bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA
Authors: Luban J , Goff S P
Published: January 2020

We previously identified blocks of sequence near the 5' end of the human immunodeficiency virus (HIV-1) genome which conferred on RNA the ability to bind specifically to the HIV-1 Gag polyprotein, Pr55gag (J. Luban and S. P. Goff, J. Virol. 65:3203-3212, 1991; R. Berkowitz, J. Luban, and S. P. Goff, J. Virol. 67:7190-7200, 1993). Here we report the use of an RNase protection assay to quantify the effect of deletion of these sequences on RNA packaging into virions. First, we demonstrated with wild-type HIV-1 sequences that in comparison with spliced viral RNA, full-length viral genomic RNA is enriched 20-fold in virions. A previously described mutation with deletion of sequences between the major splice donor and the first codon of gag (A. Lever, H. Gottlinger, W. Haseltine, and J. Sodroski, J. Virol. 63:4085-4087, 1989) disrupted these ratios such that different HIV-1 RNA forms were packaged in direct proportion to cytoplasmic concentrations. The effect of deletion mutations preceding and within gag coding sequence on packaging was then tested in competition with RNAs containing wild-type packaging sequences. Using this system, we were able to demonstrate significant effects on packaging of RNAs with mutations immediately preceding the first codon of gag. The greatest reduction in packaging was seen with RNAs lacking the first 40 nucleotides of gag coding sequence, although sequences more 3' had slight additional effects.
The middle hepatitis B virus envelope protein is not necessary for infectivity of hepatitis delta virus
Authors: Sureau C , Guerra B , Lee H
Published: January 2020

The hepatitis delta virus (HDV) envelope contains the large (L), middle (M), and small (S) surface proteins encoded by coinfecting hepatitis B virus. Although HDV-like particles can be assembled with only the S protein in the envelope, the L protein is essential for infectivity in vitro (C. Sureau, B. Guerra, and R. Lanford, J. Virol. 67:366-372, 1993). Here, we demonstrate that the M protein, previously described as carrying a site for binding to polymerized human albumin, is not necessary for infectivity. HDV-like particles coated with the S plus L or the S plus M plus L proteins are infectious in primary cultures of chimpanzee hepatocytes. We conclude that the S and L proteins serve two essential functions in the HDV replication cycle; the S protein ensures the export of the HDV genome from an infected cell by forming a particle, and the L protein ensures its import into a human hepatocyte.
Expression and characterization of virus-like particles containing rubella virus structural proteins
Authors: Qiu Z , Ou D , Hobman T C , Gillam S
Published: January 2020

Rubella virus (RV) virions contain two envelope glycoproteins (E1 and E2) and a capsid protein (C). Noninfectious RV-like particles (VLPs) containing three structural proteins were expressed in a BHK cell line (BHK-24S) by using an inducible promoter. These VLPs were found to resemble RV virons in terms of their size, their morphology, and some biological activities. In immunoblotting studies, VLPs were found to bind similarly to native RV virions with 10 of a panel of 12 RV-specific murine monoclonal antibodies. Immunization of mice with VLPs induced specific antibody responses against RV structural proteins as well as virus-neutralizing and hemagglutination-inhibiting antibodies. After immunization of mice with VLPs, in vitro challenge of isolated lymphocytes with inactivated RV and individual RV structural proteins stimulated proliferation. Our data suggest the possibility of using VLPs as immunogens for serodiagnostic assays and RV vaccines.
Human immunodeficiency virus type 1 Tat upregulates interleukin-2 secretion in activated T cells
Authors: Westendorp M O , Li-Weber M , Frank R W , Krammer P H
Published: January 2020

Dysregulation of cytokines secreted by T cells may play an important role in the pathogenesis of AIDS. To investigate the effects of human immunodeficiency virus type 1 (HIV-1) Tat on interleukin-2 (IL-2) expression, we used IL-2 promoter-chloramphenicol acetyltransferase constructs and IL-2-secreting Jurkat T cells as a model system. Transient expression of HIV-1 Tat induced a five- to eightfold increase in IL-2 promoter activity in Jurkat T cells stimulated with phytohemagglutinin and phorbol myristate acetate. IL-2 secretion was increased more than twofold in both Jurkat T cells and primary T cells stimulated by extracellular HIV-1 Tat protein. Analysis of mRNA suggested that Tat exerts its effect on IL-2 primarily at the transcriptional level. The NF-kappa B site at positions -206 to -195 of the IL-2 promoter was required but not sufficient for the Tat effect. The Tat-mediated increase in IL-2 promoter activity could selectively be blocked by antisense tat or-unlike the analogous effect of human T-cell lymphotropic virus type 1 Tax-by cyclosporin A. The observed increase in IL-2 levels might facilitate virus spread from or to T cells. Furthermore, it might contribute to the hypergammaglobulinemia or, together with other cytokines found to be dysregulated, the T-helper cell dysfunctions observed in AIDS patients.
Amino acid substitutions in the human immunodeficiency virus type 1 gp120 V3 loop that change viral tropism also alter physical and functional properties of the virion envelope
Authors: Willey R L , Theodore T S , Martin M A
Published: January 2020

