bluuebunny/crossref_metadata_2025 huggingface dataset:
Human immunodeficiency virus integration in a cell-free system
Authors: Ellison V , Abrams H , Roe T , Lifson J , Brown P
Published: January 2020

Integration of the viral genome into the nuclear DNA of a host cell plays a pivotal role in the replication of retroviruses. We have developed an in vitro method for studying the biochemistry of human immunodeficiency virus (HIV) integration by using extracts from HIV-infected cells. Analysis of the reaction products showed that HIV integration in vitro accurately reproduces the in vivo process. Integration occurred without apparent specificity for the target sequence, and the integrated provirus was directly flanked by a 5-base-pair duplication of DNA from the target site. HIV integration did not require a high-energy cofactor, and the enzymatic activities required for integration were recovered with the viral DNA when cell extracts were fractionated by gel exclusion chromatography.
Structural requirements in the herpes simplex virus type 1 transactivator Vmw65 for interaction with the cellular octamer-binding protein and target TAATGARAT sequences
Authors: Greaves R F , O'Hare P
Published: January 2020

Herpes simplex virus type 1 virion protein Vmw65 forms a complex (TRF.C) with TAATGARAT sequences and the cellular transcription factor oct-1, which has been implicated as an intermediate in the activation of gene expression by Vmw65. To examine structural requirements within Vmw65 for this interaction, we analyzed extracts of transfected cells that express mutant Vmw65 proteins by gel retardation assay and identified two regions in the primary sequence of Vmw65 which are necessary for in vitro assembly of TRF.C. The amino-terminal boundary for complex assembly and trans activation mapped between residues 49 and 75. At the carboxyl terminus, deletion as far as residue 388 did not affect in vitro TRF.C assembly, although trans-activating activity was abolished. Deletion beyond residue 388 rapidly impaired the ability of the protein to participate in the TRF.C complex, such that a truncated mutant of 380 residues was completely inactive. These requirements towards the carboxyl terminus overlap a region of strong local sequence similarity between Vmw65 and terminal protein p3 of bacteriophage phi 29. Although substitution of corresponding p3 residues into Vmw65 failed to produce a functional chimera, site-directed mutagenesis within the region of similarity identified a number of single-point mutant proteins which were completely deficient for TRF.C formation. These mutant proteins were also unable to trans activate expression from immediate-early promoters, despite the integrity of the acidic carboxyl terminus. The extreme sensitivity of both TRF.C formation and trans activation to single-residue substitutions within this region of Vmw65 suggests that it is directly involved in the protein-protein or protein-DNA interactions required for assembly of a transcriptional complex containing oct-1.
cis-dominant defect in activation of adenovirus type 5 E1b early RNA synthesis
Authors: Parks C L , Spector D J
Published: January 2020

A cis-dominant mutation in the adjacent E1a gene disrupted the accumulation of adenovirus type 5 E1b mRNA during the early phase of infection. Steady-state levels of cytoplasmic and nuclear E1b RNAs in cells infected with dl312, a strain that lacks the E1a TATA box, cap site, and much of the coding sequence, were reduced 5- to 10-fold even when the E1a activator was provided in trans. The strain was defective for early E1b RNA synthesis but not for E1b RNA made late or during prolonged incubation in the presence of an inhibitor of DNA replication. The defect in E1b RNA synthesis could not be attributed to the E1a promoter sequences missing in dl312 DNA. If the E1a protein-coding region contains cis-acting regulatory sequences, they are not part of the previously mapped E1b transcriptional control region and may represent additional regulatory elements that ensure prompt and efficient E1b expression during the early phase of infection.
Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs
Authors: Pattnaik A K , Wertz G W
Published: January 2020

Replication and amplification of RNA genomes of defective interfering (DI) particles of vesicular stomatitis virus (VSV) depend on the expression of viral proteins and have until now been attained only in cells coinfected with helper VSV. In the work described in this report, we used a recombinant vaccinia virus-T7 RNA polymerase expression system to synthesize individual VSV proteins in cells transfected with plasmid DNAs that contain cDNA copies of the VSV genes downstream of the T7 RNA polymerase promoter. In this way, we were able to examine the ability of VSV proteins, individually and in combination, to support DI particle RNA replication. VSV proteins were synthesized soon after transfection in amounts that depended on the amount of input plasmid DNA and at rates that remained constant for at least 16 h after transfection. When cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with the DI particles, rapid and efficient replication and amplification of DI particle RNA was observed. Omission of any one of the three viral proteins abrogated the replication. The maximum levels of DI particle RNA replication that were achieved in the system exceeded those seen with wild-type helper VSV by 8- to 10-fold and were observed at molar L:NS:N protein ratios of approximately 1:200:200. This replication system can be used for analysis of structure-function relationships of VSV proteins that are involved in RNA replication and has potential for use in the identification of RNA sequences in the viral genome that control transcription and replication of VSV RNA.
Rapid titration of retroviral vectors encoding intracellular antigens by flow cytometry
Authors: Sladek T L , Jacobberger J W
Published: January 2020

Flow cytometry was used to detect cells infected with retroviral vectors encoding both simian virus 40 large T antigen and G418 resistance after indirect immunofluorescence staining using a T-antigen-specific monoclonal antibody and a fluorescein-conjugated secondary antibody. Titers of viral stocks determined by flow cytometry were equivalent to those determined by quantitation of G418-resistant colonies.
Antibody-mediated neutralization in vivo of infectious papillomaviruses
Authors: Christensen N D , Kreider J W
Published: January 2020

