bluuebunny/crossref_metadata_2025 huggingface dataset:
Adenovirus E4-dependent activation of the early E2 promoter is insufficient to promote the early-to-late-phase transition
Authors: Hemström C , Virtanen A , Bridge E , Ketner G , Pettersson U
Published: January 2020

The adenovirus E4 ORF6/7 protein has been shown to activate the cellular transcription factor E2F. E2F activation leads to activation of the adenovirus early E2 promoter which controls the production of viral DNA replication proteins. In the present study an adenovirus type 5 cDNA mutant, H5ilE4L, was constructed. This mutant is capable of making the ORF6/7 polypeptide but lacks the coding sequences for all other E4 products. H5ilE4L trans activates the early E2 promoter to wild-type levels, but still it is defective for viral DNA replication. A mutant expressing ORF6 in addition to ORF6/7, H5ilE4I, is normal for viral DNA replication. This indicates that activation of the early E2 promoter is insufficient to promote efficient viral DNA replication and that another E4-encoded function is necessary. The ORF6 protein seems to provide this function. We suggest that ORF6/7-induced activation of E2F is not necessary for adenovirus growth in HeLa cells. Rather, this activation might be of importance in the normal, growth-arrested host cell, since E2F has been shown to bind to the promoter regions of a number of immediate-early genes involved in regulation of cell proliferation (M. Mudryj, S. W. Hiebert, and J. R. Nevins, EMBO J. 9:2179-2184, 1990).
Anti-adenovirus type 5 cytotoxic T lymphocytes: immunodominant epitopes are encoded by the E1A gene
Authors: Routes J M , Bellgrau D , McGrory W J , Bautista D S , Graham F L , Cook J L
Published: January 2020

Virus specific, major histocompatibility complex-restricted, cytotoxic T lymphocytes (CTL) generated in Fischer strain rats infected with human adenovirus type 5 (Ad5) were found to recognize antigenic determinants encoded within the Ad5 early region 1A (E1A) gene. Preliminary mapping studies suggest that the E1A CTL epitopes are encoded within the regions between bp 625 to 810 and 916 to 974 in the first exon of this gene. These epitope-coding regions occur within subregions of E1A that are conserved functionally, and to some extent structurally (approximately 50% sequence homology), among adenoviruses of different groups. Nevertheless, Ad5-specific CTL lysed only targets infected with adenoviruses of the same group (group C; e.g., Ad2) and not targets infected with adenoviruses of different groups (groups A, B, and E). These results suggest that virus-specific CTL may limit adenoviral dissemination by destroying virus-infected cells at an early stage in the viral replicative cycle, during E1A gene expression. Expression of other adenovirus genes does not appear to be required to target infected cells for elimination by CTL.
Origin of unenveloped capsids in the cytoplasm of cells infected with herpes simplex virus 1
Authors: Campadelli-Fiume G , Farabegoli F , Di Gaeta S , Roizman B
Published: January 2020

In cells infected with herpes simplex viruses the capsids acquire an envelope at the nuclear membrane and are usually found in the cytoplasm in structures bound by membranes. Infected cells also accumulate unenveloped capsids alone or juxtaposed to cytoplasmic membranes. The juxtaposed capsids have been variously interpreted as either undergoing terminal deenvelopment resulting from fusion of the envelope with the membrane of the cytoplasmic vesicles or undergoing sequential envelopment and deenvelopment as capsids transit the cytoplasm into the extracellular space. Recent reports have shown that (i) wild-type virus attaches to but does not penetrate cells expressing glycoprotein D (G. Campadelli-Fiume, M. Arsenakis, F. Farabegoli, and B. Roizman, J. Virol. 62:159-167, 1988) and that (ii) a mutation in glycoprotein D enables the mutant virus to productively infect cells expressing the wild-type glycoprotein (G. Campadelli-Fiume, S. Qi, E. Avitabile, L. Foa-Tomasi, R. Brandimarti, and B. Roizman, J. Virol. 64:6070-6079, 1990). If the unenveloped capsids in the cytoplasm result from fusion of the cytoplasmic membranes with the envelopes of viruses transiting the cytoplasm, cells infected with virus carrying the mutation in glycoprotein D should contain many more unenveloped capsids in the cytoplasm inasmuch as there would be little or no restriction in the fusion of the envelope with cytoplasmic membranes. Comparison of thin sections of baby hamster kidney cells infected with wild-type and mutant viruses indicated that this was the case. Moreover, in contrast to the wild-type parent, the mutant virus was not released efficiently from infected cells. The conclusion that the unenveloped capsids are arrested forms of deenveloped capsids is supported by the observation that the unenveloped capsids were unstable in that they exhibited partially extruded DNA.
VP4 monotype specificities among porcine rotavirus strains of the same VP4 serotype
Authors: Liprandi F , Rodriguez I , Piña C , Larralde G , Gorziglia M
Published: January 2020

