bluuebunny/crossref_metadata_2025 huggingface dataset:
Sp1 transcription factor is required for in vitro basal and Tat-activated transcription from the human immunodeficiency virus type 1 long terminal repeat
Authors: Suñé C , García-Blanco M A
Published: January 2020

Sp1-DNA binding sites have been reported to be essential for basal and Tat-activated transcription of the human immunodeficiency virus type 1 long terminal repeat (LTR). The role of the Sp1 transcription factor itself in regulation of the retroviral LTR, however, has not been clearly defined. It is now known, for instance, that the Sp1-DNA binding sites function also as thyroid hormone receptor response elements (V. Desay-Yajnik and H. H. Samuels, Mol. Cell. Biol. 13:5057-5069, 1993). In this report, we present data that demonstrate a strict requirement for Sp1 for both basal transcription and Tat-mediated trans activation of the human immunodeficiency virus type 1 LTR in vitro.
Cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG
Authors: Trkola A , Pomales A B , Yuan H , Korber B , Maddon P J , Allaway G P , Katinger H , Barbas C F , Burton D R , Ho D D
Published: January 2020

We have tested three human monoclonal antibodies (MAbs) IgG1b12, 2G12, and 2F5) to the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1), and a tetrameric CD4-IgG molecule (CD4-IgG2), for the ability to neutralize primary HIV-1 isolates from the genetic clades A through F and from group O. Each of the reagents broadly and potently neutralized B-clade isolates. The 2F5 MAb and the CD4-IgG2 molecule also neutralized strains from outside the B clade, with the same breadth and potency that they showed against B-clade strains. The other two MAbs were able to neutralize a significant proportion of strains from outside the B clade, although there was a reduction in their efficacy compared with their activity against B-clade isolates. Neutralization of isolates by 2F5 correlated with their possession of the LDKW motif in a segment of gp41 near the membrane-spanning domain. The other two MAbs and CD4-IgG2 recognize discontinuous binding sites on gp120, and so no comparison between genetic sequence and virus neutralization was possible. Our data show that a vaccine based on the induction of humoral immunity that is broadly active across the genetic clades is not impossible if immunogens that express the epitopes for MAbs such as 2F5, 2G12, and IgG1b12 in immunogenic configurations can be created. Furthermore, if the three MAbs and CD4-IgG2 produce clinical benefit in immunotherapeutic trials in the United States or Europe, they may also do so elsewhere in the world.
Functions of the internal pre-S domain of the large surface protein in hepatitis B virus particle morphogenesis
Authors: Bruss V , Vieluf K
Published: January 2020

The large hepatitis B virus (HBV) surface protein (L) forms two isomers which display their N-terminal pre-S domain at the internal and external side of the viral envelope, respectively. The external pre-S domain has been implicated in binding to a virus receptor. To investigate functions of the internal pre-S domain, a secretion signal sequence was fused to the N terminus of L (sigL), causing exclusive expression of external pre-S domains. A fusion construct with a nonfunctional signal (s25L), which corresponds in its primary sequence to sigL cleaved by signal peptidase, was used as a control. SigL was N glycosylated in transfected COS cells at both potential sites in pre-S in contrast to s25L or wild-type L, confirming the expected transmembrane topologies of sigL and s25L. Phenotypic characterization revealed the following points. (i) SigL lost the inhibitory effect of L or s25L on secretion of subviral hepatitis B surface antigen particles, suggesting that the retention signal mapped to the N terminus of L is recognized in the cytosol and not in the lumen of the endoplasmic reticulum. (ii) SigL was secreted into the culture medium even in the absence of the major HBV surface protein (S), while release of an L mutant lacking the retention signal was still dependent on S coexpression. (iii) s25L but not sigL could complement an L-negative HBV genome defective for virion secretion in cotransfections. This suggests that the cytosolic pre-S domain, like a matrix protein, is involved in the interaction of the viral envelope with preformed cytosolic nucleocapsids during virion assembly.
Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity
Authors: He J , Choe S , Walker R , Di Marzio P , Morgan D O , Landau N R
Published: January 2020

The Vpr accessory gene product of human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus is believed to play a role in permitting entry of the viral core into the nucleus of nondividing cells. A second role for Vpr was recently suggested by Rogel et al. (M. E. Rogel, L. I. Wu, and M. Emerman, J. Virol. 69:882-888, 1995), who showed that Vpr prevents the establishment in vitro of chronically infected HIV producer cell lines, apparently by causing infected cells to arrest in the G2/M phase of the cell cycle. In cycling cells, progression from G2 to M phase is driven by activation of the p34cdc2/cyclin B complex, an event caused, in part, by dephosphorylation of two regulatory amino acids of p34cdc2 (Thr-14 and Tyr-15). We show here that Vpr arrests the cell cycle in G2 by preventing the activation of the p34cdc2/cyclin B complex. Vpr expression in cells caused p34cdc2 to remain in the phosphorylated, inactive state, p34cdc2/cyclin B complexes immunoprecipitated from cells expressing Vpr were almost completely inactive in a histone H1 kinase assay. Coexpression of a constitutively active mutant p34cdc2 molecule with Vpr relieved the G2 arrest. These findings strongly suggest that Vpr arrests cells in G2 by preventing the activation of the p34cdc2/cyclin B complex that is required for entry into M phase. In vivo, Vpr might, by preventing p34cdc2 activation, delay or prevent apoptosis of infected cells. This would increase the amount of virus each infected cell produced.
Quantitative measurement of paramyxovirus fusion: differences in requirements of glycoproteins between simian virus 5 and human parainfluenza virus 3 or Newcastle disease virus
Authors: Bagai S , Lamb R A
Published: January 2020