Specific antibody-mediated neutralization of infectious human papillomavirus type 11 (HPV-11) was achieved in the athymic mouse xenograft system, in which HPV-11 induced morphological transformation of human foreskin. Virus-specific neutralization was demonstrated by the ability of an HPV-11-specific polyclonal antiserum to neutralize HPV-11 infectivity and not bovine papillomavirus type 1 (BPV-1) or cottontail rabbit papillomavirus (CRPV) infectivity. In all three virus infectivity systems, neutralization was detected by the failure of the virus suspension to induce morphological transformation of the appropriate skin xenografts placed under the renal capsule of athymic mice. Rabbit polyclonal antisera were also generated against intact virions of both BPV-1 and CRPV, and neutralizing activity was tested in the xenograft system with BPV-1 and fetal bovine skin and with CRPV and rabbit ear skin. The three polyclonal antisera contained virus-specific neutralizing antibodies, demonstrating that neutralizing epitopes existed on all three papillomaviruses and that these epitopes were antigenically non-cross-reactive. The athymic mouse xenograft system was a useful model for detecting papillomavirus-specific neutralizing antibodies and offers the only opportunity for the analysis of neutralizing antibodies to human papillomaviruses.
Presence of mitochondrial D-loop DNA in scrapie-infected brain preparations enriched for the prion protein
Authors: Aiken J M , Williamson J L , Borchardt L M , Marsh R F
Published: January 2020

The prion preparation has, in recent years, been the focal point of scrapie research. The inability to identify agent-specific nucleic acids in this sample has led to the formulation of the infectious protein or prion hypothesis. In this study, we analyzed three different prion protein-enriched preparations and found all to contain significant amounts of mitochondrial nucleic acid. Southern blot analyses indicated that they are enriched for a specific component of the mitochondrial genome, the single-stranded displacement loop fragment. Our results suggest that if mitochondrial nucleic acids are involved in scrapie infection, it is the displacement loop fragment that is specifically responsible.
T-cell-induced expression of human immunodeficiency virus in macrophages
Authors: Schrier R D , McCutchan J A , Venable J C , Nelson J A , Wiley C A
Published: January 2020

Macrophages are major reservoirs of human immunodeficiency virus (HIV) in the tissues of infected humans. As monocytes in the peripheral blood do not show high levels of infection, we have investigated the expression of HIV in T-cell-activated, differentiated macrophages. Peripheral blood mononuclear cells were isolated from HIV-seropositive individuals and stimulated with antigens or mitogens, and the nonadherent fraction was removed. Macrophages were cultured alone for 2 weeks, and HIV expression was assessed. Results from p24 antigen capture assays demonstrated that the presence of autologous T cells and concanavalin A or autologous T cells and allogeneic cells for the initial 24 h of culture induced HIV expression in 35 of 47 (74%) HIV-seropositive patients tested. The macrophage monolayers could be immunostained with anti-HIV antibodies to reveal discrete infectious centers, indicating that complete virus replication was occurring in the macrophages and that infection of adjacent cells was mediated by cell-cell contact. Time course studies of the interval of coculture of the adherent and nonadherent cells indicated that 24 h (but not 2 h) was sufficient for induction of HIV in the macrophages. Direct contact between the adherent cells and activated T cells was required as well. Since the presence of autologous T cells also appeared to be necessary, induction of HIV expression in macrophages may be genetically restricted. HIV-seronegative nonadherent cells were able to induce HIV expression in macrophages from HIV-seropositive donors, demonstrating that the virus originated in the monocytes and was reactivated in the context of a classic T-cell-mediated immune reaction. The high percentage of monocytes from HIV-seropositive donors which can be induced to replicate HIV by activated T cells suggests that infection of monocytes may be critical to the pathogenesis of this lentivirus infection.
Interference with human immunodeficiency virus (HIV) replication by CD8+ T cells in peripheral blood leukocytes of asymptomatic HIV carriers in vitro
Authors: Kannagi M , Masuda T , Hattori T , Kanoh T , Nasu K , Yamamoto N , Harada S
Published: January 2020

A long asymptomatic period is one of the characteristics of human immunodeficiency virus (HIV) infection, despite its fatal consequences. Antiviral defense in HIV-infected individuals controls viral replication during this period. In the present study, we demonstrate that peripheral blood leukocytes (PBL) of asymptomatic HIV-1 carriers, following exogenous HIV-1 infection in vitro, do not support viral replication. These cells do not produce detectable amounts of reverse transcriptase or accumulate unintegrated proviral DNA. This is a striking contrast to the behavior of HIV-1-infected PBL of seronegative individuals, which produce large amounts of RT and unintegrated DNA. Such resistance to HIV-1 replication is not seen in PBL of patients with advanced disease. Since the binding of HIV-1 to CD4 molecule is not impaired in PBL of asymptomatic carriers, the interference with HIV replication must occur after the stage of virus binding. PBL lose their resistance when CD8+ lymphocytes are removed. In addition, these PBL are not resistant to an exogenous infection with HIV-2. These observations suggest that certain populations of CD8+ lymphocytes of asymptomatic HIV-1 carriers operate on the target cells in PBL to block viral replication in an HIV-1-specific manner. Such CD8+ lymphocyte-mediated interference with HIV replication could play an important role in the maintenance of the period of disease latency.
Ping-pong amplification of a retroviral vector achieves high-level gene expression: human growth hormone production
Authors: Kozak S L , Kabat D
Published: January 2020