The third variable (V3) region within the gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) has been reported to be an important determinant of viral tropism. In this study a series of isogenic recombinant HIV-1 viruses, containing V3 regions from fresh isolates, were examined to ascertain if a relationship exists between viral tropism and specific properties of the virion-associated envelope. All of the viruses were able to infect CD4+ primary lymphocytes, although with different infection kinetics. Several recombinants, however, were unable to infect a continuous CD4+ T-cell line permissive for the parental virus and exhibited a marked decrease in the kinetics of virion-associated gp120 binding to a soluble form of CD4. A known macrophage-tropic HIV-1 isolate, also unable to infect the T-cell line, bound CD4 with similarly slow reaction kinetics. Although the inability to infect T-cell lines is a commonly observed property of macrophage-tropic isolates of HIV-1, the loss of T-cell line tropism by the V3 recombinants was not accompanied by a substantial infectivity for monocyte-derived macrophages, as monitored by reverse transcriptase production. Additional analyses of the recombinant virion gp120s indicated that most of the V3 substitutions increased the inherent stability of the virion gp120-gp41 envelope complex. These results indicate that V3-induced alterations in viral tropism are associated with changes in physical and functional properties of the virion envelope.
Mutations in accessory DNA replicating functions alter the relative mutation frequency of herpes simplex virus type 1 strains in cultured murine cells
Authors: Pyles R B , Thompson R L
Published: January 2020

The contribution of the herpes simplex virus type 1 (HSV-1)-encoded uracil DNA glycosylase (UNG), thymidine kinase (TK), and dUTPase to the relative mutant frequency (RMF) of the virus in cultured murine cells was examined. A panel of HSV-1 mutants that lacked singly or doubly the UNG, TK, or dUTPase activity were generated by disruption of the enzyme coding regions with the Escherichia coli beta-galactosidase (beta-gal) gene in strain 17syn+. To establish a baseline RMF of strain 17syn+, the beta-gal gene was inserted into the UL3 locus. In all of the viruses, the beta-gal insert served as a phenotypic marker of RMF. A mutant plaque was identified by the lack of beta-gal activity and, in selected cases, positive in situ hybridization for beta-gal sequences. Replication kinetics in NIH 3T3 cells demonstrated that all of the mutants replicated efficiently, generating stocks with equivalent titers. Two independently generated UL3-beta-gal viruses were examined and established a baseline RMF of approximately 0.5% in both NIH 3T3 and LM TK- cells. Loss of dUTPase activity resulted in viruses with fivefold-increased RMFs, indicating that the HSV-1 dUTPase has an antimutator function. The RMF observed for the tk- viruses was reduced as much as 40-fold (RMF of 0.02%), suggesting that the viral TK is a mutator activity. The RMF of two independent UNG- viruses showed no significant difference from the baseline RMF in limited passage; however, following successive passage, the data suggested that UNG activity serves as an antimutator. These results have implications for the natural history of HSV and the development of antiviral therapies.
The membrane-associated and secreted forms of the respiratory syncytial virus attachment glycoprotein G are synthesized from alternative initiation codons
Authors: Roberts S R , Lichtenstein D , Ball L A , Wertz G W
Published: January 2020

Respiratory syncytial (RS) virus synthesizes two mature forms of its attachment glycoprotein G: an anchored type II integral membrane form and a smaller form that is secreted into the medium. Here we demonstrate that these two forms are synthesized as distinct primary translation products of a single species of G protein mRNA by initiation at either of two different AUGs. Mutant cDNAs which eliminated one of the other of the two AUG codons near the 5' end of the G gene open reading frame were constructed. Analysis of the proteins synthesized from these cDNAs, either by translation of transcripts in a cell-free system or in cells infected with recombinant vaccinia viruses containing either one of the mutant cDNAs, showed that elimination of either the first or the second of these AUG codons abrogated the synthesis of the membrane-anchored or the secreted form of the protein, respectively. Additionally, two unglycosylated forms of G protein which comigrated with the unglycosylated G proteins expressed by these recombinant viruses were detected in RS virus-infected cells. Since the second AUG encodes a methionine residue that lies near the middle of the signal/anchor domain, initiation at this codon resulted in a protein with a hydrophobic amino terminus. This form of the glycoprotein was efficiently secreted from cells infected with the vaccinia virus recombinant, and the amino-terminal sequence of this protein was identical to that of G protein secreted from RS virus-infected cells. Our results demonstrate that the secreted form of RS virus G protein is produced by initiation at the second AUG codon of the G open reading frame, followed by proteolytic removal of the signal/anchor domain.
Unprocessed foot-and-mouth disease virus capsid precursor displays discontinuous epitopes involved in viral neutralization
Authors: Sáiz J C , Cairó J , Medina M , Zuidema D , Abrams C , Belsham G J , Domingo E , Vlak J M
Published: January 2020