To compare the requirements for paramyxovirus-mediated cell fusion, the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of simian virus 5 (SV5), human parainfluenza virus 3 (HPIV-3), and Newcastle disease virus (NDV) were expressed individually or coexpressed in either homologous or heterologous combinations in CV-1 or HeLa-T4 cells, using the vaccinia virus-T7 polymerase transient expression system. The contribution of individual glycoproteins in virus-induced membrane fusion was examined by using a quantitative assay for lipid mixing based on the relief of self-quenching (dequenching) of fluorescence of the lipid probe octadecyl rhodamine (R18) and a quantitative assay for content mixing based on the cytoplasmic activation of a reporter gene, beta-galactosidase. In these assays, expression of the individual F glycoproteins did not induce significant levels of cell fusion and no cell fusion was observed in experiments when cells individually expressing homologous F or HN proteins were mixed. However, coexpression of homologous F and HN glycoproteins resulted in extensive cell fusion. The kinetics of fusion were found to be very similar for all three paramyxoviruses studied. With NDV and HPIV-3, no cell fusion was detected when F proteins were coexpressed with heterologous HN proteins or influenza virus hemagglutinin (HA). In contrast, SV5 F protein exhibited a considerable degree of fusion activity when coexpressed with either NDV or HPIV-3 HN or with influenza virus HA, although the kinetics of fusion were two- to threefold higher when the homologous SV5 F and HN proteins were coexpressed. Thus, these data indicate that among the paramyxoviruses tested, SV5 has different requirements for cell fusion.
High-level expression of adeno-associated virus (AAV) Rep78 or Rep68 protein is sufficient for infectious-particle formation by a rep-negative AAV mutant
Authors: Hölscher C , Kleinschmidt J A , Bürkle A
Published: January 2020

Adeno-associated virus (AAV) codes for four closely related nonstructural proteins (Rep) required for AAV DNA replication and gene regulation. In vitro studies have revealed that either Rep78 or Rep68 alone is sufficient for AAV DNA replication. Rep52 and Rep40 are not required for DNA replication but have been reported to enhance the efficiency of accumulation of single-stranded progeny DNA. Previous studies on rep-expressing cell lines had indicated that only a subset of the four Rep proteins are required for the production of infectious AAV. We therefore set out to determine the minimal set of Rep proteins sufficient for the generation of infectious AAV. Transient cotransfections in HeLa cells of constructs for high-level expression of individual Rep proteins with a rep-negative AAV genome revealed that either Rep78 or Rep68 alone could complement for a full replication cycle yielding infectious virus. This result was confirmed by transfection studies in the cell line HeM2, which selectively expresses Rep78 at rather low levels under the control of the glucocorticoid-responsive mouse mammary tumor virus long terminal repeat (C. Hölscher, M. Hörer, J. A. Kleinschmidt, H. Zentgraf, A. Bürkle, and R. Heilbronn, J. Virol. 68:7169-7177, 1994). Increasing the level of Rep78 expression by transfection of a glucocorticoid receptor expression construct resulted in a higher level of DNA replication of a cotransfected rep-negative AAV genome and in the production of infectious rep-negative AAV particles. We further report on the generation of a new rep-expressing cell line, HeCM1, which was obtained by stable supertransfection of a construct for constitutive Rep40 expression into HeM1 cells (Hölscher et al., J. Virol. 68:7169-7177). Transfection of rather large amounts of rep-negative AAV DNA led to detectable virus production in HeCM1 cells even in the absence of the cotransfected glucocorticoid receptor expression construct, but higher yields were obtained after increasing the Rep78 level by coexpression of the glucocorticoid receptor. These data demonstrate that all Rep functions required for the productive replication of AAV in HeLa cells are contained within both Rep78 and Rep68.
The cytoplasmic tail of CD4 is required for inhibition of human immunodeficiency virus type 1 replication by antibodies that bind to the immunoglobulin CDR3-like region in domain 1 of CD4
Authors: Benkirane M , Schmid-Antomarchi H , Littman D R , Hirn M , Rossi B , Devaux C
Published: January 2020

Monoclonal antibodies (MAb) directed against the immunoglobulin complementary determining region 3 (CDR3)-like region of the CD4 molecule inhibit human immunodeficiency virus type 1 (HIV-1) transcription. We report here data showing that the cytoplasmic tail of CD4 is required for such inhibition to be achieved. To this aim, we studied the effect of MAb 13B8-2 treatment on (i) HIV-1 production in A2.01 cells, which express different forms of the CD4 gene, (ii) Tat-induced HIV-1 promoter activation, and (iii) mitogen-activated protein kinase (MAPK) activation, which is induced in CD4-positive cells by HIV-1 cross-linking of CD4. Inhibition of HIV production by 13B8-2 MAb treatment was consistently observed in cells expressing wild-type CD4 and cells expressing a hybrid CD4-CD8 molecule (amino acids 1 to 177 of CD4 fused to the hinge, transmembrane, and cytoplasmic domains of CD8). However, no delay in HIV-1 production was observed in cells expressing a truncated CD4 which lacks the cytoplasmic domain (CD4.401). Chloramphenicol acetyltransferase assays demonstrated that Tat-dependent activation of the HIV-1 long terminal repeat promoter was inhibited by MAb 13B8-2 in A2.01/CD4 and A2.01/CD4-CD8 but not in A2.01/CD4.401 cells. Finally, we found that MAb 13B8-2 treatment inhibited the activation of MAPK induced in A2.01/CD4 and A2.01/CD4-CD8 following cross-linking of CD4 by HIV-1.
Differences in the capacity of reovirus strains to induce apoptosis are determined by the viral attachment protein sigma 1
Authors: Tyler K L , Squier M K , Rodgers S E , Schneider B E , Oberhaus S M , Grdina T A , Cohen J J , Dermody T S
Published: January 2020

