bluuebunny/crossref_metadata_2025 huggingface dataset:
A 37-kilodalton glycoprotein from a baculovirus of Orgyia pseudotsugata is localized to cytoplasmic inclusion bodies
Authors: Gross C H , Wolgamot G M , Russell R L , Pearson M N , Rohrmann G F
Published: January 2020

The gene encoding a 37-kDa glycoprotein (gp37) of Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was located and sequenced. gp37 of OpMNPV was found to have 62 and 37% amino acid sequence identity with gp37 of Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) and with a protein reported to be a component of occlusion bodies from Choristoneura biennis entomopoxvirus, respectively. The mRNA start site of the OpMNPV gp37 gene was mapped within a late promoter sequence (TTAAG). A TrpE fusion protein containing 55% of the OpMNPV gp37 gene amino acid sequence was used to generate a monospecific antiserum. Western immunoblot analysis of OpMNPV-infected Lymantria dispar cells detected gp37 beginning at 24 h postinfection. Immunoelectron microscopy indicated that the protein is concentrated in cytoplasmic inclusion bodies late in infection. In contrast to gp37 of AcMNPV which was present in the matrix of occlusion bodies, OpMNPV gp37 was not observed in this location. Neither OpMNPV nor AcMNPV gp37 was associated with the polyhedron envelope.
Neutralizing monoclonal antibodies that distinguish three antigenic sites on human cytomegalovirus glycoprotein H have conformationally distinct binding sites
Authors: Simpson J A , Chow J C , Baker J , Avdalovic N , Yuan S , Au D , Co M S , Vasquez M , Britt W J , Coelingh K L
Published: January 2020

Seven neutralizing murine monoclonal antibodies specific for the glycoprotein H of human cytomegalovirus were produced and used to construct a topological map of two nonoverlapping antigenic sites that are bridged by a third antigenic site. Neutralization assays with 15 laboratory or clinical human cytomegalovirus strains indicated that the monoclonal antibodies recognize three antigenically variable and three conserved epitopes within the three antigenic sites. The variable-domain genes encoding monoclonal antibodies representing each of the three antigenic sites were cloned and sequenced, and molecular models of their binding sites were generated. Conformational differences in the antibody-binding sites suggested a structural basis for experimentally observed differences in gH epitope recognition.
Cell fusion induced by the murine leukemia virus envelope glycoprotein
Authors: Jones J S , Risser R
Published: January 2020

To determine whether ecotropic murine leukemia virus (MuLV) envelope glycoproteins are sufficient to cause cell-to-cell fusion when expressed in the absence of virus production, we used an ecotropic MuLV, AKV, to construct env expression vectors that lack the gag and pol genes. The rat cell line XC, which undergoes cell-to-cell fusion upon infection with ecotropic MuLV, was transfected with wild-type env expression vectors, and high levels of syncytium formation resulted. Transfection of the murine cell line NIH 3T3 with expression vectors containing the wild-type or mutated env region did not result in syncytium formation. Immunoprecipitation analysis of the envelope glycoproteins expressed in NIH 3T3 and XC cells showed that the mature surface glycoprotein expressed in XC cells was of a much lower apparent molecular weight than that expressed in NIH 3T3 cells. Further characterization showed that most if not all of this difference was the result of differences in glycosylation. Finally, site-directed mutagenesis was used to introduce several conservative and nonconservative changes into the amino-terminal region of the transmembrane protein. Analysis of the effect of these mutations confirmed that this region is a fusion domain.
Modulating effects of the extracellular sequence of the human insulinlike growth factor I receptor on its transforming and tumorigenic potential
Authors: Liu D , Rutter W J , Wang L H
Published: January 2020

We reported previously that an N-terminally truncated insulinlike growth factor I receptor (IGFR) fused to avian sarcoma virus UR2 gag p19 had a greater transforming potential than did the native IGFR, but it failed to cause tumors in vivo. To investigate whether the 36 amino acids (aa) of the IGFR extracellular (EC) sequence in the gag-IGFR fusion protein encoded by the retrovirus UIGFR have a modulatory effect on the biological and biochemical properties of the protein, four mutants, NM1, NM2, NM3, and NM4 of the EC sequence were constructed. NM1 lacks the entire 36 aa residues; NM2 lacks the N-terminal 16 aa residues (aa 870 to 885), including two potential N-linked glycosylation sites of the EC sequence; NM3 contains a deletion of the C-terminal 20 aa residues (aa 886 to 905) of the EC sequence; and NM4 contains N-to-Q substitutions at both N-linked glycosylation sites. NM1 was the strongest of the four mutants in promoting anchorage-independent growth of transfected chicken embryo fibroblasts, while NM2 and NM4 had weaker transforming potential than did the original UIGFR virus. Only NM1 and NM3 were able to induce sarcomas in chickens. The four NM mutant-transformed cells expressed the expected proteins with comparable steady-state levels. The in vitro tyrosine kinase activity of P53NM1 was about fourfold higher than that of the parental P57-75UIGFR, whereas NM2 and NM4 proteins exhibited four- to fivefold-lower kinase activities. Despite lacking the IGFR EC sequence, P53NM1 formed covalent dimers similar to those formed by the parental P57-75UIGFR. Increased phosphatidylinositol (PI) 3-kinase activity was found to be associated with the mutant IGFR proteins. Among NM4 proteins. Elevated tyrosine phosphorylation of cellular proteins of 35, 120, 140, 160, and 170 kDa was detected in all mutant IGFR-transformed cells. We conclude that the EC 36-aa sequence of IGFR in the gag-IGFR fusion protein exerts intricate modulatory effects on the protein's transforming and tumorigenic potential. The 20 aa residues immediately upstream of the transmembrane domain have an inhibitory effect on the tumorigenic potential of gag-IGFR, whereas N-linked glycosylation within the EC sequence appears to have a positive effect on the transforming potential of UIGFR. Increased in vitro kinase activity and, to a lesser extent, in vivo tyrosine phosphorylation as well as the elevated association of PI 3-kinase activity with IGFR proteins seem to be correlated with the transforming potential of IGFR mutant proteins.
Delayed cytopathicity of a feline leukemia virus variant is due to four mutations in the transmembrane protein gene
Authors: Thomas E , Overbaugh J
Published: January 2020