The porcine rotavirus OSU strain was used to produce monoclonal antibodies (MAbs) directed against the outer capsid protein VP4. From two separate fusions, eight MAbs that inhibited hemagglutination activity of the OSU strain were selected. All MAbs immunoprecipitated both the OSU VP4 protein derived from a lysate of infected MA104 cells and the OSU VP4 protein expressed in Sf9 cells by a recombinant baculovirus. By immunoprecipitation of in vitro-translated OSU gene 4 transcripts of different length, the eight MAbs were found to be specific for the VP8 subunit of VP4. All MAbs neutralized the OSU strain but failed to neutralize human, bovine, and simian rotavirus strains. Antiserum to the expressed OSU VP4 protein was used to study the distribution of VP4 antigenicity among porcine rotaviruses. At least two distinct specificities were identified among 14 rotavirus strains that had been previously assigned to four distinct VP7 serotypes. Five groups of monotype specificities of the VP4 protein were identified by the eight anti-VP4 MAbs among 11 porcine strains that share the same VP4 serotype.
Identification of the region including the epitope for a monoclonal antibody which can neutralize human parvovirus B19
Authors: Sato H , Hirata J , Furukawa M , Kuroda N , Shiraki H , Maeda Y , Okochi K
Published: January 2020

In this study, we identified a region in the human parvovirus structural protein which involves the neutralization of the virus by a monoclonal antibody and site-specific synthetic peptides. A newly established monoclonal antibody reacted with both viral capsid proteins VP1 and VP2. The epitope was found in six strains of independently isolated human parvovirus B19. The monoclonal antibody could protect colony-forming unit erythroid in human bone marrow cell culture from injury by the virus. The monoclonal antibody reacted with only 1 of 12 peptides that were synthesized according to a predicted amino acid sequence based on nucleotide sequences of the coding region for the structural protein of B19 virus. The sequence recognized by the antibody was a site corresponding to amino acids 328 to 344 from the amino-terminal portion of VP2. This evidence suggests that the epitope of the viral capsid protein is located on the surface of the virus and may be recognized by virus-neutralizing antibodies.
Measles virus nucleocapsid protein protects rats from encephalitis
Authors: Bankamp B , Brinckmann U G , Reich A , Niewiesk S , ter Meulen V , Liebert U G
Published: January 2020

Lewis rats immunized with recombinant vaccinia virus expressing the nucleocapsid (N) protein of measles virus were protected from encephalitis when subsequently challenged by intracerebral infection with neurotropic measles virus. Immunized rats revealed polyvalent antibodies to the N protein of measles virus in the absence of any neutralizing antibodies as well as an N protein-specific proliferative lymphocyte response. Depletion of CD8+ T lymphocytes did not abrogate the protective potential of the N protein-specific cell-mediated immune response in rats, while protection could be adoptively transferred with N protein-specific CD4+ T lymphocytes. These results indicate that a CD4+ cell-mediated immune response specific for the N protein of measles virus is sufficient to control measles virus infections of the central nervous system.
Mapping the anatomy of the immunodominant domain of the human immunodeficiency virus gp41 transmembrane protein: peptide conformation analysis using monoclonal antibodies and proton nuclear magnetic resonance spectroscopy
Authors: Oldstone M B , Tishon A , Lewicki H , Dyson H J , Feher V A , Assa-Munt N , Wright P E
Published: January 2020

Thirty-six monoclonal antibodies from mice and three from rats were raised against a peptide corresponding to the immunodominant domain of the transmembrane gp41 protein of human immunodeficiency virus (HIV) type 1 (LGLWGCSGKLIC; amino acid residues 598 to 609). Of these, three monoclonal antibodies from the mice and one from a rat also reacted with the corresponding peptide derived from the HIV type 2 transmembrane gp41 protein (amino acid residues 593 to 603; NSWGCAFRQVC). Immunochemical studies using a variety of synthetic peptides indicated that the cross-reactivity was due to antibody binding to CSGKLIC of HIV type 1 or CAFRQVC of HIV type 2. Single amino acid substitutions for a cysteine at either the amino or the carboxy end of the peptide interrupted antibody binding, indicating that the site recognized was the Cys-XXXXX-Cys loop. Similar results were obtained when the 11-mer HIV type 2 gp41 peptide (amino acids 593 to 603) was inoculated into mice to raise monoclonal antibodies. In this instance, of 30 monoclonal antibodies developed, 4 reacted with both HIV type 1 and HIV type 2 peptides. The conformation of a seven-residue peptide, CSGKLIC, corresponding to residues 603 to 609 of the gp41 immunodominant epitope of HIV-1 was investigated by proton nuclear magnetic resonance spectroscopy. The immunologically active form of CSGKLIC contains an intramolecular disulfide bond and maintains a preference for a folded conformation, apparently including a type I reverse turn about the residues SGKL. No such preference is observed for the reduced form of the peptide, which contains two thiol groups. The presence of the disulfide bond is thus integral to the formation of the structure of the loop in solution. In agreement with this finding, elimination of the possibility of loop formation by substitution of S for C at the amino or carboxy termini of the 7-mer is accompanied by the failure of antibody binding to this peptide.
Selection, recombination, and G----A hypermutation of human immunodeficiency virus type 1 genomes
Authors: Vartanian J P , Meyerhans A , Asjö B , Wain-Hobson S
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) isolates are genetically so heterogeneous that they must be described in terms of populations of related but distinct genomes called quasispecies. A recent study of the influence of ex vivo culturing on HIV-1 quasispecies demonstrated that usually low-abundance genomes outgrew the more prominent forms. Here it is shown that multiple passages of an HIV-1 isolate on peripheral blood mononuclear cells resulted in the outgrowth of very minor forms. A single passage of equal proportions of supernatants to either of the established lymphocyte and monocyte cell lines Molt-3 and U937-2, respectively, resulted in the isolation of different sets of minor forms. Recombination between component sequences was observed. Extensive and monotonous base substitutions of G----A (G----A hypermutation) were evident in many sequences. A strong preference for the transition within the GpA dinucleotide was observed. Dislocation mutagenesis, in this case, a -1 slippage or dislocation of the primer with respect to the template, during DNA synthesis by the HIV-1 reverse transcriptase would explain this bias. When the consequences of polymerase errors, recombination, hypermutation, and instability are added to the genetic description of HIV-1, the real complexity of this virus starts to become apparent.
Induction of self-reactive T cells after murine coronavirus infection
Authors: Kyuwa S , Yamaguchi K , Toyoda Y , Fujiwara K
Published: January 2020