Reoviruses are important models for studies of viral pathogenesis; however, the mechanisms by which these viruses produce cytopathic effects in infected cells have not been defined. In this report, we show that murine L929 (L) cells infected with prototype reovirus strains type 1 Lang (TIL) and type 3 Dearing (T3D) undergo apoptosis and that T3D induces apoptosis to a substantially greater extent than T1L. Using T1L x T3D reassortant viruses, we found that differences in the capacity of T1L and T3D to induce apoptosis are determined by the viral S1 gene segment, which encodes the viral attachment protein sigma 1 and the non-virion-associated protein sigma 1s. Apoptosis was induced by UV-inactivated, replication-incompetent reovirus virions, which do not contain sigma 1s and do not mediate its synthesis in infected cells. Additionally, T3D-induced apoptosis was inhibited by anti-reovirus monoclonal antibodies that inhibit T3D cell attachment and disassembly. These results indicate that sigma 1, rather than sigma 1s, is required for induction of apoptosis by the reovirus and suggest that interaction of virions with cell surface receptors is an essential step in this mechanism of cell killing.
Effects of anti-E2 monoclonal antibody on sindbis virus replication in AT3 cells expressing bcl-2
Authors: Després P , Griffin J W , Griffin D E
Published: January 2020

Antibodies directed to Sindbis virus (SV) envelope protein E2 are able to control virus replication in vivo and in persistently infected cultures of neurons in vitro. We investigated the mechanisms by which anti-E2 monoclonal antibody (MAb) alters virus replication by using AT3 rat prostatic carcinoma cells expressing the inhibitor of apoptosis bcl-2. Treatment of SV-infected AT3-bcl-2 cells with anti-E2 MAb G5 for 2 h decreased the rate of virus release for 6 to 8 h after removal of the antibody. Electron microscopic analysis of MAb-treated cells revealed that failure of virus release was linked to a defect in the budding process. The decrease in extracellular virus particles occurred despite continued formation of nucleocapsids and synthesis of envelope glycoproteins. MAb treatment delayed the inhibition of K+ influx and shutoff of host cell protein synthesis by SV infection in a dose-dependent manner. Synthesis of host cell factors and of nonstructural polyprotein precursors required for the formation of initial replication complexes was also prolonged, causing a slower shutdown of overall viral RNA synthesis. We conclude that one mechanism by which anti-E2 MAb treatment down-regulates SV replication is by reestablishing certain critical host cell functions in infected cells.
Glycoproteins E and I facilitate neuron-to-neuron spread of herpes simplex virus
Authors: Dingwell K S , Doering L C , Johnson D C
Published: January 2020

Two herpes simplex virus (HSV) glycoproteins E and I (gE and gI) form a heterooligomer which acts as an Fc receptor and also facilitates cell-to-cell spread of virus in epithelial tissues and between certain cultured cells. By contrast, gE-gI is not required for infection of cells by extracellular virus. HSV glycoproteins gD and gJ are encoded by neighboring genes, and gD is required for both virus entry into cells and cell-to-cell spread, whereas gJ has not been shown to influence these processes. Since HSV infects neurons and apparently spreads across synaptic junctions, it was of interest to determine whether gD, gE, gI and gJ are also important for interneuronal transfer of virus. We tested the roles of these glycoproteins in neuron-to-neuron transmission of HSV type 1 (HSV-1) by injecting mutant viruses unable to express these glycoproteins into the vitreous body of the rat eye. The spread of virus infection was measured in neuron-rich layers of the retina and in the major retinorecipient areas of the brain. Wild-type HSV-1 and a gJ- mutant spread rapidly between synaptically linked retinal neurons and efficiently infected major retinorecipient areas of the brain. gD mutants, derived from complementing cells, infected only a few neurons and did not spread in the retina or brain. Mutants unable to express gE or gI were markedly restricted in their ability to spread within the retina, produced 10-fold-less virus in the retina, and spread inefficiently to the brain. Furthermore, when compared with wild-type HSV-1, gE- and gI- mutants spread inefficiently from cell to cell in cultures of neurons derived from rat trigeminal ganglia. Together, our results suggest that the gE-gI heterooligomer is required for efficient neuron-to-neuron transmission through synaptically linked neuronal pathways.
Release of the catalytic domain N(o) from the herpes simplex virus type 1 protease is required for viral growth
Authors: Matusick-Kumar L , McCann P J , Robertson B J , Newcomb W W , Brown J C , Gao M
Published: January 2020