Two molecularly cloned, replication-defective variants of feline leukemia virus, called 61B and 61C, have both been shown to cause fatal immunodeficiency in cats when coinfected with a replication-competent, minimally pathogenic helper virus, but 61B exhibits a longer latency period between infection and disease (J. Overbaugh, E. A. Hoover, J. I. Mullins, D. P. W. Burns, L. Rudensey, S. L. Quackenbush, V. Stallard, and P. R. Donahue, Virology 188:558-569, 1992). Infection of the 3201 feline T-cell line with 61B plus helper virus also results in longer time from infection to cytopathic effect compared with 61C plus helper virus, providing an in vitro system with which to study the mechanism for this difference. We report that the primary determinant of cytopathicity of 61B maps to gp70, the extracellular envelope glycoprotein. The long latency of 61B, on the other hand, maps to the extracellular portion of the envelope transmembrane protein, in which there are only four predicted amino acid differences between 61B and 61C. These differences render 61B replication defective, and two of the predicted amino acid changes lie in a region that is highly conserved among many retroviruses. The eventual onset of 61B cytopathicity in cell culture was associated with the outgrowth of an apparent recombinant virus that encodes the pathogenic gp70 of 61B and replaces the transmembrane protein of 61B with that of the helper virus. Thus, during in vitro infection, a cytopathic virus evolved from a replication-defective virus and a nonpathogenic virus, suggesting that recombination between multiple variants in natural infection may influence progression of feline leukemia virus-associated immunodeficiency disease.
Severity of neurological signs and degree of inflammatory lesions in the brains of rats with Borna disease correlate with the induction of nitric oxide synthase
Authors: Zheng Y M , Schäfer M K , Weihe E , Sheng H , Corisdeo S , Fu Z F , Koprowski H , Dietzschold B
Published: January 2020

The putative role of nitric oxide in the neuropathogenesis of Borna disease was investigated by determining changes in the expression of inducible nitric oxide synthase (iNOS) mRNA and constitutively expressed NOS (cNOS) mRNA in brains of Borna disease virus (BDV)-infected rats. iNOS mRNA was not detected in normal rat brain but was identified in BDV-infected brain at 14 days postinfection (p.i.), reaching maximum levels at 21 days p.i., when neurological signs and inflammatory reactions in the brain were also at a peak. cNOS mRNA was expressed in both normal brain and infected brain, increasing markedly at 17 days p.i. and reaching a peak at 21 days p.i. In situ hybridization analysis revealed iNOS mRNA in some, but not all, BDV-infected regions of the brain, particularly in the basolateral cortex and the hippocampus. iNOS-positive cells, as identified immunohistologically, were preferentially localized in perivascular areas of the hippocampus and in outer cortical layers. These iNOS-positive cells resembled monocytes/macrophages in morphology and distribution pattern but were significantly fewer. The correlation of iNOS and cNOS mRNA expression with the development of neurological disease, as well as the enhanced expression of iNOS within brain regions with inflammatory lesions, strongly suggests that NO may contribute to pathogenesis of Borna disease.
Sequence and temporal appearance of the early transcribed baculovirus gene HE65
Authors: Becker D , Knebel-Mörsdorf D
Published: January 2020

We have identified the early transcribed HE65 gene by screening a cDNA library from polyadenylated RNA which was isolated at 1 h after infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus (AcNPV). Nucleotide sequencing analysis of the HE65-specific cDNA clone reveals one open reading frame of 1,662 nucleotides from which a protein of 65 kDa in size can be predicted. The HE65 gene is located downstream of the late transcribed p80 gene and upstream of the homologous region hr4left, which overlaps the 5' sequences of the HE65 gene. An HE65-specific transcript of about 1,800 nucleotides is detectable 2 h postinfection and remains stable during the late phases of infection. RNase protection and primer extension analyses demonstrate that transcripts from the early start site of HE65 continue to accumulate from 2 to 48 h postinfection, even in the presence of aphidicolin. Furthermore, transcriptional analysis of the HE65 gene indicates a lower intensity of early transcription in comparison with the very early transcribed genes IEN, PE38, and ME53.
Differential replication of human immunodeficiency virus type 1 in CD8- and CD8+ subsets of natural killer cells: relationship to cytokine production pattern
Authors: Tóth F D , Mosborg-Petersen P , Kiss J , Aboagye-Mathiesen G , Zdravkovic M , Hager H , Ebbesen P
Published: January 2020