Retroviral vectors offer major advantages for gene transfer studies but have not been useful for producing proteins in large quantities. This deficiency has resulted in part from interference to superinfection, which limits the numbers of active proviruses in cells. Recently, we found that these vectors amplify when they are added as calcium phosphate precipitates to cocultures of cells that package retroviruses into ecotropic and amphotropic host range envelopes. Helper-free virions from either cell type can infect the other without interference, resulting in theoretically limitless back-and-forth (ping-pong) vector replication. In initial studies, however, amplifications of a vector that contained the human growth hormone gene ceased when the hormone produced was 0.3% or less of cellular protein synthesis. This limit was caused by two factors. First, recombinant shutoff viruses that are replication defective and encode envelope glycoproteins form at a low probability during any round of the vector replication cycle and these spread in cocultures, thereby establishing interference. Single cells in shutoff cocultures therefore synthesize both ecotropic and amphotropic envelope glycoproteins, and they release promiscuous (presumably hybrid) virions. The probability of forming shutoff viruses before the vector had amplified to a high multiplicity was reduced by using small cocultures. Second, cells with large numbers of proviruses are unhealthy and their proviral expression can be unstable. Stable expresser cell clones were obtained by selection. Thereby, cell lines were readily obtained that stably produce human growth hormone as 4 to 6% of the total protein synthesis. A ping-pong retroviral vector can be used for high-level protein production in vertebrate cells.
The growth of simian virus 40 (SV40) host range/adenovirus helper function mutants in an African green monkey cell line that constitutively expresses the SV40 agnoprotein
Authors: Stacy T P , Chamberlain M , Carswell S , Cole C N
Published: January 2020

The simian virus 40 T-antigen carboxy-terminal mutants, dlA2459 and dlA2475, are cell line and temperature dependent for growth and plaque formation in monkey kidney cells. Although these mutants did form plaques on BSC-1 cells at 37 degrees C, they were about fivefold less efficient for plaque formation than wild-type simian virus 40. These mutants did not grow in CV-1 cells and did not synthesize agnoprotein in those cells. CV-1 cells which constitutively express the agnoprotein were permissive for mutant plaque formation. However, late mRNAs, virion proteins, and progeny virion yields did not accumulate to wild-type levels during mutant infection of the agnoprotein-producing cells.
Mutagenic analysis of the v-crk oncogene: requirement for SH2 and SH3 domains and correlation between increased cellular phosphotyrosine and transformation
Authors: Mayer B J , Hanafusa H
Published: January 2020

We have constructed a series of mutants with deletion, linker insertion, and point mutations in the v-crk oncogene of avian sarcoma virus CT10. The v-crk gene contains no apparent catalytic domain, but does contain two blocks of homology to putative regulatory domains, termed SH2 and SH3, found in a variety of proteins implicated in signal transduction. Infection with CT10 causes a dramatic increase in the level of tyrosine phosphorylation of several cellular proteins. We found that mutation of either the SH2 or SH3 domain of v-crk reduced or eliminated transforming activity, whereas mutation of regions outside the conserved domains had no effect. Deletion of amino-terminal gag sequences caused a partial loss of transforming activity and a change in subcellular distribution of the crk protein. In all cases, there was an absolute correlation between increased cellular phosphotyrosine and transformation.
Kinetics of poliovirus uncoating in HeLa cells in a nonacidic environment
Authors: Gromeier M , Wetz K
Published: January 2020

Lysis of HeLa cells infected with poliovirus revealed intact virus; 135S particles, devoid of VP4 but containing the viral RNA; and 80S empty capsids. During infection the kinetics of poliovirus uncoating showed a continuous decrease of intact virus, while the number of 135S particles and empty shells increased. After 1.5 h of infection conformational transition to altered particles resulted in complete disappearance of intact virions. To investigate the mechanism of poliovirus uncoating, which has been suggested to depend on low pH in endosomal compartments of cells, we used lysosomotropic amines to raise the pH in these vesicles. In the presence of ammonium chloride, however, the kinetics of uncoating were similar to those for untreated cells, whereas in cells treated with methylamine, monensin, or chloroquine, uncoating was merely delayed by about 30 min. This effect could be attributed to a delay of virus entry into cells after treatment with methylamine and monensin, whereas chloroquine stabilized the viral capsid itself. Thus, elevation of endosomal pH did not affect virus uncoating. We therefore propose a mechanism of poliovirus uncoating which is independent of low pH.
A conformational change in Sindbis virus glycoproteins E1 and E2 is detected at the plasma membrane as a consequence of early virus-cell interaction
Authors: Flynn D C , Meyer W J , Mackenzie J M , Johnston R E
Published: January 2020