We studied the mechanism of in vitro spontaneous lymphokine production by spleen cells from mice injected intraperitoneally with murine coronavirus stain JHM 1 month after infection, when infectious virus had already been cleared from the spleens. Removal of either CD4+ T cells or Ia+ antigen-presenting cells (APC) from the spleen cells abrogated interleukin-2 (IL-2) production. Addition of anti-CD4 or anti-Iad monoclonal antibodies to the culture suppressed IL-2 production. These results suggest that the response involved typical receptor-mediated activation of T cells. Surprisingly, reciprocal mixing experiments with a coculture of T cells from infected mice and APC from either infected or naive mice resulted in the production of IL-2. The absence of viral antigens in spleen cells 1 month after infection, as indicated by their inability to induce the proliferation of T-cell clones specific for the viral antigens, suggest that the T cells from mice 1 month after infection were not responding to the viral antigens. The inoculum components other than the virus did not induce this immune response. We also found that the frequency of self-reactive but not alloreactive IL-2-producing T cells in the spleens of infected mice was 3- to 10-fold higher than that in naive mice. These findings suggest that an increased frequency of self-reactive T cells which secrete IL-2 occurs following murine coronavirus infection. This may have important implications in the development of autoimmunelike phenomena following murine coronavirus infection.
Immunoglobulin A mediation of murine nasal anti-influenza virus immunity
Authors: Renegar K B , Small P A
Published: January 2020

Most mice which have recovered from influenza virus infection are immune to reinfection with the same influenza virus. This immunity could be abrogated by the intranasal instillation of anti-immunoglobulin A (anti-IgA) but not of anti-IgG or anti-IgM antiserum. Thus, IgA is the major, if not the sole, mediator of nasal immunity to influenza virus in immunocompetent mice.
EBNA-2 transactivates a lymphoid-specific enhancer in the BamHI C promoter of Epstein-Barr virus
Authors: Sung N S , Kenney S , Gutsch D , Pagano J S
Published: January 2020

Among the few Epstein-Barr virus (EBV) genes expressed during latency are the Epstein-Barr nuclear antigens (EBNAs), at least one of which contributes to the ability of the virus to transform B lymphocytes. We have analyzed a promoter located in the BamHI-C fragment of EBV which is responsible for the expression of EBNA-1 in some cell lines. Deletion analysis of a 1.4-kb region 5' of the RNA start site has identified a 700-bp fragment that is required for optimal promoter activity in latently infected B lymphocytes, as shown by promoter constructs linked to the chloramphenicol acetyltransferase reporter gene. This fragment is also able to enhance activity, in an orientation-independent manner, of the simian virus 40 early promoter linked to the chloramphenicol acetyltransferase gene. The enhancer element has some constitutive activity in EBV-negative lymphoid cells, which is increased in the presence of the EBNA-2 gene product. Further deletions have shown that the EBNA-2-responsive region requires a 98-bp region that contains a degenerate octamer-binding motif. In epithelial cells there was no enhancer activity regardless of the presence of EBNA-2. These results demonstrate that BamHI-C promoter activity may be dependent not on an enhancer contained in the ori-P, as was previously assumed, but rather on EBNA-2 transactivation of this more proximal enhancer located in the upstream region of the BamHI C promoter itself.
Amplification of human papillomavirus genomes in vitro is dependent on epithelial differentiation
Authors: Bedell M A , Hudson J B , Golub T R , Turyk M E , Hosken M , Wilbanks G D , Laimins L A
Published: January 2020

Human papillomaviruses (HPVs) infect squamous epithelium and establish their genomes as episomes in proliferating basal cells. As infected cells differentiate, the viral DNA is amplified to high copy number and infectious virus is produced. Viral production has not yet been observed in vitro due to the inability of standard culture methods to duplicate most stages of epithelial differentiation. In this study, we have examined a cell line derived from a low-grade cervical lesion and found that it contained episomal copies of an HPV-31 subtype, HPV-31b, at approximately 50 copies per cell. When allowed to stratify at the air-liquid interface of in vitro raft cultures, this cell line differentiates in a manner which histologically resembles a low-grade cervical lesion in vivo. We have observed the amplification of HPV-31b genomes in distinct foci in the upper portion of the in vitro-stratified epithelium similar to that found in productive HPV infections in vivo. Although transcripts from the late region of HPV-31b were also detected specifically in stratified raft cultures, no capsid protein was found. These studies duplicate one important aspect of a productive HPV infection in vitro, the differentiation-dependent amplification of papillomavirus genomes.
Characterization and sequence analyses of antibody-selected antigenic variants of herpes simplex virus show a conformationally complex epitope on glycoprotein H
Authors: Gompels U A , Carss A L , Saxby C , Hancock D C , Forrester A , Minson A C
Published: January 2020