A foot-and-mouth disease virus (FMDV) cDNA cassette containing sequences encoding the capsid precursor P1, peptide 2A and a truncated 2B (abbreviated P1-2A) of type C FMDV, has been modified to generate the authentic amino terminus and the myristoylation signal. This construct has been used to produce a recombinant baculovirus (AcMM53) which, upon infection of Spodoptera frugiperda insect cells, expressed a recombinant P1-2A precursor with a high yield. This polyprotein reacted with neutralizing monoclonal antibodies (MAbs) that bind to continuous epitopes of the major antigenic site A (also termed site 1) of capsid protein VP1. Unexpectedly, it also reacted with neutralizing MAbs which define complex, discontinuous epitopes previously identified on FMDV particles. The reactivity of MAbs with P1-2A was quantitatively similar to their reactivity with intact virus and, in both cases, the reactivity with MAbs that recognized discontinuous epitopes was lost upon heat denaturation of the antigen. The finding that a capsid precursor may fold in such a way as to maintain discontinuous epitopes involved in virus neutralization present on the virion surface opens the possibility of using unprocessed capsid precursors as novel antiviral immunogens.
A novel, glycan-dependent epitope in the V2 domain of human immunodeficiency virus type 1 gp120 is recognized by a highly potent, neutralizing chimpanzee monoclonal antibody
Authors: Warrier S V , Pinter A , Honnen W J , Girard M , Muchmore E , Tilley S A
Published: January 2020

An anti-gp120 monoclonal antibody (MAb), C108G (gamma 1, kappa), was isolated from a chimpanzee that had been infected with strain IIIB of human immunodeficiency virus type 1 (HIV-1IIIB) and subsequently immunized with the recombinant glycoprotein rgp160MN. This MAb is specific for the IIIB strain of HIV-1 and related clones and exhibits very potent neutralization of these viruses; e.g., 100% neutralization of approximately 8 x 10(3) infectious units of HXB2 was achieved with 125 ng of C108G per ml. Commensurate with this potent neutralizing activity, the apparent affinity of C108G for rgp160LAI was very high, i.e., approximately 3 x 10(10) liters/mol. The C108G epitope was not destroyed by reduction of gp120 disulfide bonds but was profoundly disrupted by removal of N-linked sugars from gp120. Despite the importance of a glycan(s) in forming the C108G epitope, specific binding of C108G to synthetic peptides overlapping in amino acids 162 to 169 of the V2 region was detected, albeit with an affinity approximately 2,000-fold lower than that of C108G's binding to glycosylated envelope protein. This epitope mapping correlated with results of competition assays using MAbs of known epitope specificities. To our knowledge, this is the first description of an anti-V2 MAb raised in response to HIV-1 infection. Its potent neutralizing activity and epitope specificity indicate that the V2 domain of gp120 may be an effective target of the protective immune response and, therefore, potentially an important component of HIV vaccines.
Genetic drift in hypervariable region 1 of the viral genome in persistent hepatitis C virus infection
Authors: Kato N , Ootsuyama Y , Sekiya H , Ohkoshi S , Nakazawa T , Hijikata M , Shimotohno K
Published: January 2020

The hypervariable region 1 (HVR1) of the putative second envelope glycoprotein (gp70) of hepatitis C virus (HCV) contains a sequence-specific immunological B-cell epitope that induces the production of antibodies restricted to the specific viral isolate, and anti-HVR1 antibodies are involved in the genetic drift of HVR1 driven by immunoselection (N. Kato, H. Sekiya, Y. Ootsuyama, T. Nakazawa, M. Hijikata, S. Ohkoshi, and K. Shimotohno, J. Virol. 67:3923-3930, 1993). We further investigated the sequence variability of the HCV genomic region that entirely encodes the envelope proteins (gp35 and gp70); these sequences were derived from virus isolated during the acute and chronic phases of hepatitis in one patient, and we found that HVR1 was a major site for genetic mutations in HCV after the onset of hepatitis. We carried out epitope-mapping experiments using the HVR1 sequence derived from the acute phase of hepatitis and identified two overlapping epitopes which are each composed of 11 amino acids (positions 394 to 404 and 397 to 407). The presence of two epitopes within HVR1 suggested that epitope shift happened during the course of hepatitis. Four of six amino acid substitutions detected in HVR1 were located within the two epitopes. We further examined the reactivities of anti-HVR1 antibodies to the substituted amino acid sequences within the two epitopes. HVR1 variants in both epitopes within the HVR1 escaped from anti-HVR1 antibodies that were preexisting in the patient's serum.
Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1
Authors: Roben P , Moore J P , Thali M , Sodroski J , Barbas C F , Burton D R
Published: January 2020

