authors: Hutchings D L , Campos M , Qualtiere L , Babiuk L A
Published: January 2020

The mechanism by which bovine herpesvirus 1 (BHV-1) predisposes cattle to bacterial pneumonia was investigated by using an in vitro system to demonstrate immunosuppression. At a multiplicity of infection of 0.001, live or inactivated BHV-1 induced a 50% inhibition of the proliferative response of peripheral blood mononuclear leukocytes to antigen (vaccinia virus in vaccinia virus-immunized cattle which were BHV-1 negative) or interleukin-2. At this same multiplicity of infection, the mitogen-induced proliferation of peripheral blood mononuclear leukocytes was unaffected. This inhibition of antigen and interleukin-2-induced proliferative responses could not be reversed by the addition of excess amounts of interleukin-2 and could not be prevented by the addition of indomethacin to block prostaglandin production. Antibodies to BHV-1, especially those specific for glycoproteins gI and gIV, were able to block the inhibitory effect of BHV-1 in these in vitro assays. These results showed that antibody to BHV-1 blocks the immunosuppressive effect of the virus in vitro and suggested that an appropriate antibody response to BHV-1 could protect cattle from virus-induced immunosuppression leading to secondary bacterial pneumonia.
Infection of nonlymphoid cells by human immunodeficiency virus type 1 or type 2
Authors: Ikeuchi K , Kim S , Byrn R A , Goldring S R , Groopman J E
Published: January 2020

Human epithelial cells (L132) derived from embryonic lung and human lung fibroblasts (MRC5) were infected by human immunodeficiency virus type 1 (HIV-1) or type 2 (HIV-2). Surface CD4 protein was detected on these cells, and recombinant soluble CD4 (sCD4) blocked infection, indicating that HIV infection was mediated by the cell surface CD4 protein. In contrast, infection of human primary chondrocyte cells (C23), synovial cells (HSA), and foreskin fibroblasts (F13) was apparently independent of cell CD4-mediated mechanisms. Surface CD4 protein could not be detected on these cells, and sCD4 did not block the infection. F13 cells could be infected only by HIV-2, not by HIV-1, under our experimental conditions. In cells of mesenchymal orgin, viral production could be detected only after cocultivation with the human T-lymphoid H9 cells but not by conventional viral assays, including reverse transcriptase and p24 antigen assays in cell culture supernatant and immunofluorescence of host cells. Our DNA transfection studies indicated that this lack of detectable viral production was not due to the inefficient use of the HIV long terminal repeat or the Tat protein in these cells. These mesenchymal and epithelial cells were susceptible to HIV infection but differed in mechanism of virus entry compared with hematopoietic cells such as T lymphocytes. These observations may provide insights into clinical syndromes such as lung dysfunction in HIV-infected newborns and connective tissue disorders in HIV-infected adults.
Recovery from chronic rotavirus infection in mice with severe combined immunodeficiency: virus clearance mediated by adoptive transfer of immune CD8+ T lymphocytes
Authors: Dharakul T , Rott L , Greenberg H B
Published: January 2020

Severe combined immunodeficient (SCID) mice lack both functional T and B cells. These mice develop chronic rotavirus infection following an oral inoculation with the epizootic diarrhea of infant mice (EDIM) rotavirus. Reconstitution of rotavirus-infected SCID mice with T lymphocytes from immunocompetent mice allows an evaluation of a role of T-cell-mediated immunity in clearing chronic rotavirus infection. Complete rotavirus clearance was demonstrated in C.B-17/scid mice 7 to 9 days after the transfer of immune CD8+ splenic T lymphocytes from histocompatible BALB/c mice previously immunized intraperitoneally with the EDIM-w strain of murine rotavirus. The virus clearance mediated by T-cell transfer was restricted to H-2d-bearing T cells and occurred in the absence of rotavirus-specific antibody as determined by enzyme-linked immunosorbent assay, neutralization, immunohistochemistry, and radioimmunoprecipitation. Temporary clearance of rotavirus was observed after the transfer of immune CD8+ T cells isolated from the intestinal mucosa (intraepithelial lymphocytes [IELs]) or the spleens of BALB/c mice previously infected with EDIM by the oral route. Chronic virus shedding was transiently eliminated 7 to 11 days after spleen cell transfer and 11 to 12 days after IEL transfer. However, recurrence of rotavirus infection was detected 1 to 8 days later in all but one SCID recipient receiving cells from orally immunized donors. The viral clearance was mediated by IELs that were both Thy1+ and CD8+. These data demonstrated that the clearance of chronic rotavirus infection in SCID mice can be mediated by immune CD8+ T lymphocytes and that this clearance can occur in the absence of virus-specific antibodies.
Functional elements of the steroid hormone-responsive promoter of mouse mammary tumor virus
Authors: Toohey M G , Lee J W , Huang M , Peterson D O
Published: January 2020