A conformational change in the structure of Sindbis (SB) virus was detected after virion attachment to baby hamster kidney cells but before internalization. The alteration was manifested as increased virion binding of certain glycoprotein E1 and E2 monoclonal antibodies (MAbs) that recognized transitional epitopes. These epitopes were inaccessible to MAb on native virions but became accessible to their cognate MAbs in the early stages of infection. Transit of virions through a low-pH compartment apparently was not required for the conformational change. Exposure of transitional epitopes was unaffected by treatment of BHK cells with NH4Cl and occurred normally in Chinese hamster ovary cells temperature sensitive for endosomal acidification. However, the rearrangement was correlated with both the time course and temperature dependence of SB virus penetration, and the rearrangement occurred earlier with an SB virus mutant having an accelerated penetration phenotype. In addition, MAb to a transitional epitope, a probe specific for rearranged particles, retarded penetration of infectious virions. These results suggested that the SB virus E1/E2 glycoprotein spike undergoes a structural rearrangement as a consequence of virion interaction with the cell surface and that this altered virion form may be an important early intermediate in an entry pathway leading to productive infection.
Immunization with a live, attenuated simian immunodeficiency virus (SIV) prevents early disease but not infection in rhesus macaques challenged with pathogenic SIV
Authors: Marthas M L , Sutjipto S , Higgins J , Lohman B , Torten J , Luciw P A , Marx P A , Pedersen N C
Published: January 2020

An infectious, virulence-attenuated molecular clone of simian immunodeficiency virus (SIV), SIVMAC-1A11, was derived from an SIV isolate that causes fatal immunodeficiency in rhesus macaques. When inoculated intravenously in rhesus macaques, SIVMAC-1A11 induced transient viremia (1 to 6 weeks) without clinical disease and a persistent humoral antibody response. The antibodies were directed mainly against the viral envelope glycoproteins, as determined by immunoblots and virus neutralization. The potential of this virulence-attenuated virus to protect against intravenous challenge with a pathogenic SIVMAC strain was assessed. Five rhesus macaques were each given two intravenous inoculations with SIVMAC-1A11 7 months apart. Three of the five immunized monkeys and four naive control animals were then challenged with 100 to 1,000 100% animal infectious doses of pathogenic SIVMAC. All seven animals became persistently viremic following the challenge. Four of four unimmunized animals developed severe clinical signs of simian acquired immunodeficiency syndrome by 38 to 227 days after challenge and were euthanatized 91 to 260 days postchallenge. However, no signs of illness were seen in immunized monkeys until 267 to 304 days postchallenge, when two of three immunized animals developed mild thrombocytopenia and lymphopenia; one of these animals died with clinical signs of simian immunodeficiency disease at 445 days after challenge. The two SIVMAC-1A11-immunized monkeys that were not challenged were healthy and antibody positive 22 months after the initial immunization. Thus, although live SIVMAC-1A11 was immunogenic and did not induce any disease, it failed to protect rhesus macaques against infection with a moderately high dose of pathogenic virus. However, immunization prevented severe, early disease and prolonged the lives of monkeys subsequently infected with pathogenic SIV.
Combined infection by Moloney murine leukemia virus and a mink cell focus-forming virus recombinant induces cytopathic effects in fibroblasts or in long-term bone marrow cultures from preleukemic mice
Authors: Li Q X , Fan H
Published: January 2020

We described previously a preleukemic state in mice inoculated with Moloney murine leukemia virus (M-MuLV) characterized by generalized hematopoietic hyperplasia in the spleen. To investigate this further, long-term bone marrow cultures (LTBMC) from preleukemic mice were established. Surprisingly, LTBMC from M-MuLV-inoculated preleukemic mice showed less hematopoiesis than LTBMC from control mice. This resulted from a quantitative defect in establishment of bone marrow stromal cells in the LTBMC. This phenomenon could also be observed in LTBMC from normal mice infected in vitro with a stock of M-MuLV containing a mink cell focus-forming virus (MCF) derivative (M-MCF), but not in LTBMC infected with M-MuLV alone. This implicated MCF derivatives in the reduction in bone marrow stromal cells. The phenomenon could also be detected in infected NIH 3T3 cells. Combined infection of M-MuLV plus M-MCF resulted in fewer cells, in comparison to uninfected cells or cells infected with either virus alone. Further studies indicated that this was predominantly due to an inhibition in cell growth rather than to cell lysis. The cytopathic effect did not appear to result from overreplication of viral DNA, as measured by Southern blots. Thus, combined infection with M-MuLV and an MCF derivative had cytostatic effects on cell growth. This phenomenon might also contribute to the leukemogenic process in vivo.
Substitution of the U3 long terminal repeat region of the neurotropic Cas-Br-E retrovirus affects its disease-inducing potential
Authors: Paquette Y , Kay D G , Rassart E , Robitaille Y , Jolicoeur P
Published: January 2020