Thirteen antigenic variants of herpes simplex virus which were resistant to neutralization by monoclonal antibody 52S or LP11 were isolated and characterized. The antibodies in the absence of complement potently neutralize infectivity of wild-type virus as well as inhibit the transfer of virus from infected to uninfected cells ("plaque inhibition") and decrease virus-induced cell fusion by syncytial strains. The first variant isolated arose in vivo. Of 66 type 1 isolates analyzed from typing studies of 100 clinical isolates, one was identified as resistant to neutralization by LP11 antibody. The glycoprotein H (gH) sequence was derived and compared with those of wild-type and syncytial laboratory strains SC16, strain 17, and HFEM. The sequences were highly conserved in contrast to the diversity observed between gH sequences from herpesviruses of different subgroups. Only four coding changes were present in any of the comparisons, and only one unique coding change was observed between the laboratory strains and the clinical isolate (Asp-168 to Gly). These sequences were compared with those of antigenic variants selected by antibody in tissue culture. Twelve variants were independently selected with antibody LP11 or 52S from parent strain SC16 or HFEM. For each variant, the gH nucleotide sequence was derived and a point mutation was identified giving rise to a single amino acid substitution. The LP11-resistant viruses encoded gH sequences with amino acid substitutions at sites distributed over one-half of the gH external domain, Glu-86, Asp-168, or Arg-329, while the 52S-resistant mutant viruses had substitutions at adjacent positions Ser-536 and Ala-537. One LP11 mutant virus had a point mutation in the gH gene that was identical to that of the clinical isolate, giving rise to a substitution of Asp-168 with Gly. Both LP11 and 52S appeared to recognize distinct gH epitopes as mutant virus resistant to neutralization and immunoprecipitation with LP11 remained sensitive to 52S and the converse was shown for the 52S-resistant mutant virus. This is consistent with previous studies which showed that while the 52S epitope could be formed in the absence of other virus products, virus gene expression was required for stable presentation of the LP11 epitope, and for transport of gH to the cell surface (Gompels and Minson, J. Virol. 63:4744-4755, 1989). All mutant viruses produced numbers of infectious particles that were similar to those produced by the wild-type virus, with the exception of one variant which produced lower yields.(ABSTRACT TRUNCATED AT 400 WORDS)
Site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin
Authors: Veit M , Kretzschmar E , Kuroda K , Garten W , Schmidt M F , Klenk H D , Rott R
Published: January 2020

The hemagglutinin (HA) of influenza virus is a type I transmembrane glycoprotein which is acylated with long-chain fatty acids. In this study we have used oligonucleotide-directed mutagenesis of cloned cDNA and a simian virus 40 expression system to determine the fatty acid binding site in HA and to examine possible functions of covalently linked fatty acids. The results show that the HA is acylated through thioester linkages at three highly conserved cysteine residues located in the cytoplasmic domain and at the carboxy-terminal end of the transmembrane region, whereas a cysteine located in the middle of the membrane-spanning domain is not acylated. Mutants lacking fatty acids at individual or all three attachment sites acquire endoglycosidase H-resistant oligosaccharide side chains, are cleaved into HA1 and HA2 subunits, and are transported to the plasma membrane at rates similar to that of wild-type HA. All mutants are membrane bound and not secreted into the medium. These results exclude transport signal and membrane-anchoring functions of covalently linked fatty acids for this integral membrane glycoprotein. Furthermore, lack of acylation has no obvious influence on the biological activities of HA: cells expressing fatty acid-free HA bind to and, after brief exposure to mildly acidic pH, fuse with erythrocytes; the HA-induced polykaryon formation is not impaired, either. Other possible functions of covalently linked fatty acids in integral membrane glycoproteins which cannot be examined in conventional cDNA expression systems are discussed.
trans rescue of a mutant poliovirus RNA polymerase function
Authors: Charini W A , Burns C C , Ehrenfeld E , Semler B L
Published: January 2020

A series of three-nucleotide insertions was engineered into the P2 and P3 coding regions of the T7 expression plasmid pT7(tau)-PV1, which encodes a full-length copy of poliovirus type 1 (Mahoney) cDNA. When RNA derived in vitro from these mutated templates was used to transfect HeLa cells, viable virus mutants were recovered. One mutant, Sel-3D-18, which contained a single amino acid insertion in the 3Dpol coding region, was temperature sensitive for growth at 39 degrees C and showed defects in both RNA synthesis and P1 protein processing at the nonpermissive temperature. The RNA replication defect in Se1-3D-18 was identified at the level of RNA chain elongation. A highly specific and sensitive method was developed for analyzing the ability of mutant RNA templates to replicate in the presence or absence of helper functions provided in trans. This approach was used to demonstrate that RNA synthesis in Se1-3D-18 can be rescued by helper functions provided in trans.
Polyomavirus middle tumor antigen increases responsiveness to growth factors
Authors: Raptis L
Published: January 2020