Transcription from the promoter of mouse mammary tumor virus is subject to induction by several classes of steroid hormones as well as to repression by a negative regulatory element present in the long terminal repeats of proviral DNA. In order to characterize the functional elements of the promoter that in some way must respond to these regulatory signals, a number of promoter mutations were constructed, including a set of linker-scanning mutations across the entire promoter region. Analysis of these mutated promoters with a transient-transfection assay defined at least three mutation-sensitive promoter elements that are required for both basal and hormone-induced transcription. One mutation-sensitive region contains a TATA element located at approximately position -30 with respect to the start of transcription. A second mutation-sensitive region contains two 10-base-pair direct repeats located between positions -60 and -38, within which are embedded three copies of octamer-related sequences; complete disruption of this region of the promoter leads to a more severe decrease in transcription than do any of the linker-scanning mutations, suggesting that the repeated sequences may be at least partially functionally redundant. Gel electrophoresis mobility shift assays were used to demonstrate specific binding of a nuclear protein to this region of the promoter. A third mutation-sensitive region contains a binding site for nuclear factor 1 (NF-1) located between positions -77 and -63. Site-directed mutations in the NF-1-binding site which increase the apparent affinity of NF-1 for the promoter in vitro do not decrease the hormone dependence of transcription, suggesting that transcriptional activation mediated by steroid hormone-receptor complexes cannot be explained by facilitation or stabilization of the interaction of promoter sequences with NF-1 and consistent with the idea that binding of NF-1 is not rate determining in transcription from the mouse mammary tumor virus promoter. None of the promoter mutations functionally separates basal from glucocorticoid-induced transcription, suggesting that hormone induction does not make the promoter independent of any of the DNA-binding factors required for its basal activity.
Pathogenesis of human parainfluenza virus 3 infection in two species of cotton rats: Sigmodon hispidus develops bronchiolitis, while Sigmodon fulviventer develops interstitial pneumonia
Authors: Porter D D , Prince G A , Hemming V G , Porter H G
Published: January 2020

Human parainfluenza virus 3 replicates well in the noses and lungs of two species of cotton rats, Sigmodon hispidus and Sigmodon fulviventer. Peak viral titers of nearly 10(6) PFU/g are reached 2 days after infection in both tissues, are maintained through day 5, and are equivalent in the two species. Infectious virus is eliminated by day 8 after infection. Both species produce a strong neutralizing antibody response with titers of 1:10,000 4 weeks after infection. Viral replication in the nasal epithelium results in only minor histological changes, and viral antigen is found only in the apical portion of epithelial cells. Infection of S. hispidus causes a bronchiolitis with a peribronchiolar lymphoid cell infiltration that reaches a peak 6 days after infection, and there is only a minor component of interstitial pneumonia. In contrast, infection of S. fulviventer causes an interstitial pneumonia, and this lesion reaches its maximal extent by 6 days after infection. There is minimal peribronchiolar lymphoid cell infiltration in infected S. fulviventer. Lung lesions in both species of cotton rats are largely healed 9 days after infection, and the lungs are indistinguishable from those of uninfected controls 16 days after infection. These species of cotton rats offer separate models for the two major pulmonary manifestations of human parainfluenza virus 3 infection. The models may be useful for basic studies of the pathogenesis of this infection and for initial evaluation of candidate vaccines.
Two signals mediate nuclear localization of influenza virus (A/WSN/33) polymerase basic protein 2
Authors: Mukaigawa J , Nayak D P
Published: January 2020

Polymerase basic protein 2 (PB2), a component of the influenza virus polymerase complex, when expressed alone from cloned cDNA in the absence of other influenza virus proteins, is transported into the nucleus. In this study, we have examined the nuclear translocation signal of PB2 by making deletions and mutations in the PB2 sequence. Our studies showed that two distant regions in the polypeptide sequence were involved in the nuclear translocation of PB2. In one region, four basic residues (K-736 R K R) played a critical role in the nuclear translocation of PB2, since the deletion or mutation of these residues rendered the protein totally cytoplasmic. However, seven residues (M K R K R N S) of this region, including the four basic residues, failed to translocate a cytoplasmic reporter protein into the nucleus, suggesting that these sequences were necessary but not sufficient for nuclear translocation. Deletion of another region (amino acids 449 to 495) resulted in a mutant protein which was cytoplasmic with a perinuclear distribution. This novel phenotype suggests that a perinuclear binding step was involved prior to translocation of PB2 across the nuclear pore and that a signal might be involved in perinuclear binding. Possible involvement of these two signal sequences in the nuclear localization of PB2 is discussed.
Expression of bovine herpesvirus 1 glycoprotein gIV by recombinant baculovirus and analysis of its immunogenic properties
Authors: van Drunen Littel-van den Hurk S , Parker M D , Fitzpatrick D R , Zamb T J , van den Hurk J V , Campos M , Harland R , Babiuk L A
Published: January 2020

The gene encoding the gIV glycoprotein of bovine herpesvirus 1 has been inserted into the genome of Autographa californica baculovirus in lieu of the coding region of the A. californica baculovirus polyhedrin gene. Recombinant protein was identified by its reactivity with gIV-specific monoclonal antibodies and expressed at high levels (about 85 micrograms per 2.5 x 10(6) cells) in Spodoptera frugiperda (SF9) cells. The recombinant glycoprotein had an apparent molecular mass of 63 kDa, indicating that it was incompletely glycosylated. However, it was transported to and expressed on the cell surface of infected SF9 cells. Furthermore, reactivity with polyclonal and monoclonal antibodies specific for gIV suggested that most epitopes were functionally unaltered on the recombinant gIV. Immunization of cattle with recombinant gIV in crude, partially purified, or pure form resulted in the induction of neutralizing antibodies to BHV-1, which were reactive with authentic gIV. However, the neutralizing antibody titers were lower than those elicited by an equivalent amount of affinity-purified authentic gIV, which appeared to be mainly due to reduced recognition of one of the neutralizing antigenic domains of gIV, designated domain I. The potential use of this recombinant gIV glycoprotein as a vaccine to bovine herpesvirus 1 infection in cattle is discussed.