The Cas-Br-E and ts-Mo BA-1 murine leukemia viruses (MuLV) induce a spongiform neurodegenerative disease with different clinical manifestations, namely, either hind limb paralysis (Cas-Br-E) or tremors, spasticity, and hind limb weakness (ts-Mo Ba-1). We constructed the chimeric NEBA-1 MuLV by replacing the long terminal repeat of Cas-Br-E MuLV with that of ts-Mo BA-1 MuLV. In SWR/J or CFW/D mice, NEBA-1 MuLV induced an ataxic neurological disease characterized by clinical signs different from those induced by both parents. Although NEBA-1 MuLV did not induce lesions in novel brain areas, the spongiform lesions were more severe in deep cerebellar nuclei and in the spinal cord than those found in paralyzed mice inoculated with Cas-Br-E MuLV. By in situ hybridization, we found that the distribution of the spongiform lesions closely correlated with the distribution of the infected central nervous system cells. In the spinal cord, a close correlation was found between the number of infected cells and the severity of the spongiform degeneration. Sequencing of the substituted ts-BA-1 MuLV fragment and comparison with homologous sequences of Cas-Br-E and Moloney MuLV showed differences mainly in the U3 tandem direct repeats. Our results show that a few modifications within the U3 long terminal repeat allow the virus to cause more severe lesions in some central nervous system regions and that the severity of the spongiform degeneration correlates with the level of viral replication.
The soluble viral glycoprotein of vesicular stomatitis virus efficiently sensitizes target cells for lysis by CD4+ T lymphocytes
Authors: Browning M , Reiss C S , Huang A S
Published: January 2020

The soluble glycoprotein Gs of vesicular stomatitis virus (VSV), at approximately 10(4) molecules per cell, sensitized target cells for lysis by clones of CD4+ cytolytic T lymphocytes (CTL). In addition to lysis, the clones responded by proliferation and interleukin-2 release. Targets sensitized by Gs competed effectively with VSV-infected cells for recognition. Immune cytolysis by these CD4+ CTLs was restricted by class II major histocompatibility complex (MHC) antigens and was specific to VSV. The specific class II MHC antigen which was restricting for each clone remained the same whether the targets were sensitized by infection with VSV or by exogenously added soluble antigen. Sensitization by Gs appeared to require prior processing because the antigen-presenting cells that were fixed prior to exposure to Gs failed to be recognized by the CTL clones. The high efficiency of this uptake and processing was suggested by the inability of Gs at concentrations up to 10(7) per cell to block superinfection by VSV or to effect the RNA-synthetic machinery of uninfected cells. Also, Gs failed to hemolyze sheep erythrocytes when there was hemolysis by virions or an amino-terminal peptide of the VSV glycoprotein. Extrapolation of these results to viral diseases was possible because soluble viral glycoproteins were naturally synthesized during many viral infections and class II MHC antigens were inducible in cells of nonlymphoid origin. Therefore, CD4+ CTLs may be important participants in increasing virus-induced pathology, especially among adjacent uninfected cells.
Proteolytic dissection of Sindbis virus core protein
Authors: Strong R K , Harrison S C
Published: January 2020

Mild trypsin treatment of the Sindbis virus nucleocapsid protein yields a fragment with a molecular mass of approximately 18.5 kilodaltons with its N terminus at residue 105. The fragment, which is stable to further digestion, appears by gel exclusion chromatography to be monomeric. These data are consistent with a model for the alphavirus core proteins, consisting of an extended and flexible N-terminal arm (residues 1 to 103) and a compactly folded C-terminal domain (residues 104 to 274), as previously suggested on the basis of sequence characteristics.
Serologic type conversion of a herpes simplex virus type 1 (HSV-1) to an HSV-2 epitope caused by a single amino acid substitution in glycoprotein C
Authors: Kimmel K A , Dolter K E , Toth G M , Levine M , Glorioso J C
Published: January 2020

A monoclonal antibody to herpes simplex virus type 2 glycoprotein C (gC-2) did not recognize wild-type herpes simplex virus type 1 gC (gC-1) but did recognize a mutant gC-1 molecule. This conversion from a type 1 to a type 2 epitope was shown to be due to a single amino acid substitution in gC-1.
Inducible nuclear factor binding to the kappa B elements of the human immunodeficiency virus enhancer in T cells can be blocked by cyclosporin A in a signal-dependent manner
Authors: Schmidt A , Hennighausen L , Siebenlist U
Published: January 2020

Cyclosporin A (CsA) is thought to exert its immunosuppressive effects by inhibiting the expression of a distinct set of lymphokine genes which are induced upon T-cell activation, among them the gene coding for interleukin-2. In addition, the activation of the human immunodeficiency virus (HIV) is partially suppressed. To better understand the molecular mechanisms underlying suppression by CsA, we have investigated the effects of this drug on transcription factors in T cells. Here we report that the formation of two distinct mitogen-inducible DNA-binding complexes, the kappa B complex within the HIV enhancer and the NFAT-1 complex within the interleukin-2 enhancer, is inhibited in the presence of CsA. The kappa B-binding activity with the HIV enhancer is inhibited only if it is activated via the mitogen phytohemagglutinin whereas phorbol myristate acetate-mediated activation is completely insensitive to the drug. This suggests a model in which functionally indistinguishable kappa B complexes can be activated via two separate pathways of signal transduction distinguishable by CsA.
Characterization of hepatitis delta antigen: specific binding to hepatitis delta virus RNA
Authors: Lin J H , Chang M F , Baker S C , Govindarajan S , Lai M M
Published: January 2020