CD8+ and CD8- subsets of peripheral blood natural killer (NK) cells were examined for susceptibility to infection with human immunodeficiency virus type 1 (HIV-1) and for the ability to produce various types of interferon (IFN) and tumor necrosis factor (TNF). HIV-1 was preferentially grown in CD8+ NK cells. The ability of CD8- NK cells to suppress HIV-1 replication was related to their ability to produce alpha IFN (IFN-alpha) upon viral induction. Induction with interleukin-2 resulted in IFN-gamma production in both subsets of NK cells. In the CD8+ subset, IFN-gamma and HIV-1 mutually enhanced the production of TNF alpha, leading to hyperactivation of viral replication, whereas in CD8- NK cells IFN-gamma primed HIV-induced IFN-alpha production. The dichotomous effects of IFN-gamma on HIV-1 replication were dependent on the IFN-alpha-producing ability of the cellular targets. These findings can explain the selective depletion of the CD16+ CD8+ subset that begins early in the in vivo HIV-1 infection.
Effects of temperature and lipophilic agents on poliovirus formation and RNA synthesis in a cell-free system
Authors: Molla A , Paul A V , Wimmer E
Published: January 2020

The translation and primary processing events of poliovirus polyproteins in HeLa cell extracts were more efficient at 34 degrees C than at 30 or 36 degrees C. The cleavage products of P2 such as 2Apro, 2BC, and 2C appeared early in the reaction before the appearance of the cleavage products of P1 and of 3CDpro, an observation suggesting that P2 was cleaved in cis by 3CDpro. Proteolytic processing of the capsid precursor P1 into VP0, VP1, and VP3 was also more efficient at 34 degrees C than at either 30 or 32 degrees C. Surprisingly, processing of 3CDpro to 3Cpro and 3Dpol was almost completely inhibited at 36 degrees C. The synthesis of virus in the cell extract was greatly enhanced at 34 degrees C over that at 30 or 32 degrees C, whereas incubation at 36 degrees C yielded very little virus. Cerulenin, an inhibitor of lipid synthesis, did not appear to affect virus-specific translation or protein processing, but it almost completely inhibited viral synthesis in vitro. Oleic acid drastically inhibited in vitro translation at 100 microM and in vitro poliovirus synthesis at 25 microM. Addition of HeLa cell smooth membranes partially restored translation but not virus formation. Our observations suggest that in vitro translation, proteolytic processing, and virus formation require intact membranes. Analysis of the in vitro translation products revealed that viral RNA polymerase activity increased linearly during incubation of the translation mixture. RNA polymerase in the crude mixture was inhibited by oleic acid but not by cerulenin. Surprisingly, oleic acid had no direct effect on oligo(U)-primed, poly(A)-dependent poly(U) synthesis catalyzed by purified 3Dpol.
Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene
Authors: Dolja V V , Herndon K L , Pirone T P , Carrington J C
Published: January 2020

The RNA genome of tobacco etch potyvirus (TEV) was engineered to express bacterial beta-glucuronidase (GUS) fused to the virus helper component proteinase (HC-Pro). It was shown previously that prolonged periods (approximately 1 month) of TEV-GUS propagation in plants resulted in the appearance of spontaneous deletion variants. Nine deletion mutants were identified by nucleotide sequence analysis of 40 cDNA clones obtained after polymerase chain reaction amplification. The mutants were missing between 1,741 and 2,074 nucleotides from TEV-GUS, including the sequences coding for most of GUS and the N-terminal region of HC-Pro. This region of HC-Pro contains determinants involved in helper component activity during aphid transmission, as well as a highly conserved series of cysteine residues. The deletion variants were shown to replicate and move systemically without the aid of a helper virus. Infectious viruses harboring the two largest HC-Pro deletions (termed TEV-2del and TEV-7del) were reconstructed by subcloning the corresponding mutated regions into full-length DNA copies of the TEV genome. Characterization of these and additional variants derived by site-directed mutagenesis demonstrated that deletion of sequences coding for the HC-Pro N-terminal domain had a negative effect on accumulation of viral RNA and coat protein. The TEV-2del variant possessed an aphid-nontransmissible phenotype that could be rescued partially by prefeeding of aphids on active HC-Pro from another potyvirus. These data suggest that the N-terminal domain of HC-Pro or its coding sequence enhances virus replication or genome expression but does not provide an activity essential for these processes. The function of this domain, as well as a proposed deletion mechanism involving nonhomologous recombination, is discussed.
Intracerebral hemorrhages and syncytium formation induced by endothelial cell infection with a murine leukemia virus
Authors: Park B H , Lavi E , Blank K J , Gaulton G N
Published: January 2020