The herpes simplex virus type 1 (HSV-1) protease and its substrate, ICP35, are involved in the assembly of viral capsids and required for efficient viral growth. The full-length protease (Pra) consists of 635 amino acid (aa) residues and is autoproteolytically processed at the release (R) site and the maturation (M) site, releasing the catalytic domain No (VP24), Nb (VP21), and a 25-aa peptide. To understand the biological importance of cleavage at these sites, we constructed several mutations in the cloned protease gene. Transfection assays were performed to determine the functional properties of these mutant proteins by their abilities to complement the growth of the protease deletion mutant m100. Our results indicate that (i) expression of full-length protease is not required for viral replication, since a 514-aa protease molecule lacking the M site could support viral growth; and that (ii) elimination of the R site by changing the residue Ala-247 to Ser abolished viral replication. To better understand the functions that are mediated by proteolytic processing at the R site of the protease, we engineered an HSV-1 recombinant virus containing a mutation at this site. Analysis of the mutant A247S virus demonstrated that (i) the mutant protease retained the ability to cleave at the M site and to trans process ICP35 but failed to support viral growth on Vero cells, demonstrating that release of the catalytic domain No from Pra is required for viral replication; and that (ii) only empty capsid structures were observed by electron microscopy in thin sections of A247S-infected Vero cells, indicating that viral DNA was not encapsidated. Our results demonstrate that processing of ICP35 is not sufficient to support viral replication and provide genetic evidence that the HSV-1 protease has nuclear functions other than enzymatic activity.
Selective infection of human T-lymphotropic virus type 1 (HTLV-1)-infected cells by chimeric human immunodeficiency viruses containing HTLV-1 tax response elements in the long terminal repeat
Authors: Lin H C , Bodkin M , Lal R B , Rabson A B
Published: January 2020

Previous studies have suggested that the human immunodeficiency virus long terminal repeat (HIV LTR) enhancer/promoter sequences contribute to the replication ability of HIV in different T-cell lines; mutation of these sequences can alter HIV tropism. We have utilized site-specific mutagenesis to generate variants of HIV that exhibit specific tropism for human T-lymphotropic virus type 1 (HTLV-1) Tax-expressing CD4+ T cells. The wild-type HIV LTR NF-kappa B and Sp1 sites in an infectious molecular clone of HIV type 1 were replaced with sequences derived from the 21-bp Tax response elements (TRE) from the HTLV-1 LTR to generate TRE-containing chimeric HIVs (TRE-HIVs). The TRE-HIVs exhibit selective replication and cell killing in HTLV-infected human CD4+ T cells, but not in HTLV-negative T cells. Transient transfections suggested that Tax-TRE interactions could account for the observed replication specificity. The TRE-containing HIV LTRs were synergistically activated by the HIV Tat and HTLV-1 Tax transactivators. These results demonstrate that it is possible to specifically target HIV replication and cytotoxicity to HTLV-1+, CD4+ human T cells, on the basis of Tax-TRE interactions, and provide a model for the development of specific, cytotoxic, retroviral gene therapy vectors for HTLV-1-infected cells based on alterations of the LTR transcriptional regulatory elements. They also suggest that HIV Tat can cooperate with heterologous transcriptional activators, such as Tax, which act through upstream binding sites without directly binding to DNA.
Gamma interferon is critical for resistance to Theiler's virus-induced demyelination
Authors: Rodriguez M , Pavelko K , Coffman R L
Published: January 2020

Administration of neutralizing monoclonal antibody to gamma interferon increased Theiler's virus-induced demyelination and virus antigen persistence in the spinal cord in susceptible SJL/J mice and completely abrogated resistance such that all C57BL/10SNJ mice developed demyelination. These experiments support the hypothesis that gamma interferon is critically important for resistance to Theiler's virus-induced disease but is not required for myelin destruction.
The packaging phenotype of the SE21Q1b provirus is related to high proviral expression and not trans-acting factors
Authors: Anderson D J , Stone J , Lum R , Linial M L
Published: January 2020

The avian packaging cell line SE21Q1b produces particles which encapsidate cellular RNAs. Such RNAs can be reverse transcribed by endogenous polymerase and integrated into the genomes of newly infected cells (M. Linial, Cell 49:93-102, 1987). Genomic RNA is not packaged because the packaging (psi) region of the provirus is deleted. The provirus also lacks the negative-strand primer binding site, which prevents efficient reverse transcription of randomly packaged genomic RNA. Previous work from our laboratory suggested that the trans-acting defect which allows packaging of cellular mRNA mapped to the provirus but did not map to the nucleocapsid region of the gag gene (D.J. Anderson, P. Lee, K. L. Levine, J. Sang, S. A. Shah, O. O. Yang, P. R. Shank, and M. L. Linial, J. Virol. 66:204-216, 1992). We have found, using proviral recombinants between SE21Q1b and wild-type Rous sarcoma virus, that packaging of cellular RNAs does not map to the gag gene. Rather, the propensity of SE21Q1b particles to package cellular mRNA is a function of the high level of particle production in these cells and not of any specific viral structural proteins.
The bovine herpesvirus 1 maturational proteinase and scaffold proteins can substitute for the homologous herpes simplex virus type 1 proteins in the formation of hybrid type B capsids
Authors: Haanes E J , Thomsen D R , Martin S , Homa F L , Lowery D E
Published: January 2020