Six recombinant human Fab fragments that were derived from the same human immunodeficiency virus type 1 (HIV-1)-infected individual and are directed against the CD4 binding site (CD4bs) of the gp120 envelope glycoprotein were studied. A range of neutralizing activity against the HIV-1 (HXBc2) isolate was observed, with Fab b12 exhibiting the greatest potency among the Fabs tested. The neutralizing potency of Fab b12 was better than that of monoclonal whole antibodies directed against the third variable (V3) region of gp120. To explore the basis for the efficient neutralizing activity of b12, the recognition of a panel of HIV-1 gp120 mutants by the six Fabs was studied. The patterns of sensitivity to particular gp120 amino acid changes were similar for all six Fabs to those seen for anti-CD4bs monoclonal antibodies derived from HIV-1-infected individuals by conventional means. In addition, recognition by Fab b12 demonstrated an atypical sensitivity to changes in the V1 and V2 variable regions. Next, the binding of the Fabs to monomeric gp120 and to the envelope glycoprotein complex was examined. Neither the binding properties of the b12 Fab to monomeric gp120 nor the ability of the Fab to compete with soluble CD4 for monomeric gp120 binding appeared to account for the greater neutralizing potency. However, both quantitative and qualitative differences between the binding of b12 and that of less potent Fabs to the cell surface envelope glycoprotein complex were observed. Relative to less potently neutralizing Fabs, Fab b12 exhibited a higher affinity for a subpopulation of cell surface envelope glycoproteins, the conformation of which was best approximated by the mature gp120 glycoprotein. Apparently, subtle differences in the gp120 epitope recognized allow some members of the group of anti-CD4bs antibodies to bind to the functionally relevant envelope glycoprotein complex and to neutralize virus more efficiently.
Significance of the immune response to a major, conformational B-cell epitope on the hepatitis C virus NS3 region defined by a human monoclonal antibody
Authors: Mondelli M U , Cerino A , Boender P , Oudshoorn P , Middeldorp J , Fipaldini C , La Monica N , Habets W
Published: January 2020

The nonstructural protein NS3 of hepatitis C virus (HCV) possesses two enzymatic domains which are thought to be essential for the virus life cycle: an N-terminal serine-type proteinase, responsible for the processing of nonstructural polypeptides, and a C-terminal nucleoside triphosphatase/helicase, presumably involved in the unwinding of the viral genome. The human antibody response to NS3 usually appears early in the course of HCV infection and is predominantly directed against the carboxyl-terminal portion; however, its fine specificity and clinical significance are largely unknown. We have generated a human monoclonal antibody (hMAb), designated CM3.B6, from a cloned B-cell line obtained from the peripheral blood of a patient with chronic HCV infection, which selectively recognized the purified NS3 protein expressed in bacteria or in eukaryotic cells transfected with full-length or NS3 cDNA. Fine-specificity studies revealed that CM3.B6 recognized a 92-amino-acid sequence (clone 8, amino acids 1363 to 1454) selected from an NS3 DNase fragment library but failed to bind to 12-mer peptides synthesized from the same region, suggesting recognition of a conformational B-cell epitope. Experiments using deletion mutants of clone 8 and competitive inhibition studies using a panel of NS3 peptide-specific murine MAbs indicated that limited N-terminal and C-terminal deletions resulted in a significant reduction of hMAb binding to clone 8, thus identifying a minimal antibody binding domain within clone 8. Competition experiments showed that binding of CM3.B6 to the NS3 protein was efficiently inhibited by 39 of 44 (89%) sera from HCV-infected patients, suggesting that the hMAb recognized an immunodominant epitope within the NS3 region. More importantly, recognition of the sequence defined by CM3.B6 appeared to accurately discriminate between viremic and nonviremic anti-HCV positive sera, suggesting potentially relevant clinical applications in the diagnosis and treatment of HCV infection.
Genes in the HindIII J fragment of the murine cytomegalovirus genome are dispensable for growth in cultured cells: insertion mutagenesis with a lacZ/gpt cassette
Authors: Vieira J , Farrell H E , Rawlinson W D , Mocarski E S
Published: January 2020

The organization and function of the genes encoded within the HindIII J region of the murine cytomegalovirus genome were analyzed by transcript mapping and cDNA isolation, nucleotide sequence analysis and identification of open reading frames (ORFs), and construction of recombinant viruses carrying insertions disrupting five of the seven ORFs. This region was found to encode five beta transcripts and one gamma transcript in addition to two beta transcripts previously mapped to the sgg1 locus. Seven open reading frames were identified, and one was recognized as a homolog of a human cytomegalovirus US22 gene family. The five largest ORFs contained within the HindIII J fragment (sgg1, HJ4, HJ5, HJ6, and HJ7) were each disrupted by the insertion of a lacZ/gpt genetic marker cassette. The growth kinetics of all recombinant viruses were investigated and found to be the same as wild-type parental virus, indicating that these five ORFs were dispensable for growth in cell culture.
Differential regulation of cellular tropism and sensitivity to soluble CD4 neutralization by the envelope gp120 of human immunodeficiency virus type 1
Authors: Stamatatos L , Werner A , Cheng-Mayer C
Published: January 2020

Using recombinant and mutant viruses generated between two human immunodeficiency virus type 1 isolates that display differences in cell tropism and sensitivity to soluble CD4 neutralization, we show that these two properties of the virus are regulated by different mechanisms. Whereas there is an association between V3 loop conformation and a particular cellular tropism, soluble CD4 neutralization sensitivity appears to be determined by amino acid differences in the C2 domain of the envelope gp120 that modulate the stability of gp120-gp41 association. Our findings further illustrate the importance of functional interactions among different regions of the envelope gp120 in regulating the biological phenotypes of human immunodeficiency virus and suggest that additional probing of the V3 loop with monoclonal antibodies may identify specific structural features of this loop that determine cell tropism.
RNA splicing in Borna disease virus, a nonsegmented, negative-strand RNA virus
Authors: Schneider P A , Schneemann A , Lipkin W I
Published: January 2020