The mechanisms of endothelial cell damage that lead to cerebral hemorrhage are not completely understood. In this study, a cloned murine retrovirus, TR1.3, that uniformly induced stroke in neonatal BALB/c mice is described. Restriction digest mapping suggests that TR1.3 is part of the Friend murine leukemia virus (FMuLV) family. However, unlike mice exposed to other FMuLVs, mice infected with TR1.3 virus developed tremors and seizures within 8 to 18 days postinoculation. This was uniformly followed by paralysis and death within 1 to 2 days. Postmortem examination of TR1.3-inoculated mice revealed edematous brain tissue with large areas of intracerebral hemorrhage. Histologic analysis revealed prominent small vessel pathology including syncytium formation of endothelial cells. Immunohistochemical analysis of frozen brain sections using double fluorescence staining demonstrated that TR1.3 virus specifically infected small vessel endothelial cells. Although infection of vessel endothelial cells was detected in several organs, only brain endothelial cells displayed viral infection associated with hemorrhage. The primary determinant of TR1.3-induced neuropathogenicity was found to reside within a 3.0-kb fragment containing the 3' end of the pol gene, the env gene, and the U3 region of the long terminal repeat. The restricted tropism and acute pathogenicity of this cloned murine retrovirus provide a model for studying virus-induced stroke and for elucidating the mechanisms involved in syncytium formation by retroviruses in vivo.
Viral determinants of simian immunodeficiency virus (SIV) virulence in rhesus macaques assessed by using attenuated and pathogenic molecular clones of SIVmac
Authors: Marthas M L , Ramos R A , Lohman B L , Van Rompay K K , Unger R E , Miller C J , Banapour B , Pedersen N C , Luciw P A
Published: January 2020

To identify viral determinants of simian immunodeficiency virus (SIV) virulence, two pairs of reciprocal recombinants constructed from a pathogenic (SIVmac239) and a nonpathogenic (SIVmac1A11) molecular clone of SIV were tested in rhesus macaques. A large 6.2-kb fragment containing gag, pol, env, and the regulatory genes from each of the cloned (parental) viruses was exchanged to produce one pair of recombinant viruses (designated SIVmac1A11/239gag-env/1A11 and SIVmac239/1A11gag-env/239 to indicate the genetic origins of the 5'/internal/3' regions, respectively, of the virus). A smaller 1.4-kb fragment containing the external env domain of each of the parental viruses was exchanged to create the second pair (SIVmac1A11/239env/1A11 and SIVmac239/1A11env/239) of recombinant viruses. Each of the two parental and four recombinant viruses was inoculated intravenously into four rhesus macaques, and all 24 animals were viremic by 4 weeks postinoculation (p.i.). Virus could not be isolated from peripheral blood mononuclear cells (PBMC) of any animals infected with SIVmac1A11 after 6 weeks p.i. but was consistently isolated from all macaques inoculated with SIVmac239 for 92 weeks p.i. Virus isolation was variable from animals infected with recombinant viruses; SIVmac1A11/239gag-env/1A11 and SIVmac239/1A11env/239 were isolated most frequently. Animals inoculated with SIVmac239 had 10 to 100 times more virus-infected PBMC than those infected with recombinant viruses. Three animals infected with SIVmac239 died with simian AIDS (SAIDS) during the 2-year observation period after inoculation, and the fourth SIVmac239-infected animal had clinical signs of SAIDS. Two animals infected with recombinant viruses died with SAIDS; one was infected with SIVmac239/1A11gag-env/239, and the other was infected with SIVmac1A11/239gag-env/1A11. The remaining 18 macaques remained healthy by 2 years p.i., and 13 were aviremic. One year after inoculation, peripheral lymph nodes of some of these healthy, aviremic animals harbored infected cells. All animals seroconverted within the first few weeks of infection, and the magnitude of antibody response to SIV was proportional to the levels and duration of viremia. Virus-suppressive PBMC were detected within 2 to 4 weeks p.i. in all animals but tended to decline as viremia disappeared. There was no association of levels of cell-mediated virus-suppressive activity and either virus load or disease progression. Taken together, these results indicate that differences in more than one region of the viral genome are responsible for the lack of virulence of SIVmac1A11.
Distinct repertoire of antigenic variants of foot-and-mouth disease virus in the presence or absence of immune selection
Authors: Borrego B , Novella I S , Giralt E , Andreu D , Domingo E
Published: January 2020

Antigenic variants of foot-and-mouth disease virus (FMDV) were generated and frequently became dominant in clonal populations of FMDV (clone C-S8c1) grown in the absence of anti-FMDV antibodies. We have now passaged eight samples of the same FMDV clone in the presence of a limited amount of neutralizing polyclonal antibodies directed to the major antigenic site A of capsid protein VP1. Complex populations of variants showing increased resistance to polyclonal sera and to site A-specific monoclonal antibodies were selected. Some populations exhibited marked decreases in viral fitness. Multiple amino acid replacements within site A--and also elsewhere in VP1--accumulated upon passage of the virus in either the absence or the presence of neutralizing antibodies. However, antigenically critical replacements at one position in site A occurred repeatedly in FMDV passaged under antibody selection, but they were never observed in many passages carried out either in the absence of antiviral antibodies or in the presence of an irrelevant antiviral serum. Thus, even though antigenic variation of FMDV can occur in the absence or presence of immune selection, critical replacements which lead to important changes in antigenic specificity were observed only as a result of selection by neutralizing antibodies.
Transactivation of the P2 promoter of parathyroid hormone-related protein by human T-cell lymphotropic virus type I Tax1: evidence for the involvement of transcription factor Ets1
Authors: Dittmer J , Gitlin S D , Reid R L , Brady J N
Published: January 2020