The middle tumor antigen (mT) of polyomavirus is unable to transform a clone of NIH 3T3 cells to anchorage independence (L. Raptis and J.B. Bolen, J. Virol. 63:753-758, 1989). However, this oncogene increased the responsiveness of these cells to the growth factors (alpha-like and beta-type transforming growth factors) produced by cells possessing the whole transforming region of polyomavirus. This resulted in the growth of NIH 3T3 cells, expressing mT under control of the dexamethasone-regulatable mouse mammary tumor virus promoter, in agar medium supplemented with these growth factors upon addition of the inducer. Therefore, mT, a transforming oncogene, is able to enhance the responsiveness of established cells to growth factors, a property previously attributed primarily to myc and other establishment type oncogenes.
The viral transcriptional regulatory region upstream of the E6 and E7 genes is a major determinant of the differential immortalization activities of human papillomavirus types 16 and 18
Authors: Romanczuk H , Villa L L , Schlegel R , Howley P M
Published: January 2020

The human papillomavirus types 16 (HPV-16) and 18 (HPV-18) can immortalize primary human keratinocytes. The region of the viral genome responsible for this function maps to the E6 and E7 genes and their respective upstream transcriptional regulatory sequences, the long control regions (LCRs). The HPV-18 LCR/E6/E7 is more efficient in this immortalization function than the analogous region of the HPV-16 genome, resembling the difference in the immortalization potentials of the two full-length viral genomes. This study was designed to examine the basis for the difference in HPV-16 and HPV-18 immortalization efficiencies. The E6 and E7 genes of either HPV-16 or HPV-18, when expressed from the same heterologous promoter, immortalized primary human keratinocytes with the same low efficiency, suggesting that the difference in immortalization activities was not due to the different E6 or E7 genes themselves but rather to a difference in the transcriptional regulatory regions upstream of these genes. The analysis of a series of chimeric HPV-16 and HPV-18 LCR/E6/E7 constructs confirmed this observation and further mapped the viral element responsible for the major difference in immortalization efficiency to the transcriptional regulatory region upstream of the E6 and E7 genes.
Identification and characterization of human immunodeficiency virus type 1 gag-pol fusion protein in transfected mammalian cells
Authors: Peng C , Chang N T , Chang T W
Published: January 2020

Three human immunodeficiency virus type 1 (HIV-1) mutants were constructed with mutations in their protease genes: AH2-pSVL, with an in-phase deletion; BH27-pSVL, with an out-of-phase deletion creating a stop codon immediately after the deletion site; and CA-pSVL, with a point mutation creating an Asp-to-Ala substitution at the putative protease active site. The wild-type, HXB2-pSVL, and the mutated viral genomes were used to transfect COS-M6 cells and to produce virions. Immunoblotting assays with a monoclonal antibody (MAb) specific for p24 showed that all three mutant contained a gag precursor, Pr56gag, with AH2 and CA expressing an extra band of about 160 kDa. Similar assays with a MAb specific for HIV-1 reverse transcriptase (RT) also revealed a 160-kDa protein from AH2 and CA virions and two mature p66 and p51 RT subunits from HXB2 virions. In addition, HXB2, AH2, and CA but not BH27 virions exhibited RT activity. The same protein in the 160-kDa band seemed to possess both p24 and RT components, since the MAb against p24 was able to immunoadsorb RT antigen and enzymatic activity. These results indicate that the HIV-1 gag-pol fusion protein produced in mammalian cells expressed significant RT activity.
Single amino acid substitutions in the hemagglutinin can alter the host range and receptor binding properties of H1 strains of influenza A virus
Authors: Aytay S , Schulze I T
Published: January 2020

We have previously characterized an influenza A (H1N1) virus which has host-dependent growth and receptor binding properties and have shown that a mutation which removes an oligosaccharide from the tip of the hemagglutinin (HA) by changing Asn-129 to Asp permits this virus to grow to high titer in MDBK cells, (C. M. Deom, A. J. Caton, and I. T. Schulze, Proc. Natl. Acad. Sci. USA 83:3771-3775, 1986). We have now isolated monoclonal antibodies specific for the mutant HA and have used escape mutants to identify alterations in HA sequence which reduce virus yields from MDBK cells without reducing those from chicken embryo fibroblasts. Two types of escape mutants which grow equally well in chicken embryo fibroblasts were obtained. Those with the parent phenotype contain Asn at residue 129 and are glycosylated at that site. Those with the mutant phenotype are unchanged at residue 129 but have a Gly to Glu substitution at residue 158, which is close to residue 129 on the HA1 subunit. Binding assays with neoglycoproteins containing N-acetylneuraminic acid in either alpha 2,3 or alpha 2,6 linkage to galactose showed that the MDBK-synthesized oligosaccharides at Asn-129 reduce binding to both of these receptors, leaving the HA's preference for alpha 2,6 linkages unchanged. Glu at residue 158 greatly reduces binding to both receptors without reducing virus yields from MDBK cells. We conclude that changes in the receptor binding properties of the HA can result either from direct alteration of the HA protein by host cell glycosylation or from mutations in the HA gene and that these changes generate heterogeneity that can contribute to the survival of influenza A virus populations in nature.
Rapid activation and subsequent down-regulation of the human immunodeficiency virus type 1 promoter in the presence of Tat: possible mechanisms contributing to latency
Authors: Drysdale C M , Pavlakis G N
Published: January 2020