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus related to rhabdoviruses and paramyxoviruses. Unlike animal viruses of these two families, BDV transcribes RNAs in the nuclei of infected cells and produces high levels of transcripts containing multiple open reading frames. Previous Northern blot analysis of RNA from BDV-infected rat brain tissue has shown that two viral transcripts, a 6.1-kb RNA and a 1.5-kb RNA, lack regions that are internal to two otherwise identical transcripts, the 7.1-kb RNA and the 2.8-kb RNA, respectively (T. Briese, A. Schneemann, A. Lewis, Y. Park, S. Kim, H. Ludwig, and W. I. Lipkin, Proc. Natl. Acad. Sci. USA 91:4362-4366, 1994). To determine the precise location of this deletion, we performed reverse transcription PCR analysis using total RNA from BDV-infected rat brain tissue. This investigation resulted in the identification of two introns in the 7.1- and 2.8-kb RNAs, which can be alternatively spliced to yield additional RNA species, including the 6.1- and 1.5-kb RNAs. Transient transfection of COS-7 cells with a cDNA clone of the 2.8-kb RNA resulted in the production of both the 2.8-kb RNA and the 1.5-kb RNA, confirming the theory that the 2.8-kb RNA is a sufficient substrate for splicing in mammalian cells. Splicing has not previously been observed in nonsegmented, negative-strand RNA viruses and presumably serves as a mechanism by which expression of BDV proteins is regulated in infected cells.
Transcription stimulation of the adenovirus type 12 E1a gene in vitro by the 266-amino-acid E1A protein
Authors: Kawamura H , Wada N , Makino Y , Tamura T A , Koikeda S , Shiroki K , Masamune Y , Nakanishi Y
Published: January 2020

We previously showed that the 13S but not the 12S mRNA product of the E1a gene of the highly oncogenic type 12 adenovirus (Ad12) stimulates the expression of its own gene. In this study, the mechanism for the autoregulation of the Ad12 E1a gene was investigated in vitro. The 266-amino-acid E1A protein of Ad12 was synthesized in yeast cells and purified as a 57-kDa polypeptide. The purified Ad12 E1A protein stimulated transcription from the proximal promoter of its own gene but had almost no effect on that from the distal promoter. A 35-bp upstream region including a TATA box for the proximal promoter seemed to be sufficient for transcription stimulation by the E1A protein. The Ad12 E1A protein formed a complex with a TATA box-binding protein (TBP), as does the E1A protein of nononcogenic Ad serotypes. Moreover, the E1A protein significantly reduced the binding of TBP to a TATA sequence, while it did not affect the DNA-binding activity of nuclear factor I, a stimulatory protein of the distal transcription of the Ad12 E1a gene. These results suggest that the 13S mRNA product of the Ad12 E1a gene regulates the transcription of its own gene by modulating the activity of TBP.
Site-directed mutagenesis of a conserved hexapeptide in the paramyxovirus hemagglutinin-neuraminidase glycoprotein: effects on antigenic structure and function
Authors: Mirza A M , Deng R , Iorio R M
Published: January 2020

The sequence NRKSCS constitutes the longest linear stretch in the amino acid sequence of the hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxoviruses that is completely conserved among all viruses in the group. We have used site-directed mutagenesis and expression of the mutated HN protein of one member of the group, Newcastle disease virus, to explore the role of this highly conserved sequence in the structure and function of the protein. Any substitution introduced for each of four residues in the sequence, N-234, R-235, K-236, or S-237, results in a drastic decrease in neuraminidase activity relative to that of the wild-type protein. Only substitutions for the terminal serine residue in the sequence had comparatively little effect on this activity. These findings are consistent with prior computer-based predictions of protein secondary structure which had suggested that this domain corresponds to one in the beta-sheet propeller structure of the neuraminidase protein of influenza virus closest to the center of the sialic acid binding site and forms part of the enzyme active site. Four of the substitutions, N-234-->Y and K-236-->E, -->Q, and -->S, apparently cause a local alteration in the antigenic structure of the protein. This is evidenced by (i) the diminished recognition of the protein only by monoclonal antibodies thought to bind at the neuraminidase active site, among an extensive panel of conformation-specific antibodies, and (ii) the slower rate of migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for all except the K-236-->Q mutation. One of the mutations, K-236-->S, completely abolishes the ability of the protein to promote cellular fusion when coexpressed with the fusion protein. The latter cannot be explained by a decrease in the relative hemadsorption activity of the protein and suggests that the globular head of the protein may contribute to this process beyond providing receptor recognition.
Animal-derived antigenic variants of foot-and-mouth disease virus type A12 have low affinity for cells in culture
Authors: Rieder E , Baxt B , Mason P W
Published: January 2020