It has previously been shown that human hepatitis virus delta antigen has an RNA-binding activity (Chang et al., J. Virol. 62:2403-2410, 1988). In the present study, the specificity of such an RNA-protein interaction was demonstrated by expressing various domains of the delta antigen in Escherichia coli as TrpE fusion proteins and testing their RNA-binding activities in a Northwestern protein-RNA immunoblot assay and RNA gel mobility shift assay. Hepatitis delta virus (HDV) RNA bound specifically to the delta antigen in the presence of an excess amount of unrelated RNAs and a relatively high salt concentration. Both genome- and antigenome-sense HDV RNAs and at least two different regions of HDV genomic RNA bound to the delta antigen. Surprisingly, these two different regions of HDV genomic RNA could compete with each other for delta antigen binding, although they do not have common nucleotide sequences. In contrast, this binding could not be competed with by other viral or cellular RNA. Since both the genomic and antigenomic HDV RNAs had strong intramolecular complementary sequences, these results suggest that the binding of delta antigen is probably specific for a secondary structure unique to the HDV RNA. By expressing different subdomains of the delta antigen, we found that the middle one-third of delta antigen was responsible for binding HDV RNA. Neither the N-terminal nor the C-terminal domain bound HDV RNA. Binding between the delta antigen and HDV RNA was also demonstrated within the HDV particles isolated from the plasma of a human delta hepatitis patient. This in vivo binding resisted treatment with 0.1% sodium dodecyl sulfate and 0.5% Nonidet P-40. In addition, we showed that the antiserum from a human patient with delta hepatitis reacted with all three subdomains of the delta antigen, indicating that all of the domains are immunogenic in vivo. These studies demonstrated the specific interaction between delta antigen and HDV RNA.
Alternative splice acceptor utilization during human immunodeficiency virus type 1 infection of cultured cells
Authors: Guatelli J C , Gingeras T R , Richman D D
Published: January 2020

The utilization of alternative splice acceptors for excision of the 5' major intron of human immunodeficiency virus type 1 RNA was observed after infection in vitro. Specific splice events were monitored by a cDNA-polymerase chain reaction. These splice events shared a common splice donor but utilized several alternative splice acceptors. In addition to identifying the previously documented splice acceptors for tat and nef (S. K. Arya, C. Guo, S. F. Josephs, and F. Wong-Staal, Science 229:69-73, 1985), nucleotide sequence analysis of cDNA-polymerase chain reaction fragments also revealed the following: (i) two splice acceptors 15 and 9 nucleotides upstream from the rev start codon, which are utilized to create transcripts suitable for specific rev expression; and (ii) use of the splice acceptor previously attributed to nef to generate a singly spliced, env-encoding transcript. Hybridization signals representing the nef/env, tat, and rev splice events increased in intensity between 6 and 12 h after infection of CEM cells with the LAV-1BRU strain of human immunodeficiency virus type 1. In contrast, the signal for utilization of the nef/env splice acceptor for the singly spliced env transcript appeared first at 12 h and increased to maximum intensity by 24 h. The nef/env splice acceptor was dominant at all time points examined. We propose that this dominance ensures efficient downstream splicing proximal to the env initiation codon in singly spliced transcripts. However, early after infection, the dominance of the nef/env splice acceptor appears to divert primary transcripts away from tat- and rev-specific processing paths. The relative proportions of hybridization signals representing these alternative splice events remained constant throughout the viral replicative cycle. This result suggests that trans-acting factors that might influence splice choices are not induced during infection, but rather that cis-acting, sequence-specific splice preferences determine the relative efficiency of alternative acceptor utilization.
Temperature-sensitive mutants of Abelson murine leukemia virus deficient in protein tyrosine kinase activity
Authors: Engelman A , Rosenberg N
Published: January 2020

The effect of two missense mutations in abl on transformation by Abelson murine leukemia virus was evaluated. These mutations led to the substitution of a histidine for Tyr-590 and a glycine for Lys-536. Both changes gave rise to strains that were temperature dependent for transformation of both NIH 3T3 cells and lymphoid cells when expressed in the context of a truncated Abelson protein. In the context of the prototype P120 v-abl protein, the Gly-536 substitution generated a host range mutant that induced conditional transformation in lymphoid cells but had only a subtle effect on NIH 3T3 cells. The combination of both substitutions gave rise to a P120 strain that was temperature sensitive for both NIH 3T3 and lymphoid cell transformation. The Abelson proteins encoded by the temperature-sensitive strain displayed in vitro kinase activities that were reduced when compared with those of wild-type proteins. In vivo, levels of phosphotyrosine were reduced only at the restrictive temperature. Analysis of cells expressing either the wild-type P160 v-abl protein or the P210 bcr/abl protein and an Abelson protein encoded by a temperature-sensitive strain failed to correct this defect, suggesting either that tyrosine phosphorylation in vivo is an intramolecular reaction or that the protein encoded by the temperature-sensitive strain is a poor substrate for tyrosine phosphorylation in vivo. These results raise the possibility that tyrosine phosphorylation of Abelson protein plays a role in transformation.
Linear B-cell epitopes of the major core protein of human immunodeficiency virus types 1 and 2
Authors: Janvier B , Archinard P , Mandrand B , Goudeau A , Barin F
Published: January 2020