The mechanism of induction of gene expression of the human immunodeficiency virus type 1 long terminal repeat (LTR) by the Tat transactivator protein was studied in a cell fusion assay. Tat causes a rapid activation of both transcription from the LTR and accumulation of hybrid LTR-chloramphenicol acetyltransferase mRNAs. Approximately 4 h after induction by Tat, expression from the LTR promoter is down-regulated, resulting in a decrease in the accumulation of LTR mRNA. This down-regulation of expression occurs in the continued presence of Tat. Protein synthesis inhibitors can block this down-regulation; therefore, the postinduction repression of expression is dependent upon de novo protein synthesis. We propose that a labile cellular protein(s) is responsible for the low levels of human immunodeficiency virus type 1 expression, possibly contributing to the establishment of a latent state of viral expression.
The zinc finger region of simian virus 40 large T antigen is needed for hexamer assembly and origin melting
Authors: Loeber G , Stenger J E , Ray S , Parsons R E , Anderson M E , Tegtmeyer P
Published: January 2020

Simian virus 40 large T antigen contains a single sequence element with an arrangement of cysteines and histidines that is characteristic of a zinc finger motif. The finger region maps from amino acids 302 through 320 and has the sequence C-302 L K C-305 I K K E Q P S H Y K Y H-317 E K H-320. Previous genetic analysis has shown that the cysteine and histidine sequences and the contiguous S H Y K Y region in the finger are important for DNA replication in vivo. We show here that representative mutations in either of these elements of the finger prevent the assembly of large T antigen into stable hexamers in vitro. These same mutations have a characteristic effect on the interaction of T antigen with the simian virus 40 core origin of replication. The mutant T antigens bind to the central pentanucleotide domain of the core origin but fail to melt the adjacent inverted repeat domain and to untwist the adenine-thymine domain. These defects would prevent the formation of a replication bubble and the initiation of DNA replication. Finger mutations have lesser effects on the helicase function of T antigen and no observable effect on binding of T antigen to the mouse p53 protein. We propose that the zinc finger region contributes to protein-protein interactions essential for the assembly of stable T-antigen hexamers at the origin of replication and that hexamers are needed for subsequent alterations in the structure of origin DNA. We cannot exclude the possibility that the zinc finger region also makes specific contacts with components of origin DNA.
Serotype-specific epitope(s) present on the VP8 subunit of rotavirus VP4 protein
Authors: Larralde G , Li B G , Kapikian A Z , Gorziglia M
Published: January 2020

cDNA clones representing the VP8 and VP5 subunits of VP4 of symptomatic human rotavirus strain KU (VP7 serotype 1 and VP4 serotype 1A) or DS-1 (VP7 serotype 2 and VP4 serotype 1B) or asymptomatic human rotavirus strain 1076 (VP7 serotype 2 and VP4 serotype 2) were constructed and inserted into the pGEMEX-1 plasmid and expressed in Escherichia coli. Immunization of guinea pigs with the VP8 or VP5 protein of each strain induced antibodies that neutralized the rotavirus from which the VP4 subunits were derived. In a previous study (M. Gorziglia, G. Larralde, A.Z. Kapikian, and R. M. Chanock, Proc. Natl. Acad. Sci. USA 87:7155-7159, 1990), three distinct serotypes and one subtype of VP4 outer capsid protein were identified among 17 human rotavirus strains that had previously been assigned to five distinct VP7 serotypes. The results obtained by cross-immunoprecipitation and by neutralization assay with antisera to the VP8- and VP5-expressed proteins suggest that the VP8 subunit of VP4 contains the major antigenic site(s) responsible for serotype-specific neutralization of rotavirus via VP4, whereas the VP5 subunit of VP4 is responsible for much of the cross-reactivity observed among strains that belong to different VP4 serotypes.
The Q300 gene: a novel transcription unit induced in simian virus 40-infected and -transformed mouse cells
Authors: Wagner S , Cullmann G , Knippers R
Published: January 2020

The Q300 element is a single-copy 233-bp genomic mouse DNA fragment carrying a high-affinity binding site for the simian virus 40 (SV40) large T antigen. This element was used to screen an EMBL3 mouse genomic library. We could identify a genomic clone containing an approximately 500-bp transcribed region flanking the Q300 element. The transcribed region, termed the Q300 transcription unit or Q300 gene, is overexpressed in acutely SV40-infected or SV40-transformed mouse and rat cells. The Q300 gene includes an open reading frame which has the coding potential for a small polypeptide with an extremely hydrophobic N terminus and a hydrophilic C terminus. The deduced polypeptide has some similarity with the papillomavirus E5 oncoprotein.
Herpes simplex virus origin-binding protein (UL9) loops and distorts the viral replication origin
Authors: Koff A , Schwedes J F , Tegtmeyer P
Published: January 2020