We recently have shown that binding of foot-and-mouth disease virus (FMDV) to cells in culture requires an arginine-glycine-aspartic acid (RGD) sequence in the G-H loop of the capsid protein VP1 (P. W. Mason, E. Rieder, and B. Baxt, Proc. Natl. Acad. Sci. USA 91:1932-1936, 1994). In this report, we show that FMDV type A12 viruses found in infected bovine tongue tissue (BTT) differ from their tissue culture-grown derivatives at amino acid residues near the RGD. Viruses genetically engineered to contain VP1 sequences found in animal tissue (BTT viruses) were antigenically different from their tissue culture derivatives and bound to BHK cells more poorly than did the tissue culture-adapted viruses. Passage of the genetically engineered BTT viruses in BHK cells resulted in the rapid selection of variants with cell-binding properties, antigenic characteristics, and sequences typical of tissue culture-adapted viruses. These data indicate that residues near the RGD are critical for cell binding and that interpretations of antigenic variation of FMDV can be affected by virus cultivation in vitro.
Single amino acid changes in the human immunodeficiency virus type 1 matrix protein block virus particle production
Authors: Freed E O , Orenstein J M , Buckler-White A J , Martin M A
Published: January 2020

The matrix protein of human immunodeficiency virus type 1 is encoded by the amino-terminal portion of the Gag precursor and is postulated to be involved in a variety of functions in the virus life cycle. To define domains and specific amino acid residues of the matrix protein that are involved in virus particle assembly, we introduced 35 amino acid substitution mutations in the human immunodeficiency virus type 1 matrix protein. Using reverse transcriptase and radioimmunoprecipitation analyses and transmission electron microscopy, we assessed the mutants for their ability to form virus particles and to function in the infection process. This study has identified several domains of the matrix protein in which single amino acid substitutions dramatically reduce the efficiency of virus particle production. These domains include the six amino-terminal residues of matrix, the region of matrix between amino acids 55 and 59, and the region between amino acids 84 and 95. Single amino acid substitutions in one of these domains (between matrix amino acids 84 and 88) result in a redirection of the majority of virus particle formation to sites within cytoplasmic vacuoles.
The size and symmetry of B capsids of herpes simplex virus type 1 are determined by the gene products of the UL26 open reading frame
Authors: Desai P , Watkins S C , Person S
Published: January 2020

Herpes simplex virus type 1 (HSV-1) B capsids are composed of seven proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26 in order of decreasing molecular weight. Three proteins (21, 22a, and VP24) are encoded by a single open reading frame (ORF), UL26, and include a protease whose structure and function have been studied extensively by other investigators. The protease encoded by this ORF generates VP24 (amino acids 1 to 247), a structural component of the capsid and mature virions, and 21 (residues 248 to 635). The protease also cleaves C-terminal residues 611 to 635 of 21 and 22a, during capsid maturation. Protease activity has been localized to the N-terminal 247 residues. Protein 22a and probably the less abundant protein 21 occupy the internal volume of capsids but are not present in virions; therefore, they may form a scaffold that is used for B capsid assembly. The objective of the present study was to isolate and characterize a mutant virus with a null mutation in UL26. Vero cells were transformed with plasmid DNA that encoded ORF UL25 through UL28 and screened for their ability to support the growth of a mutant virus with a null mutation in UL27 (K082). Four of five transformants that supported the growth of the UL27 mutant also supported the growth of a UL27-UL28 double mutant. One of these transformants (F3) was used to isolate a mutant with a null mutation in UL26. The UL26 null mutation was constructed by replacement of DNA sequences specifying codons 41 through 593 with a lacZ reporter cassette. Permissive cells were cotransfected with plasmid and wild-type virus DNA, and progeny viruses were screened for their ability to grow on F3 but not Vero cells. A virus with these growth characteristics, designated KUL26 delta Z, that did not express 21, 22a, or VP24 during infection of Vero cells was isolated. Radiolabeled nuclear lysates from infected nonpermissive cells were layered onto sucrose gradients and subjected to velocity sedimentation. A peak of radioactivity for KUL26 delta Z that sedimented more rapidly than B capsids from wild-type-infected cells was observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the gradient fractions showed that the peak fractions contained VP5, VP19C, VP23, and VP26. Analysis of sectioned cells and of the peak fractions of the gradients by electron microscopy revealed sheet and spiral structures that appear to be capsid shells.(ABSTRACT TRUNCATED AT 400 WORDS)
Fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virus-based assay quantitating cell fusion-dependent reporter gene activation
Authors: Nussbaum O , Broder C C , Berger E A
Published: January 2020