Expression of the parathyroid hormone-related protein (PTHrP), a protein that plays a primary role in the development of the humoral hypercalcemia of malignancy, is regulated by two distinct promoters, P1 and P2. PTHrP is overexpressed in lymphocytes from adult T-cell leukemia patients. We now demonstrate that in the human T-cell lymphotropic virus type I-transformed cell line MT-2, RNA synthesis is initiated primarily at the P2 promoter. Furthermore, in cotransfection experiments, Tax1 transactivates the P2 promoter 10- to 12-fold. By using deletion and site-specific point mutations, we have identified a promoter-proximal sequence (positions -72 to -40) which is important for Tax1 transactivation. The PTHrP promoter-proximal element contains two potential overlapping Ets1 binding sites, EBS I and EBS II. Gel shift analysis demonstrated that Ets1 binds specifically to both EBS I and EBS II. Mutation of the consensus GGAA core motif in EBS I abolished binding and Tax1 transactivation in Jurkat T lymphocytes. In Ets1-deficient cells, cotransfection of Tax1 and Ets1 expression plasmids stimulates PTHrP promoter activity. In the absence of Ets1, minimal transactivation of the PTHrP promoter is observed. These data suggest that Ets1 binds to EBS I and cooperates with Tax1 to transactivate the PTHrP P2 promoter.
Mapping of functionally important residues of a cysteine-histidine box in the human immunodeficiency virus type 1 nucleocapsid protein
Authors: Dorfman T , Luban J , Goff S P , Haseltine W A , Göttlinger H G
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein contains two copies of a sequence motif, the cysteine-histidine box, that is conserved among retroviruses. To identify the functionally relevant positions of a cysteine-histidine box, each amino acid in the proximal copy of the motif was individually substituted by site-directed mutagenesis. Mutations at 5 of 14 positions abolished virus replication and reduced the viral RNA content of mutant particles to between 10 and 20% of parental levels. Mutations at other positions had either no or only a minor effect on virus replication and virion RNA content. In vitro binding of RNA to bacterially expressed mutant Pr55gag polyprotein correlated well with the effects of the mutations on particle-associated viral RNA levels. The two different copies of the motif in the HIV-1 nucleocapsid protein are not functionally equivalent, since the conversion of the proximal motif to an exact copy of the distal motif results in a defect in virus replication and a reduction in the viral RNA content of mutant particles. The simultaneous substitution of functionally relevant positions in both motifs led to a significant decline in gag protein export, indicating that the nucleocapsid domain of the gag precursor is also required for efficient assembly or release of the virion.
Strain specificity and binding affinity requirements of neutralizing monoclonal antibodies to the C4 domain of gp120 from human immunodeficiency virus type 1
Authors: Nakamura G R , Byrn R , Wilkes D M , Fox J A , Hobbs M R , Hastings R , Wessling H C , Norcross M A , Fendly B M , Berman P W
Published: January 2020

The binding properties of seven CD4-blocking monoclonal antibodies raised against recombinant gp120 of human immunodeficiency virus type 1 strain MN (HIV-1MN) and two CD4-blocking monoclonal antibodies to recombinant envelope glycoproteins gp120 and gp160 of substrain IIIB of HIVLAI were analyzed. With a panel of recombinant gp120s from seven diverse HIV-1 isolates, eight of the nine antibodies were found to be strain specific and one was broadly cross-reactive. Epitope mapping revealed that all nine antibodies bound to epitopes located in the fourth conserved domain (C4) of gp120. Within this region, three distinct epitopes could be identified: two were polymorphic between HIV-1 strains, and one was highly conserved. Studies with synthetic peptides demonstrated that the conserved epitope, recognized by antibody 13H8, was located between residues 431 and 439. Site-directed mutagenesis of gp120 demonstrated that residue 429 and/or 432 was critical for the binding of the seven antibodies to gp120 from HIV-1MN. Similarly, residues 423 and 429 were essential for the binding of monoclonal antibody 5C2 raised against gp120 from HIV-1IIIB. The amino acids located at positions 423 and 429 were found to vary between strains of HIV-1 as well as between molecular clones derived from the MN and LAI isolates of HIV-1. Polymorphism at these positions prevented the binding of virus-neutralizing monoclonal antibodies and raised the possibility that HIV-1 neutralization serotypes may be defined on the basis of C4 domain sequences. Analysis of the binding characteristics of the CD4-blocking antibodies demonstrated that their virus-neutralizing activity was directly proportional to their gp120-binding affinity. These studies account for the strain specificity of antibodies to the C4 domain of gp120 and demonstrate for the first time that antibodies to this region can be as effective as those directed to the principal neutralizing determinant (V3 domain) in neutralizing HIV-1 infectivity.
Two cis-acting signals control ribosomal frameshift between human T-cell leukemia virus type II gag and pro genes
Authors: Falk H , Mador N , Udi R , Panet A , Honigman A
Published: January 2020