We determined the nucleotide sequence of a 3.5-kb region of the bovine herpesvirus 1 (BHV-1) genome which contained the complete BHV-1 homologs of the herpes simplex virus type 1 (HSV-1) UL26 and UL26.5 genes. In HSV-1, the UL26 and UL26.5 open reading frames encode scaffold proteins upon which viral capsids are assembled. The UL26-encoded protein is also a proteinase and specifically cleaves both itself and the UL26.5-encoded protein. The overall BHV-1-encoded amino acid sequence showed only 41% identity to the HSV-1 sequences and was most divergent in the regions defined to be involved in the scaffolding function. We substituted the proteins encoded by the BHV-1 homologs of the UL26 and UL26.5 open reading frames, expressed in baculovirus, for the corresponding HSV-1 proteins in an in vitro HSV-1 capsid assembly system. The proteins expressed from the BHV-1 UL26 and UL26.5 homologs facilitated the formation of hybrid type B capsids indistinguishable from those formed entirely with HSV-1-encoded proteins.
Immunodominance of major histocompatibility complex class I-restricted influenza virus epitopes can be influenced by the T-cell receptor repertoire
Authors: Daly K , Nguyen P , Woodland D L , Blackman M A
Published: January 2020

We have used T-cell receptor beta-chain transgenic mice to determine the effects of a limited T-cell receptor repertoire on major histocompatibility complex class I-restricted epitope selection during the course of an influenza virus infection. Analysis of T-cell hybridomas generated from wild-type and transgenic mice demonstrated that the viral epitope recognized depended on the available T-cell receptor repertoire. Wild-type T-cell hybridomas recognized epitopes derived from the nucleoprotein and basic polymerase molecules, whereas hybridomas generated from transgenic mice recognized epitopes derived from the nonstructural protein and the matrix protein. There was no overlap in specificity between the two panels of hybridomas. This reciprocal pattern of specificity was also apparent in cytoxicity assays with brochoalveolar lavage cells isolated from the lungs of influenza virus-infected mice. T-cell receptor usage in the transgenic hybridomas was very restricted, with only one V alpha element used for ech of the two viral epitopes recognized. In the case of the hybridomas reactive to the nonstructural protein, sequence analysis showed that they all expressed V alpha 4J alpha 32 chains associated with the same junctional amino acids (Leu-Leu) that were encoded by five different nucleotide sequences, indicating a strong selection for T-cell receptor usage. Taken together, these data demonstrate that the available T-cell receptor repertoire can have a profound effect on the immunodominance of class I-restricted epitopes during a viral infection.
High-titer packaging cells producing recombinant retroviruses resistant to human serum
Authors: Cosset F L , Takeuchi Y , Battini J L , Weiss R A , Collins M K
Published: January 2020

Novel retroviral protein expression constructs were designed to retain minimal retroviral sequences and to express dominant selectable markers by reinitiation of translation after expression of the viral genes. HT1080 cells were selected as producer cells for their ability to release high-titer viruses that are resistant to inactivation by human serum. Two HT1080-based packaging cell lines which produce Moloney murine leukemia virus cores with envelope glycoproteins of either amphotropic murine leukemia virus (FLYA13 line) or cat endogenous virus RD114 (FLYRD18 line) are described. Direct comparison with previous retroviral packaging systems indicated that 100-fold-higher titers of helper-free recombinant viruses were released by the FLYA13 and FLYRD18 lines.
Role of mutations G-480 and C-6203 in the attenuation phenotype of Sabin type 1 poliovirus
Authors: McGoldrick A , Macadam A J , Dunn G , Rowe A , Burlison J , Minor P D , Meredith J , Evans D J , Almond J W
Published: January 2020

Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from its neurovirulent progenitor (P1/Mahoney), two have been strongly implicated by previous studies as determinants of the attenuation phenotype. A change of an A to a G at position 480, located within the 5' noncoding region, has been suggested to be the major attenuating mutation, analogous to the mutations at positions 481 and 472 in poliovirus types 2 and 3, respectively. In addition, the change of a U to a C at position 6203, resulting in an amino acid change in the polymerase protein 3D, has also been implicated as a determinant of attenuation, albeit to a lesser extent. To assess the contributions of these mutations to attenuation and temperature sensitivity, reciprocal changes were generated at these positions in infectious cDNA clones of Sabin 1 and P1/Mahoney. Assays in tissue culture and primates indicated that the two mutations make some contribution to the temperature sensitivity of the Sabin 1 strain but that neither is a strong determinant of attenuation.
Binding of cellular repressor protein or the IE2 protein to a cis-acting negative regulatory element upstream of a human cytomegalovirus early promoter
Authors: Huang L , Stinski M F
Published: January 2020

We have previously shown that the human cytomegalovirus early UL4 promoter has upstream negative and positive cis-acting regulatory elements. In the absence of the upstream negative regulatory region, the positive element confers strong transcriptional activity. The positive element contains a CCAAT box dyad symmetry and binds the cellular transcription factor NF-Y. The effect of the negative regulatory element is negated by the viral IE2 protein (L. Huang, C.L. Malone, and M.F. Stinski, J. Virol. 68:2108, 1994). We investigated the binding of cellular or viral IE2 protein to the negative regulatory region. The major cis-acting negative regulatory element was located between -168 and -134 bp relative to the transcription start site. This element could be transferred to a heterologous promoter, and it functioned in either orientation. Mutational analysis demonstrated that a core DNA sequence in the cis-acting negative regulatory element, 5'-GTTTGGAATCGTT-3', was required for the binding of either a cellular repressor protein(s) or the viral IE2 protein. The cellular DNA binding activity was present in both nonpermissive HeLa and permissive human fibroblast cells but more abundant in HeLa cells. Binding of the cellular repressor protein to the upstream cis-acting negative regulatory element correlates with repression of transcription from the early UL4 promoter. Binding of the viral IE2 protein correlates with negation of the repressive effect.
Altered cytokine expression in T lymphocytes from human immunodeficiency virus Tat transgenic mice
Authors: Brady H J , Abraham D J , Pennington D J , Miles C G , Jenkins S , Dzierzak E A
Published: January 2020