Nine murine monoclonal antibodies directed to the major core protein p24 of human immunodeficiency virus type 1 (HIV-1) were obtained and then tested by using an epitope mapping system (Pepscan) covering the whole p24HIV1 protein to characterize antigenic domains. Four different linear epitopes were identified. Monoclonal antibodies recognizing three of these epitopes also reacted to p26HIV2 in Western blotting (immunoblotting). A monoclonal antibody specific for the fourth epitope, located at position 179 to 188 of the gag polyprotein p55HIV1 (human T-cell lymphotropic virus type 3B strain), did not react with HIV type 2 (HIV-2) core proteins. The corresponding sequence is constant in all known HIV-2 and simian immunodeficiency virus (SIV) isolates, including a very divergent SIV strain from African green monkeys (SIVagm/tyo). This observation may be relevant to the phylogeny of primate lentiviruses. Two of the conserved epitopes might be immunogenic during natural infection and could therefore be used for diagnosis and prognosis purposes. These two epitopes are AAEWDRVHP and EIYKRWII, starting at positions 209 and 260 of the polyprotein p55HIV1, respectively.
Purification, renaturation, and reconstituted protein kinase activity of the Sendai virus large (L) protein: L protein phosphorylates the NP and P proteins in vitro
Authors: Einberger H , Mertz R , Hofschneider P H , Neubert W J
Published: January 2020

Sodium dodecyl sulfate-solubilized Sendai virus large (L) protein was highly purified by a one-step procedure, using hydroxylapatite column chromatography. Monoclonal antibodies addressed to the carboxyl-terminal amino acid sequence of the L protein were used for monitoring L protein during purification. By removing sodium dodecyl sulfate from purified L protein, a protein kinase activity was successfully renatured. P and NP proteins served as its substrates. After immunoprecipitation with anti-L antibodies, the immunocomplex already showed protein kinase activity. In the presence of P protein, the NP protein was more highly phosphorylated. The results show that Sendai virus L protein possesses a protein kinase activity phosphorylating the other proteins of the viral nucleocapsid in vitro.
Comparison of Rous sarcoma virus RNA processing in chicken and mouse fibroblasts: evidence for double-spliced RNA in nonpermissive mouse cells
Authors: Berberich S L , Macias M , Zhang L , Turek L P , Stoltzfus C M
Published: January 2020

Rous sarcoma virus, an avian retrovirus, transforms but does not replicate in mammalian cells. To determine to what extent differences in RNA splicing might contribute to this lack of productive infection, cloned proviral DNA derived from the Prague A strain of Rous sarcoma virus was transfected into mouse NIH 3T3 cells, and the viral RNA was compared by RNase protection with viral RNA from transfected chicken embryo fibroblasts by using a tandem antisense riboprobe spanning the three major splice sites. The levels of viral RNA in NIH 3T3 cells compared with those in chicken embryo fibroblasts were lower, but the RNA was spliced at increased efficiency. The difference in the ratio of unspliced to spliced RNA levels was not due to the increased lability of unspliced RNA in NIH 3T3 cells. Although chicken embryo fibroblasts contained equal levels of src and env mRNAs, spliced viral mRNAs in NIH 3T3 cells were almost exclusively src. In NIH 3T3 cells the env mRNA was further processed by using a cryptic 5' splice site located within the env coding sequences and the normal src 3' splice site to form a double-spliced mRNA. This mRNA was identical to the src mRNA, except that a 159-nucleotide sequence from the 5' end of the env gene was inserted at the src splice junction. Smaller amounts of single-spliced RNA were also present in which only the region between the cryptic 5' and src 3' splice sites was spliced out. The aberrant processing of the viral env mRNA in NIH 3T3 cells may in part explain the nonpermissiveness of these cells to productive Rous sarcoma virus infection.
Segregation patterns of endogenous mouse mammary tumor viruses in five recombinant inbred strain sets
Authors: Lee B K , Eicher E M
Published: January 2020

We identified mouse mammary tumor proviral loci in the AKR/J, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, and SWR/J inbred mouse strains and determined their segregation patterns in the AKXD, AKXL, BXD, BXH, and SWXL recombinant inbred strain sets. Two new Mtv loci, Mtv-29 and Mtv-30, were identified. Mtv-30 was genetically mapped to chromosome 12. Additionally, two previously identified Mtv loci, Mtv-14 and Mtv-23, were genetically mapped to chromosome 4 and chromosome 6, respectively.
Preleukemic hematopoietic hyperplasia induced by Moloney murine leukemia virus is an indirect consequence of viral infection
Authors: Brightman B K , Davis B R , Fan H
Published: January 2020

We previously showed that neonatal mice inoculated with Moloney murine leukemia virus (M-MuLV) exhibit a preleukemic state characterized by splenomegaly and increased numbers of hematopoietic progenitors. An M-MuLV variant with greatly reduced leukemogenic potential, Mo+PyF101 M-MuLV, does not generally induce this preleukemic state. In order to investigate the mechanism involved in M-MuLV induction of preleukemic hyperplasia, we tested the CFU-mixed myeloid and erythroid (CFUmix) from M-MuLV- and Mo+PyF101 M-MuLV-inoculated mice for the presence of virus by antibody staining and for the release of infectious virus. The majority of CFUmix colonies from both M-MuLV- and Mo+PyF101 M-MuLV-inoculated mice contained infectious virus even though M-MuLV-inoculated mice showed elevated levels of CFUmix while the Mo+PyF101 M-MuLV-inoculated mice did not. This indicates that direct infection of hematopoietic progenitors was not sufficient to induce hyperplasia. Rather, hematopoietic hyperplasia may result indirectly from infection of some other cell type.
Characterization and expression of novel singly spliced RNA species of human immunodeficiency virus type 1
Authors: Arrigo S J , Weitsman S , Zack J A , Chen I S
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) expresses the Vif, Vpr, Vpu, and Env proteins through complex differential splicing of a single full-length RNA precursor. We used HIV-1-specific oligonucleotide primer pairs in a quantitative polymerase chain reaction procedure on RNA from fresh peripheral blood lymphocytes infected with HIV-1JR-CSF to detect and characterize the singly spliced RNA species which might encode these proteins. The nucleotide sequences at the junctions of splice donor and acceptor sites of these RNAs were determined. One of these RNAs, which has not been previously described, appears to be a novel HIV-1 RNA encoding Env and/or Vpu proteins.
Two point mutations in the transmembrane domain of P68gag-ros inactive its transforming activity and cause a delay in membrane association
Authors: Jong S M , Wang L H
Published: January 2020