The open reading frame of the human T-cell leukemia virus type II pro gene is arranged at a -1 position relative to the gag gene. Synthesis of the Gag-Pro fusion polyprotein is facilitated by ribosomal frameshift into the reading frame of the pro gene. Cloning of a synthetic 41-bp oligonucleotide corresponding to the gag-pro junction within a heterologous gene (nef of human immunodeficiency virus type I) and mutation analysis revealed that two cis-acting signals, an adenosine residue stretch and a dyad symmetry sequence, flanking the UAA termination codon, are required for efficient ribosomal frameshifting between gag and pro. The stability of the stem-loop structure is crucial for frameshifting.
Role of the cytoplasmic domains of viral glycoproteins in antibody-induced cell surface mobility
Authors: Lydy S L , Compans R W
Published: January 2020

We have investigated the role of the cytoplasmic domains of the influenza virus hemagglutinin (HA) and the parainfluenza virus type 3 (PI3) fusion (F) glycoproteins as a determinant of their ability to undergo antibody-induced redistribution on plasma membranes. The viral envelope genes were truncated in their cytoplasmic domains by using oligonucleotide-directed mutagenesis and expressed by using recombinant vaccinia viruses. In HeLa cells, the truncated HA (HAt), like the full-length HA, did not cap in response to specific antibody. In CV-1 cells, HAt showed patchy surface immunofluorescence with few caps, whereas full-length HA exhibited capping in many cells in response to bivalent antibody. Quantitation of cap formation indicated a sevenfold decrease in the frequency of capping of HAt in comparison with full-length HA. Similarly, truncated F also exhibited a significant decrease in cap formation in comparison with full-length F. These results indicate that the ability of influenza virus HA and PI3 F to undergo redistribution in response to bivalent antibody has been altered by truncation of the viral glycoproteins and suggest that capping may involve interactions between the cytoplasmic domain of the viral glycoproteins and host cell components.
Divergence between cytotoxic effector function and tumor necrosis factor alpha production for inflammatory CD4+ T cells from mice with Sendai virus pneumonia
Authors: Hou S , Fishman M , Murti K G , Doherty P C
Published: January 2020

Sendai virus pneumonia in beta 2-microglobulin-deficient [beta 2-m(-/-)] mice lacking CD8+ T cells is characterized by the development of CD4+ cytotoxic T lymphocytes that can be recovered directly from the respiratory tract. These CD4+ cytotoxic T lymphocytes are not found in beta 2-m (+/+) mice, though inflammatory CD4+ T cells from both beta 2-m (-/-) and beta 2-m (+/+) mice produce substantial amounts of tumor necrosis factor alpha. Blocking experiments with a monoclonal antibody that also inhibits tumor necrosis factor beta show that the secreted forms of these two cytokines are not responsible for virus-specific killing of class II major histocompatibility complex-compatible targets. Comparison of electron micrographs indicates that the CD4+ effectors from the beta 2-m (-/-) mice are potent inducers of apoptosis, while this is not the case for the beta 2-m (+/+) CD4+ set. These experiments further define the functional status of virus-specific CD4+ T cells responding in vivo in the presence or absence of CD8+ effectors.
Functional and genetic plasticities of the poliovirus genome: quasi-infectious RNAs modified in the 5'-untranslated region yield a variety of pseudorevertants
Authors: Gmyl A P , Pilipenko E V , Maslova S V , Belov G A , Agol V I
Published: January 2020

Poliovirus RNA species with nucleotides 564 to 571 deleted or with a secondary structure domain (positions 564 to 629) replaced by a shorter irregular oligonucleotide have been engineered previously; these RNAs have been considered quasi-infectious (yielding a single late revertant plaque) and dead, respectively (E. Pilipenko, A. Gmyl, Y. Svitkin, S. Maslova, A. Sinyakov, and V. Agol, Cell 68:119-131, 1992). By using large amounts of these RNAs for transfections, revertant clones with a great variety of genetic changes (point mutations, insertions of foreign sequences, short or extended deletions) were isolated. The pattern of these changes supported the notion that an appropriately spaced oligopyrimidine-AUG tandem is important for efficient poliovirus RNA translation. Structural features within and around this tandem modulated the initiation efficiency. The functional and genetic plasticities of the poliovirus genome are briefly discussed.
Characterization of the DNA binding properties of polyomavirus capsid protein
Authors: Chang D , Cai X , Consigli R A
Published: January 2020

The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.
Epitopes of glycoprotein G of equine herpesviruses 4 and 1 located near the C termini elicit type-specific antibody responses in the natural host
Authors: Crabb B S , Studdert M J
Published: January 2020