Examination of the interaction between human immunodeficiency virus (HIV) regulatory gene products and the host immune system is fundamental to understanding the pathogenesis of HIV and could reveal possible targets for therapeutic intervention in the treatment of AIDS. The HIV Tat gene is a potential candidate for this type of strategy. Transgenic mice can be used to investigate the in vivo effects of Tat on the developing and dynamic immune system and on cellular gene expression. Thus, we have generated transgenic mice that harbor the HIV type 1 Tat gene under the transcriptional control of the human CD2 gene regulatory elements. This expression cassette results in high-level, tissue-specific transcription of the transgene within the T-cell compartment. In this report, we demonstrate the effects of Tat on the in vivo immune system. CD2-Tat transgenic mice show no signs of aberrant thymic development and have normal levels of T-cell subsets in the thymus and peripheral lymphoid organs. However, activated T cells from transgenic mice contain increased levels of tumor necrosis factor beta mRNA as well as biologically active tumor necrosis factor protein and express elevated levels of transforming growth factor beta and interleukin-4 receptor mRNA. These increased cytokine levels do not appear to alter mitogen- or antigen-stimulated responses or induce the formation of dermal lesions in ageing mice. Such investigations should provide insight into the combination of host immune factors mediating pathogenesis in HIV infection.
Interaction of the 72-kilodalton human cytomegalovirus IE1 gene product with E2F1 coincides with E2F-dependent activation of dihydrofolate reductase transcription
Authors: Margolis M J , Pajovic S , Wong E L , Wade M , Jupp R , Nelson J A , Azizkhan J C
Published: January 2020

Three polypeptides are produced from the major immediate-early (IE) region of human cytomegalovirus by alternative splicing. The IE gene products regulate subsequent viral and cellular gene expression. We previously reported that cotransfection of a genomic clone of the major IE region stimulated transient expression of chloramphenicol acetyltransferase driven by the dihydrofolate reductase (DHFR) promoter and that an intact E2F site was required for the trans activation (M. Wade, T. F. Kowalik, M. Mudryj, E.-S. Huang, and J. C. Azizkhan, Mol. Cell. Biol. 12:4364-4374, 1992). With the availability of cDNA clones for the individual major IE proteins, we sought to determine which of these proteins exerted this effect and whether the IE protein(s) interacted with E2F. In this study, we use cotransfection to demonstrate that the 55- and 86-kDa major IE proteins from the IE2 region can each moderately trans activate the DHFR promoter and that the 72-kDa IE1 protein stimulates DHFR transcription to a much higher level. Furthermore, trans activation through the 72-kDa IE1 protein is in part E2F dependent, while activation by the 55- and 86-kDa IE proteins is E2F independent. We also demonstrate by in vitro pull-down assays that the 72-kDa IE1 protein can specifically interact with the DNA binding domain of E2F1 (amino acids 88 to 191) in the presence of nuclear extract. Moreover, antibodies to either E2F1 or IE72 will immunoprecipitate both E2F and IE72 from cells that stably express IE72, and antibody to E2F1 will immunoprecipitate IE72 from normal human fibroblast cells infected with human cytomegalovirus.
In vivo and in vitro association of hsc70 with polyomavirus capsid proteins
Authors: Cripe T P , Delos S E , Estes P A , Garcea R L
Published: January 2020

Members of the 70-kDa family of cellular stress proteins assit in protein folding by preventing inappropriate intra- and intermolecular interactions during normal protein synthesis and transport and when cells are exposed to a variety of environmental stresses. During infection of A31 mouse fibroblasts with polyomavirus, the constitutive form of hsp70, hsc70, coimmunoprecipitated with all three viral capsid proteins (VP1, VP2, and VP3). In addition, the subcellular location of hsc70 changed from cytoplasmic to nuclear late in polyomavirus infection, coincident with the nuclear localization of the viral capsid proteins. VP1 and VP2 expressed in Sf9 insect cells with recombinant baculovirus vectors also coimmunoprecipitated with an hsp70-like protein, and VP1 expressed in Escherichia coli coimmunoprecipitated with the hsp70 homolog DnaK. Capsid proteins expressed by in vitro translation coimmunoprecipitated with the hsc70 protein present in the reticulocyte translation extract. Therefore, the polyomavirus capsid proteins associate with hsc70 during virus infection as well as in recombinant protein expression systems. This association may play a role in preventing the premature assembly of capsids in the cytosol and/or in facilitating the nuclear transport of capsid protein complexes.
VP16 interacts via its activation domain with VP22, a tegument protein of herpes simplex virus, and is relocated to a novel macromolecular assembly in coexpressing cells
Authors: Elliott G , Mouzakitis G , O'Hare P
Published: January 2020