To investigate the role of the herpes simplex virus origin-binding protein (UL9) in the initiation of DNA replication, we have examined the effect of UL9 binding on the structure of the viral origin of replication. UL9 loops and alters the DNA helix of the origin regardless of the phasing of the binding sites. DNase I and micrococcal nuclease footprinting show that UL9 binds two sites in the origin and loops the AT-rich DNA between them independent of the topology of the DNA. KMnO4 and dimethyl sulfate footprinting further show that UL9 alters the DNA helix in the AT region. In contrast to the looping reaction, however, helical distortion requires the free energy of supercoiled DNA. UL9 also loops and distorts the origin DNA of a replication-defective mutant with a 6-bp insertion in the AT region. Because the helical distortion of this mutant DNA is different from that of functional origins, we conclude that an imperfect tertiary structure of the mutant DNA may contribute to its loss of replication function.
Pseudorabies virus-induced suppression of major histocompatibility complex class I antigen expression
Authors: Mellencamp M W , O'Brien P C , Stevenson J R
Published: January 2020

The ability of pseudorabies virus (PrV) to down-modulate expression of major histocompatibility complex class I antigens in murine and porcine cells was investigated. When quantified by flow cytometry, surface expression of class I Kk and Dk antigens on PrV-infected cells decreased by 60% or more. Down-modulation was associated with a decrease in total cellular class I antigens, indicating regulation at the transcriptional or posttranscriptional level. PrV did not suppress expression of transferrin receptor, suggesting a selective regulatory mechanism.
Alternative translation initiation site in the DA strain of Theiler's murine encephalomyelitis virus
Authors: Kong W P , Roos R P
Published: January 2020

Polyprotein processing studies of Theiler's murine encephalomyelitis virus (TMEV), a group of mouse picornaviruses, demonstrated synthesis of a protein we have called l during in vitro translations from the RNA of DA, a demyelinating strain of TMEV, but not GDVII, an acute neurovirulent strain. We have proposed that l is synthesized from an alternative initiation site in the DA leader (L) coding area out of phase with the polyprotein reading frame (R. P. Roos, W.-P. Kong, B. L. Semler, J. Virol. 63:5344-5353, 1989). We now provide support for this proposal from experiments involving in vitro translation of three separate mutations of an infectious DA cDNA clone: DA"l"-1, which contains a base mismatch at the putative initiation codon of l, DAL-1, which contains a base mismatch at the presumed authentic initiation site of L at the beginning of the polyprotein; and DAL:NheI, which contains nucleotides coding for a four-amino-acid insertion in the L coding area with a termination codon in the l reading frame. Our results demonstrate that the DA strain uses an alternative initiation site and reading frame to in vitro synthesize l. l may have a role in the biological activity of the virus.
An Fc receptor for human immunoglobulin G is located within the tegument of human cytomegalovirus
Authors: Stannard L M , Hardie D R
Published: January 2020

Immunogold electron microscopy has demonstrated that human immunoglobulin G (IgG) can bind to the tegument of human cytomegalovirus virions by the Fc portion of the molecule. This binding was inhibited by preincubation of the Fc probes with protein A. Treatment of AD169 virions with Triton X-100 allowed release of the Fc-binding proteins, which were precipitated and characterized by polyacrylamide gel electrophoresis (PAGE). Polypeptides of approximately 69 and 33 kDa were recovered and shown by immunoblotting to retain their capacity to bind Fc-gold after separation under both reducing and nonreducing conditions. The combined results of blocking experiments, PAGE of precipitates, and Western blots (immunoblots) indicate that the tegument proteins which bind IgG-Fc are identical to those which bind beta 2 microglobulin.
Identification of lentivirus tat functional domains through generation of equine infectious anemia virus/human immunodeficiency virus type 1 tat gene chimeras
Authors: Carroll R , Martarano L , Derse D
Published: January 2020

The structural regions that comprise the functional domains of lentivirus Tat proteins were examined. Chimeric tat genes and chimeric viral promoters were constructed between the distantly related human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV). These exchange experiments revealed that the EIAV Tat-responsive element recognition domain is formed by two distinct structural regions. Activation domains of both HIV-1 and EIAV Tat contain a conserved core element, but at least HIV-1 Tat requires the presence of additional structural regions. The interchangeable nature of Tat activation domains suggests that these domains act through a common or ubiquitous cellular transcription factor.
Mapping of a neutralizing antigenic site of Coxsackievirus B4 by construction of an antigen chimera
Authors: Reimann B Y , Zell R , Kandolf R
Published: January 2020