Specific serological diagnosis of equine herpesvirus 4 (EHV4; equine rhinopneumonitis virus) and EHV1 (equine abortion virus) hitherto has not been possible because of extensive antigenic cross-reactivity between these two closely related but distinct viruses. Recently, we identified EHV4 glycoprotein G (gG) and characterized it as a type-specific, secreted glycoprotein (B. S. Crabb, H. S. Nagesha, and M. J. Studdert, Virology 190:143-154, 1992). This paper shows that EHV1 gG also possesses type-specific epitopes and describes the localization of strong, type-specific epitopes to the apparently corresponding and highly variable regions comprising amino acids 287 to 382 of EHV4 gG and 288 to 350 of EHV1 gG. Fusion proteins expressing these variable regions reacted strongly and type specifically with sera from four foals, three of which were colostrum-deprived, specific-pathogen-free foals, whose history with respect to exposure to EHV4 or EHV1 was well-defined. These antigens provided the basis for the development of a single-well diagnostic enzyme-linked immunosorbent assay to distinguish horses infected with EHV4, EHV1, or both. Such a type-specific test provides for the first time the opportunity to differentiate antibodies to these viruses, and it has, therefore, important implications for understanding the epidemiology of these equine pathogens. Evidence for the existence of EHV1 in Australia 10 years prior to the first confirmed case of EHV1 abortion is presented.
Productive infection of a cervical epithelial cell line with human immunodeficiency virus: implications for sexual transmission
Authors: Tan X , Pearce-Pratt R , Phillips D M
Published: January 2020

The human cervix-derived epithelial cell line (ME180) used in this study displays a characteristics epithelial morphology, including numerous desmosomes, tonofilaments, and epidermal filaments. When T-cell lines infected with human immunodeficiency virus (HIV) are added to epithelial cultures, they rapidly adhere to the epithelial monolayer. Within a few minutes, the T cells shed numerous virions into narrow spaces formed between the epithelial cell and the adherent T cells. Virions subsequently enter the ME180 cells via large vesicles. A few days after infection, cytopathic effects and syncytium formation were observed. Infected clones of ME180 cells have remained infected for 8 months. p24 enzyme-linked immunosorbent assay and infectivity assays show that one subclone of the cell line produces virus titers equivalent to those of high-secreting HIV-infected T-cell lines. Electron microscopy reveals numerous virions budding from both the basal and apical surfaces of the epithelium. These observations suggest that cervical epithelium has the potential to serve as a site of HIV infection.
Molecular and biochemical bases for activation of the transforming potential of the proto-oncogene c-ros
Authors: Zong C S , Poon B , Chen J , Wang L H
Published: January 2020

The transforming gene of avian sarcoma virus UR2, v-ros, encodes a receptor-like protein tyrosine kinase and differs from its proto-oncogene, c-ros, in its 5' truncation and fusion to viral gag, a three-amino-acid (aa) insertion in the transmembrane (TM) domain, and changes in the carboxyl region. To explore the basis for activation of the c-ros transforming potential, various c-ros retroviral vectors containing those changes were constructed and studied for their biological and biochemical properties. Ufcros codes for the full-length c-ros protein of 2,311 aa, Uppcros has 1,661-aa internal deletion in the extracellular domain, CCros contains the 3' c-ros cDNA fused 150 aa upstream of the TM domain to the UR2 gag, CVros is the same as CCros except that the 3' region is replaced by that of v-ros, and VCros is the same as CCros except that the 5' region is replaced by that of v-ros. The Ufcros, Uppcros, CCros, and CVros are inactive in transforming chicken embryo fibroblasts, whereas VCros is as potent as UR2 in cell-transforming and tumorigenic activities. Upon passages of CCros and CVros viruses, the additional extracellular sequence in comparison with that of v-ros was delected; concurrently, both viruses (named CC5d and CV5d, respectively) attained moderate transforming activity, albeit significantly lower than that of UR2 or VCros. The native c-ros protein has a very low protein tyrosine kinase activity, whereas the ppcros protein is constitutively activated in kinase activity. The inability of CCros and CVros to transform chicken embryo fibroblasts is consistent with the inefficient membrane association, instability, and low kinase activity of their encoded proteins. The CC5d and CV5d proteins are indistinguishable in kinase activity, membrane association, and stability from the v-ros protein. The reduced transforming potency of CC5d and CV5d proteins can be attributed only to their differential substrate interaction, notably the failure to phosphorylate a 88-kDa protein. We conclude that the 5' rather than the 3' modification of c-ros is essential for its oncogenic activation; the sequence upstream of the TM domain has a negative effect on the transforming activity of CCros and CVros and needs to be deleted to activate their biological activity.
Relative affinity of the human parainfluenza virus type 3 hemagglutinin-neuraminidase for sialic acid correlates with virus-induced fusion activity
Authors: Moscona A , Peluso R W
Published: January 2020

The ability of enveloped viruses to cause disease depends on their ability to enter the host cell via membrane fusion events. An understanding of these early events in infection, crucial for the design of methods of blocking infection, is needed for viruses that mediate membrane fusion at neutral pH, such as paramyxoviruses and human immunodeficiency virus. Sialic acid is the receptor for the human parainfluenza virus type 3 (HPF3) hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, the HN must interact with its receptor. In the present report, two variants of HPF3 with increased fusion-promoting phenotypes were selected and used to study the function of the HN glycoprotein in membrane fusion. Increased fusogenicity correlated with single amino acid changes in the HN protein that resulted in increased binding of the variant viruses to the sialic acid receptor. These results suggest that the avidity of binding of the HN protein to its receptor regulates the level of F protein-mediated fusion and begin to define one role of the receptor-binding protein of a paramyxovirus in the membrane fusion process.
Incorporation of human immunodeficiency virus type 1 Gag proteins into murine leukemia virus virions
Authors: Deminie C A , Emerman M
Published: January 2020