In addition to its function as a powerful transactivator of viral immediate-early transcription, VP16 is an essential component of the herpes simplex virus (HSV) virion. As such, VP16 is introduced into cells, to effect its function in transactivation, as part of the virus tegument. Here we examine the potential for VP16 protein-protein interactions specific to virus-infected cells and show that VP16 copurifies in a highly enriched fraction with a single major polypeptide which we identify as the virus-encoded structural protein VP22. We further show that in vitro-translated VP22 binds specifically to purified VP16. The activation domain of VP16 was required and largely sufficient for this binding. Mutations within this domain, which disrupt its transactivation function, also affected VP22 binding. Furthermore, we show that while VP16 and VP22 showed distinct patterns of compartmentalization in vivo, coexpression of both proteins resulted in a profound reorganization from their normal locations to a novel macromolecular assembly. The colocalization was also dependent on the activation domain of VP16 but required additional determinants within the N terminus. These results are discussed in the context of VP16 regulation of transcription both early in infection during delivery of tegument proteins and at late times during virus assembly.
Neurons differentially control expression of a herpes simplex virus type 1 immediate-early promoter in transgenic mice
Authors: Mitchell W J
Published: January 2020

The immediate-early proteins of herpes simplex virus control the cascade of viral gene expression during lytic infection. It is not known which viral or host proteins control the reactivation of the viral genome in latently infected neurons. To determine whether neuronal proteins can regulate a herpes simplex virus immediate-early promoter in vivo, transgenic mice containing the promoter regulatory region of the herpes simplex virus type 1 immediate-early gene (ICP4) fused to the bacterial beta-galactosidase gene were generated. Two lines of mice, in the absence of viral proteins, displayed ICP4 promoter activity in neurons in specific locations in the central nervous system. The anatomic locations of these neurons were the hippocampus, cerebellar cortex, superior colliculus, indusium griseum, mammillary nucleus, cerebral cortex, and the dorsal laminae of the dorsal horns of the spinal cord. Additional subsets of neurons expressed the ICP4 promoter at lower levels; these included trigeminal ganglia and retinas. In a third line of mice, lower levels of expression were present in many of the above-described neurons. Many types of neurons, nearly all nonneuronal cells in the central nervous system, and some non-nervous system tissues were negative. Viral proteins including VP16 are not necessary to induce transcription from the ICP4 promoter in many neurons and some other cell types but may be required in most cells in vivo. An approximately 100-fold-greater number of neurons in the trigeminal ganglia expressed ICP4 promoter activity in newborn mice compared with adults. These data provide direct evidence that host proteins are sufficient to activate a herpes simplex virus immediate-early promoter in neurons in vivo and that a differential expression pattern for this promoter exists within different neuronal phenotypes and between the same neurons in different ages of mice.
In vitro selection of RNA ligands for the ribosomal L22 protein associated with Epstein-Barr virus-expressed RNA by using randomized and cDNA-derived RNA libraries
Authors: Dobbelstein M , Shenk T
Published: January 2020

The Epstein-Barr virus (EBV)-expressed RNA 1 (EBER1) associates tightly with the ribosomal protein L22. We determined the general requirements for an RNA to bind L22 in a SELEX experiment, selecting RNA ligands for L22 from a randomized pool of RNA sequences by using an L22-glutathione S-transferase fusion protein. The selected sequences all contained a stem-loop motif similar to that of the region of EBER1 previously shown to interact with L22. The nucleotides were highly conserved at three positions within the stem-loop and identical to the corresponding nucleotides in EBER1. Two independent binding sites for L22 could be identified in EBER1, and mobility shift assays indicated that two L22 molecules can interact with EBER1 simultaneously. To search for a cellular L22 ligand, we constructed a SELEX library from cDNA fragments derived from RNA that was coimmunoprecipitated with L22 from an EBV-negative whole-cell lysate. After four rounds of selection and amplification, most of the clones that were obtained overlapped a sequence corresponding to the stem-loop between nucleotides 302 and 317 in human 28S ribosomal RNA. This stem-loop fulfills the criteria for optimal binding to L22 that were defined by SELEX, suggesting that human 28S ribosomal RNA is likely to be a cellular L22 ligand. Additional L22 binding sites were found in 28S ribosomal RNA, as well as within 18S ribosomal RNA and in RNA segments not present in sequence databases. The methodology described for the conversion of a preselected cellular RNA pool into a SELEX library might be generally applicable to other proteins for the identification of cellular RNA ligands.
Preferential usage of the T-cell receptor by influenza virus hemagglutinin-specific human CD4+ T lymphocytes: in vitro life span of clonotypic T cells
Authors: Prévost-Blondel A , Chassin D , Zeliszewski D , Dorval I , Sterkers G , Pannetier C , Guillet J G
Published: January 2020

Human helper T-cell (Th) responses to influenza A virus were studied by analyzing T-cell receptor V beta gene diversity in hemagglutinin-specific Th lymphocytes. The T-lymphocyte population from peripheral blood became quickly oligoclonal when stimulated in vitro with the HA306-329 peptide, and T-cell receptor V beta 3 genes were mainly expanded. Moreover, specific junctional region oligonucleotide probes corresponding to hemagglutinin-specific clones were used to estimate temporal diversity during antigenic stimulations.
Characterization of a neurologic disease induced by a polytropic murine retrovirus: evidence for differential targeting of ecotropic and polytropic viruses in the brain
Authors: Portis J L , Czub S , Robertson S , McAtee F , Chesebro B
Published: January 2020