The fusogenic activities of enveloped-virus glycoproteins were analyzed by using a quantitative, sensitive, rapid, and highly versatile recombinant vaccinia virus-based assay measuring activation of a reporter gene upon fusion of two distinct cell populations. One population uniformly expressed vaccinia virus-encoded viral glycoproteins mediating specific binding and fusion activities; the other expressed the corresponding cellular receptor(s). The cytoplasm of one population also contained vaccinia virus-encoded bacteriophage T7 RNA polymerase; the cytoplasm of the other contained a transfected plasmid with the Escherichia coli lacZ gene linked to the T7 promoter. When the two populations were mixed, cell fusion resulted in activation of the LacZ gene in the cytoplasm of the fused cells; beta-galactosidase activity was assessed by colorimetric assay of detergent cell lysates or by in situ staining. We applied this approach to study the human immunodeficiency virus type 1 envelope glycoprotein (Env)-CD4 interaction. Beta-Galactosidase was detected within 1 h after cell mixing and accumulated over the next several hours. Cell fusion dependence was demonstrated by the strict requirement for both CD4 and functional Env expression and by the inhibitory effects of known fusion-blocking monoclonal antibodies and pharmacological agents. Quantitative measurements indicated much higher sensitivity compared with analysis of syncytium formation. The assay was used to probe mechanisms of the cell type specificity for Env-CD4-mediated fusion. In agreement with known restrictions, cell fusion occurred only when CD4 was expressed on a human cell type. Membrane vesicle transfer experiments indicated that CD4 initially produced in either human or nonhuman cells was functional when delivered to human cells, suggesting that the fusion deficiency with nonhuman cells was not associated with irreversible defects in CD4. We also demonstrated that the infectivity specificities of different human immunodeficiency virus type 1 isolates for peripheral blood lymphocytes versus continuous CD4+ cell lines were associated with corresponding fusion selectivities of the respective recombinant Env proteins. The assay enabled analysis of the fusogenic activity of the fusion glycoprotein/hemagglutinin-neuraminidase of the paramyxovirus simian virus 5. This system provides a powerful tool to study fusion mechanisms mediated by enveloped-virus glycoproteins, as well as to screen fusion-blocking antibodies and pharmacological agents.
Hepatitis B virus nucleocapsid particles do not cross the hepatocyte nuclear membrane in transgenic mice
Authors: Guidotti L G , Martinez V , Loh Y T , Rogler C E , Chisari F V
Published: January 2020

Transgenic mice that express the hepatitis B virus core protein were used to examine factors that influence the intracellular localization of nucleocapsid particles in the primary hepatocyte in vivo. In this model, viral nucleocapsid particles are strictly localized to the nucleus of the hepatocyte except when the nuclear membrane dissolves during cell division, at which time they enter the cytoplasm. The cytoplasmic nucleocapsid particles do not reenter the nucleus, however, when the nuclear membrane re-forms after cell division. The data support the notion that nucleocapsid particles can form de novo within the nucleus, and they suggest that performed nucleocapsid particles cannot be transported across the intact nuclear membrane in either direction. The results imply that nucleocapsid disassembly is probably required for entry of the hepadnaviral genome into the nucleus, and they question the role of the intranuclear viral nucleocapsid particle during the viral life cycle.
Cytokine induction during T-cell-mediated clearance of mouse hepatitis virus from neurons in vivo
Authors: Pearce B D , Hobbs M V , McGraw T S , Buchmeier M J
Published: January 2020

To investigate the mechanism by which viruses are cleared from neurons in the central nervous system, we have utilized a mouse model involving infection with a neurotropic variant of mouse hepatitis virus (OBLV60). After intranasal inoculation, OBLV60 grew preferentially in the olfactory bulbs of BALB/c mice. Using in situ hybridization, we found that viral RNA localized primarily in the outer layers of the olfactory bulb, including neurons of the mitral cell layer. Virus was cleared rapidly from the olfactory bulb between 5 and 11 days. Athymic nude mice failed to eliminate the virus, demonstrating a requirement for T lymphocytes. Immunosuppression of normal mice with cyclophosphamide also prevented clearance. Both CD4+ and CD8+ T-cell subsets were important, as depletion of either of these subsets delayed viral clearance. Gliosis and infiltrates of CD4+ and CD8+ cells were detected by immunohistochemical analysis at 6 days. The role of cytokines in clearance was investigated by using an RNase protection assay for interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, tumor necrosis factor alpha (TNF-alpha), TNF-beta, and gamma interferon (IFN-gamma). In immunocompetent mice there was upregulation of RNA for IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, and IFN-gamma at the time of clearance. Nude mice had comparable increases in these cytokine messages, with the exception of IFN-gamma. Induction of major histocompatibility complex class I (MHC-I) molecules on cells in infected brains was demonstrated by immunohistochemical analyses in normal and nude mice, suggesting that IFN-gamma may not be necessary for induction of MHC-I on neural cells in vivo.
Biological, molecular, and structural analysis of a cytopathic variant from a molecularly cloned simian immunodeficiency virus
Authors: LaBranche C C , Sauter M M , Haggarty B S , Vance P J , Romano J , Hart T K , Bugelski P J , Hoxie J A
Published: January 2020