The retroviral Gag polyprotein is necessary and sufficient for assembly and budding of viral particles. However, the exact inter- and intramolecular interactions of the Gag polyproteins during this process are not known. To locate functional domains within Gag, we generated chimeric proviruses between human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MuLV). In these chimeric proviruses, the matrix or capsid proteins of MuLV were precisely replaced with the matrix or capsid proteins of HIV-1. Although the chimeric proviruses were unable to efficiently assemble into mature viral particles by themselves, coexpression of wild-type MuLV Gag rescued the HIV proteins into virions. The specificity of the rescue of HIV proteins into MuLV virions shows that specific interactions involving homologous matrix or capsid regions of Gag are necessary for retroviral particle formation.
Simian virus 40 large T antigen associates with cyclin A and p33cdk2
Authors: Adamczewski J P , Gannon J V , Hunt T
Published: January 2020

In this paper we provide evidence that a fraction of large T antigen of simian virus 40 (SV40) interacts with cyclin A and p33cdk2 in both virus-infected and stably transformed cells. Immunoprecipitates of SV40 large T antigen from SV40-infected or SV40 large-T-antigen-transformed cells contain cyclin A, p33cdk2, and histone H1 kinase activity. Conversely, immunoprecipitates of cyclin A from these cells contain SV40 large T antigen. In this respect, SV40 large T antigen has properties similar to those of the E1A oncogene of adenoviruses and the E7 oncogene of human papillomaviruses.
Cell type-specific proteins which interact with the 5' nontranslated region of hepatitis A virus RNA
Authors: Chang K H , Brown E A , Lemon S M
Published: January 2020

The 5' nontranslated region (5'NTR) of hepatitis A virus (HAV) RNA contains structural elements which facilitate 5' cap-independent initiation of virus translation and are likely to interact with cellular proteins functioning as translation initiation factors. To define these interactions, we characterized the binding of ribosome-associated proteins from several cell types to synthetic RNAs representing segments of the 5'NTR by using a UV cross-linking/label transfer assay. Four major proteins (p30, p39, p57, and p110) were identified. p30 and p39 were present in ribosomal salt washes prepared only from HAV-permissive BS-C-1 and FRhK-4 cells, while p57 was found only in HeLa cells and rabbit reticulocyte lysates. p110 was present in all cell types. Both p30 and p39 bound to multiple sites within the 5'NTR. Efficient transfer of label to p30 occurred with minimal RNA probes representing nucleotides (nt) 96 to 155, 151 to 354, and, to a much lesser extent, 634 to 744, while label transfer to p39 occurred with probes representing nt 96 to 155 and 634 to 744. All of these probes represent regions of the 5'NTR which are rich in pyrimidines. Competitive inhibition studies indicated that both p30 and p39 bound with greater affinity to sites in the 5' half of the NTR (a probe representing nt 1 to 354) than to the more 3' site (nt 634 to 744). Binding of p39 to the probe representing nt 96 to 155 was inhibited in the presence of an equal amount of proteins derived from HeLa cells, suggesting that p39 shares binding site specificity with one or more HeLa cell proteins. A 57-kDa protein in HeLa cell protein extracts reacted with antibody to polypyrimidine tract-binding protein in immunoblots, but no immunoreactive protein was identified in a similar BS-C-1 protein fraction. These results demonstrate that ribosome-associated proteins which bind to the 5'NTR of HAV vary substantially among different mammalian cell types, possibly accounting for differences in the extent to which individual cell types support growth of the virus. Mutations in the 5'NTR which enhance the growth of HAV in certain cell types may reflect specific adaptive responses to these or other proteins.
Mutations in the cytoplasmic tail of influenza A virus neuraminidase affect incorporation into virions
Authors: Bilsel P , Castrucci M R , Kawaoka Y
Published: January 2020

The significance of the conserved cytoplasmic tail sequence of influenza A virus neuraminidase (NA) was analyzed by the recently developed reverse genetics technique (W. Luytjes, M. Krystal, M. Enami, J. D. Parvin, and P. Palese, Cell 59:1107-1113, 1989). A chimeric influenza virus A/WSN/33 NA containing the influenza B virus cytoplasmic tail rescued influenza A virus infectivity. The transfectant virus had less NA incorporated into virions than A/WSN/33, indicating that the cytoplasmic tail of influenza virus NA plays a role in incorporation of NA into virions. However, these results also suggest that the influenza A virus and influenza B virus cytoplasmic tail sequences share common features that lead to the production of infectious virus. Transfectant virus was obtained with all cytoplasmic tail mutants generated by site-directed mutagenesis of the influenza A virus tail, except for the mutant resulting from substitution of the conserved proline residue, presumably because of its contribution to the secondary structure of the tail. No virus was rescued when the cytoplasmic tail was deleted, indicating that the cytoplasmic tail is essential for production of the virus. The virulence of the transfectant viruses in mice was directly proportional to the amount of NA incorporated. The importance of the NA cytoplasmic tail in virus assembly and virulence has implications for use in developing antiviral strategies.