A variety of ecotropic murine leukemia viruses cause neurodegenerative disease. We describe here the clinical and histopathological features of a neurologic disease induced by a polytropic murine leukemia virus, FMCF98. Clinical disease was dominated by hyperexcitability and ataxia, and the histopathology was characterized primarily by astrocytosis and astrocytic degeneration. The viral envelope gene harbored the determinants of neurovirulence, since the chimeric virus Fr98E, which contained the envelope gene of FMCF98 on a background of the nonneurovirulent virus FB29, caused a similar disease. The disease caused by Fr98E differed from that induced by the coisogenic neurovirulent ecotropic virus FrCasE in clinical presentation, histopathology, and distribution of virus in the central nervous system. Since Fr98E contains a polytropic envelope gene and FrCasE contains an ecotropic envelope gene, these phenotypic differences appeared to be determined by envelope sequences and may reflect differences in virus receptor usage in the central nervous system.
Lack of detection of negative-strand hepatitis C virus RNA in peripheral blood mononuclear cells and other extrahepatic tissues by the highly strand-specific rTth reverse transcriptase PCR
Authors: Lanford R E , Chavez D , Chisari F V , Sureau C
Published: January 2020

To further explore the controversial potential for extrahepatic replication of hepatitis C virus (HCV), the highly strand-specific rTth method of reverse transcriptase PCR was used to examine sera, liver, peripheral blood mononuclear cells, and other extrahepatic tissues from HCV-infected chimpanzees and humans. Positive-strand HCV RNA was present in the liver at approximately 10-fold-higher levels than negative-strand HCV RNA. No negative-strand RNA was detected in peripheral blood mononuclear cells or other extrahepatic tissues despite the presence of abundant positive-strand RNA. These data demonstrate that within the limits of sensitivity of this highly strand-specific reverse transcriptase PCR method, no extrahepatic replication of HCV was detected.
Immunization with recombinant varicella-zoster virus expressing herpes simplex virus type 2 glycoprotein D reduces the severity of genital herpes in guinea pigs
Authors: Heineman T C , Connelly B L , Bourne N , Stanberry L R , Cohen J
Published: January 2020

Varicella-zoster virus (VZV) is an attractive candidate for a live-virus vector for the delivery of foreign antigens. The Oka vaccine strain of VZV is safe and effective in humans, and recombinant Oka VZV (ROka) can be generated by transfecting cells with a set of overlapping cosmid DNAs. By this method, the herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) gene was inserted into an intergenic site in the unique short region of the Oka VZV genome. Expression of gD2 in cells infected with the recombinant Oka strain VZV (ROka-gD2) was confirmed by antibody staining of fixed cells and by immunoblot analysis. Immune electron microscopy demonstrated the presence of gD2 in the envelope of ROka-gD2 virions. The ability of ROka-gD2 to protect guinea pigs against HSV-2 challenge was assessed by inoculating animals with three doses of uninfected human fibroblasts, fibroblasts infected with ROka VZV, or fibroblasts infected with ROka-gD2. Neutralizing antibodies specific for HSV-2 developed in animals immunized with ROka-gD2. Forty days after the third inoculation, animals were challenged intravaginally with HSV-2. Inoculation of guinea pigs with ROka-gD2 significantly reduced the severity of primary HSV-2 infection (P < 0.001). These experiments demonstrate that the Oka strain of VZV can be used as a live virus vector to protect animals from disease with a heterologous virus.
Posttranslational signal peptidase cleavage at the flavivirus C-prM junction in vitro
Authors: Stocks C E , Lobigs M
Published: January 2020

We have investigated the cleavages at the flavivirus capsid-prM protein junction in vitro. When expressed in the absence of the flavivirus proteinase, capsid and prM, which are separated by an internal signal sequence, exist as a membrane-spanning precursor protein. Here we show the induction of posttranslational signal peptidase cleavage of prM by trypsin cleavage of a cytoplasmic region of this precursor protein.
Epstein-Barr virus induction of recombinase-activating genes RAG1 and RAG2
Authors: Srinivas S K , Sixbey J W
Published: January 2020

In experimental B-cell infections, Epstein-Barr virus induced sustained expression of V(D)J recombinase-activating genes RAG1 and RAG2, whose aberrant activity has been implicated in chromosomal translocations in B-cell neoplasms. In cell lines in which RAG1 and RAG2 were detected, virus integrated into cellular DNA rather than assumed the configuration of extrachromosomal episomes. Expression of the Epstein-Barr virus nuclear antigen 1 in transient transfection assays was sufficient to induce both recombinase-activating genes.
Role of CD4 endocytosis in human immunodeficiency virus infection
Authors: Pelchen-Matthews A , Clapham P , Marsh M
Published: January 2020

We have analyzed the role of CD4 endocytosis in human immunodeficiency virus (HIV) entry by measuring the infection of HeLa cells expressing various CD4 constructs with endocytosis rates of between 0.2 and 30%/min in a quantitative infectious focus assay. For a number of laboratory-adapted HIV-1 and HIV-2 strains, the highest levels of infection were found on cells with very limited CD4 endocytosis, while cells with efficient CD4 uptake were only poorly infectable, suggesting that CD4 internalization is not required for HIV entry. This was confirmed in a modified assay involving prebinding of HIV-1LAI to HeLa-CD4 cells at 4 degrees C, synchronized virus entry during warming to 37 degrees C, and neutralization of virions remaining at the cell surface with anti-V3 loop antibodies. Warming cells in hypertonic medium inhibited CD4 endocytosis but did not affect the rate or the extent of infection. These studies confirm that HIV infection does not require endocytosis and that laboratory-adapted virus strains can enter HeLa-CD4 cells by fusion at the plasma membrane.