Some isolates of simian immunodeficiency virus (SIV) have been shown to infect Sup-T1 cells with slow kinetics and in the absence of cytopathic effects, including cell fusion or CD4 down-modulation (J. A. Hoxie, B. S. Haggarty, S. Bonser, J. Rackowski, H. Shan, and P. Kanki, J. Virol. 62:2557-2568, 1988). In the present study, we describe the isolation and characterization of a SIVmac variant, derived from the BK28 infectious molecular clone, that became highly cytopathic for Sup-T1 cells. This variant, termed CP-MAC, exhibited a number of differences from BK28, including (i) an altered tropism which largely restricted its host range to Sup-T1 cells, (ii) the ability to induce cell fusion and CD4 down-modulation, and (iii) a highly stable interaction of its external (SU) and transmembrane (TM) envelope glycoproteins. In addition, a marked increase in the level of surface envelope glycoproteins was observed both on CP-MAC-infected cells and on virions. The CP-MAC env gene was PCR amplified from infected cells, and sequence analysis identified five amino acid changes in SU and six in TM compared with BK28. The introduction of these changes into BK28 was shown to fully reconstitute the biological and morphological properties of CP-MAC. The limited number of mutations in CP-MAC should enable the molecular determinants to be more precisely defined and help to identify the underlying mechanisms responsible for the striking biological and structural alterations exhibited by this virus.
Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors
Authors: Podsakoff G , Wong K K , Chatterjee S
Published: January 2020

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.
Foot-and-mouth disease virus leader proteinase: purification of the Lb form and determination of its cleavage site on eIF-4 gamma
Authors: Kirchweger R , Ziegler E , Lamphear B J , Waters D , Liebig H D , Sommergruber W , Sobrino F , Hohenadl C , Blaas D , Rhoads R E
Published: January 2020

Many picornaviruses cause a dramatic decrease in the translation of cellular mRNAs in the infected cell, without affecting the translation of their own RNA. Specific proteolysis of protein synthesis initiation factor eIF-4 gamma occurs during infection with rhinoviruses, enteroviruses, and aphthoviruses, apparently leading to an inability of the ribosomes to bind capped mRNAs. Cleavage of eIF-4 gamma in human rhinoviruses and enteroviruses is carried out by the viral 2A proteinase; in aphthoviruses (i.e., foot-and-mouth disease viruses), the leader proteinase is responsible for this reaction. We describe here the purification to homogeneity of the Lb form of the leader proteinase expressed in Escherichia coli. The primary cleavage products of eIF-4 gamma obtained in vitro with purified leader or 2A proteinase are electrophoretically indistinguishable from those found during infection in vivo. However, additional proteolysis products of eIF-4 gamma are observed with the leader proteinase and the human rhinovirus type 2 2A proteinase in vitro. The cleavage site of the leader proteinase in eIF-4 gamma from rabbit reticulocyte was determined by sequencing the purified C-terminal cleavage product by automated Edman degradation. The cleavage site is between Gly-479 and Arg-480 and thus differs from that of rhinovirus and enterovirus 2A proteinases, which cleave between Arg-486 and Gly-487.
Detection and characterization of a functional complex of human immunodeficiency virus type 1 integrase and its DNA substrate by UV cross-linking
Authors: Yoshinaga T , Kimura-Ohtani Y , Fujiwara T
Published: January 2020

To analyze the association of the human immunodeficiency virus type 1 integrase (IN) protein with DNA substrates, we applied a shortwave UV cross-linking method to the in vitro integration system. Three photoadduct bands were detected on sodium dodecyl sulfate-polyacrylamide gels. The appearances of these photoadducts were examined with various substrates and under several incubation conditions. Our data suggest that one of these photoadducts is derived from the sequence- and strand-specific bound state of the early phase of the integration reaction before the 3' processing reaction. We also show that most of the photoadduct complexes are competent for integration even after UV irradiation.
Mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation
Authors: Zurcher T , Luo G , Palese P
Published: January 2020

The carboxy terminus of the hemagglutinin (HA) of influenza A viruses contains three cysteine residues which are highly conserved among HA subtypes. It has previously been shown for the H2, H3, and H7 subtypes of HA that these cysteine residues are modified by the covalent attachment of palmitic acid. In order to study the role of the acylated cysteines in the formation of infectious influenza viruses, we introduced mutations into the HA of influenza A/WSN/33 virus (H1 subtype) by reverse-genetics techniques. We found that the cysteine at position 563 of the cytoplasmic tail is required for infectious-particle formation. The cysteine at position 560 can be changed to alanine or tyrosine to yield virus strains that are attenuated in cell cultures. The change from cysteine at position 553 to serine or alanine does not significantly alter the phenotype of the virus. The requirement for a cysteine at position 563 suggests a functional role for palmitylation of the cytoplasmic tail. This interpretation is further supported by experiments in which two or more of the cysteine residues were mutated, eliminating potential palmitylation sites. None of these double or triple mutations resulted in infectious virus. Selection of revertants of the attenuated cysteine-to-tyrosine mutant (mutation at position 560) always resulted in reversion to cysteine rather than to other amino acids. Although our data indicate a biological role for the conserved cysteine residues in the cytoplasmic tail of the HA of influenza viruses, we cannot exclude the possibility that structural constraints in the cytoplasmic tail of the HA--rather than altered palmitylation--are the determining factors for infectious-particle formation.