The transforming protein of the avian sarcoma virus UR2 is a 68-kDa transmembrane tyrosine protein kinase. We examined the relationship between membrane localization and transforming activity of P68 by changing Val-168-Val-169 in its hydrophobic domain into Asp-168-Glu-169. The resulting transmembrane (TM) mutant (P68TM) lost transforming activity toward chicken embryo fibroblasts (CEF). We found that the mutant protein was expressed and rapidly degraded into a smaller form which was still membrane associated and kinase active. The instability of the TM mutant protein is a phenomenon only manifested in CEF, because the same mutant protein was expressed with efficiency and stability similar to those of the wild-type protein in a transient expression system in COS cells. However, there are several differences between the wild-type and the TM mutant proteins in COS cells. The wild-type protein is more heavily phosphorylated and associated with membrane fractions in a cotranslational manner. It is enzymatically active when recovered from membrane fractions. The TM mutant protein is less phosphorylated, more labile toward protease degradation, and delayed in membrane association, with a lag period of 30 min or longer, and has little kinase activity when recovered from membrane fractions. Most of the kinase-active TM mutant protein was found in the cytosol fractions. Despite the delay, most of the TM protein in COS cells was found to be membrane associated, and its orientation on the cell surface was similar to that of the wild-type protein. It is probable that loss of the CEF-transforming activity of the TM mutant protein is due to its susceptibility to protease degradation resulting from improper membrane association of the newly synthesized product. The differences in the kinetics of membrane association and the distribution of kinase activity in COS cells might not be directly applicable in explaining the inability of the TM mutant to transform CEF but are intriguing as regards protein biosynthesis and translocation. The difference between CEF and COS cells implies that different factors or pathways are involved in the biosynthesis and processing of the TM mutant protein in these two cellular environments. Changes of P68TM in the kinetics of membrane association indicate that the transmembrane domain of ros, besides functioning as a membrane anchor, also plays a role in directing initial membrane association.
Rex transregulation of human T-cell leukemia virus type II gene expression
Authors: Kim J H , Kaufman P A , Hanly S M , Rimsky L T , Greene W C
Published: January 2020

The Rex protein of the human T-cell leukemia virus type II (HTLV-II), Rex-II, plays a central role in regulating the expression of the structural genes of this retrovirus. Rex-II acts posttranscriptionally by inducing the cytoplasmic expression of the incompletely spliced viral mRNAs that encode the Gag and Env structural proteins and the enzymes derived from the pol gene. We now define a 295-nucleotide cis-acting regulatory element within the 3' long terminal repeat of HTLV-II that is required for the effects of Rex-II. This Rex-II response element (RexIIRE) corresponds to a predicted, highly stable RNA secondary structure and functions when present in the sense but not in the antisense orientation. The RexIIRE confers responsiveness not only to Rex-II but also to the Rex protein of HTLV-I. Deletion and substitution mutagenesis of the RexIIRE permitted identification of a small subregion within the larger element critically required for Rex-II responsiveness and further suggested that the structurally distinct RexIIREs generated from the 5' and 3' long terminal repeats of HTLV-II may differentially regulate the cytoplasmic expression of unspliced gag-pol and singly spliced env mRNAs. While the Rev protein of human immunodeficiency virus type 1 fails to function via the RexIIRE, the Rex-II protein, like Rex-I, can functionally replace the Rev protein of human immunodeficiency virus type 1 via its interaction with the Rev response element (RevRE).
Induction of high-titer neutralizing antibodies, using hybrid human immunodeficiency virus V3-Ty viruslike particles in a clinically relevant adjuvant
Authors: Griffiths J C , Berrie E L , Holdsworth L N , Moore J P , Harris S J , Senior J M , Kingsman S M , Kingsman A J , Adams S E
Published: January 2020

The localization of neutralization determinants within the envelope glycoproteins of human immunodeficiency virus (HIV) has been largely achieved by immunizing small animals in conjunction with Freund's adjuvant. However, for eventual use in humans, candidate HIV vaccine components must also be efficacious in a nontoxic formulation. We describe here the production of hybrid Ty viruslike particles carrying the major neutralizing domain of HIV and demonstrate the induction of high-titer virus-neutralizing antibodies and an HIV-specific T-cell proliferative response after immunization in conjunction with aluminum hydroxide. As aluminum hydroxide and aluminum phosphate are the only adjuvants currently licensed for use in humans, these observations have implications for the development of an effective vaccine against HIV.