A neutralizing antigenic site of coxsackievirus B4 (CVB4) was identified by construction of an antigen chimera between coxsackievirus B3 (CVB3) and CVB4. This chimera, designated CVB3/4, was constructed by inserting five amino acids of the putative BC loop of the structural protein VP1 of CVB4 into the corresponding loop of CVB3 by site-directed mutagenesis of infectious recombinant CVB3 cDNA. The chimeric cDNA was capable of inducing an infectious cycle upon transfection of permissive host cells. The resulting chimeric virus CVB3/4 was neutralized and precipitated by CVB4 and CVB3 serotype-specific polyclonal antisera, demonstrating that it unifies antigenic properties of both coxsackievirus serotypes. In addition, the chimera elicited antibodies in rabbits which were capable of neutralizing the two coxsackievirus serotypes CVB3 and CVB4. The insertion of the CVB4-specific antigenic site into the BC loop of CVB3 reduces the efficiency of viral replication, resulting in a small-plaque morphology of the virus chimera. In summary, these data give evidence for the presence of a serotype-specific neutralizing antigenic site in the BC loop of VP1 of CVB4 (amino acids 81 to 89). Our findings suggest that the construction of intertypic chimeras can be used as a tool for the identification of antigenic sites of coxsackieviruses. The retained immunogenicity of the mapped CVB4-specific antigenic epitope, when expressed in CVB3, indicates that CVB3 can be used as a RNA virus vector for heterologous antigenic sites.
Three envelope proteins of hepatitis B virus: large S, middle S, and major S proteins needed for the formation of Dane particles
Authors: Ueda K , Tsurimoto T , Matsubara K
Published: January 2020

The infectious particles of hepatitis B virus are called Dane particles and consist of viral nucleic acid encapsulated within a core particle that is enveloped by virus-coded surface proteins. The major S protein constitutes a significant fraction of these surface proteins. In addition, there are two other related proteins (large S and middle S), but their role in envelope formation has not yet been elucidated. We modified the translation initiation codon ATG of each of the envelope proteins by site-directed mutagenesis and found that mutant genomes that did not produce one or two of these proteins were unable to form Dane particles. The particles released into the culture medium by such mutants did not carry DNA. Synthesis of virus-coded RNA still occurred normally, and core particles carrying DNA accumulated intracellularly. The DNA in such core particles was mostly in the double-stranded open circular form, in contrast to the normal situation in which the particles contain mostly RNA and its complementary single-stranded DNA or else contain linear DNA that is partially single stranded and otherwise duplex. The role of the large S and middle S proteins in the formation of Dane particles is discussed.
Acute myeloid leukemia induction by amphotropic murine retrovirus (4070A): clonal integrations involve c-myb in some but not all leukemias
Authors: Wolff L , Koller R , Davidson W
Published: January 2020

Amphotropic murine retrovirus 4070A was demonstrated to be highly leukemogenic when inoculated intravenously into adult DBA/2 mice that were undergoing an intense chronic inflammatory response, but was nonleukemogenic in the absence of inflammation. The virus-induced promoonocytic leukemias, designated AMPH-ML, are similar morphologically and in cell surface marker expression to monocytic leukemias, called MML and MF-ML, previously shown to be induced by Moloney murine leukemia virus and MF-3 virus (a recombinant between Friend murine leukemia virus and Moloney murine leukemia virus) and resemble certain mature acute monocytic leukemias in humans (AML subtype M5). Approximately two-thirds of the AMPH-MLs (subgroup I) were demonstrated to have alterations in the 5' end of the c-myb locus, an event which occurs in 100% of MML and MF-ML. Data indicate that proviral insertions in AMPH-ML subgroup I resulted in aberrant c-myb mRNA expression and truncation of its translation product at the amino terminus. Approximately one-third of the AMPH-MLs (subgroup II) had not undergone any DNA rearrangements at the c-myb locus. In addition, their transcripts and protein products were of normal size. These latter leukemias also had not undergone DNA rearrangements in c-myc, although retroviruses expressing myc have previously been shown to induce monocyte-macrophage tumors in mice undergoing a chronic inflammation. That subgroup II leukemias had at least one clonal viral insertion suggests that there may be other sites in the cellular genome that can be activated by insertional mutagenesis in these murine acute monocytic leukemias.
An early gene maps within and is 3' coterminal with the immediate-early gene of equine herpesvirus 1
Authors: Harty R N , O'Callaghan D J
Published: January 2020

The immediate-early (IE) gene (IR1 gene) of equine herpesvirus 1 (EHV-1) encodes a single, spliced 6.0-kb mRNA during cytolytic infection. However, under early (in the presence of phosphonoacetic acid) and late (8 h postinfection; no metabolic inhibitors) conditions, in addition to the 6.0-kb IE mRNA, a 4.4-kb early (E) mRNA is transcribed from the IE gene region beginning at approximately 4 h postinfection. To map and characterize the 4.4-kb E mRNA and the protein product of this early gene (IR2 gene), Northern (RNA) blot hybridization, S1 nuclease, primer extension, and in vitro transcription and translation analyses were used. The data from RNA mapping analyses revealed that the 4.4-kb E IR2 mRNA (i) maps at nucleotides 4481 to 635 within each of the inverted repeats of the short region and thus is encoded by sequences that lie entirely within the IE gene, (ii) is transcribed in the same direction as the IE mRNA, initiating at nucleotide 4481, which lies 25 bp downstream of a putative TATA-like sequence and 1,548 bp downstream of the transcription initiation site of the IE mRNA, and (iii) is 3' coterminal with the IE mRNA which terminates at nucleotide 635 of the inverted repeats. The IR2 open reading frame was inserted into the transcription expression vector pGEM-3Z, and the RNA transcribed from this construct (pGEM44) was shown to be a 4.2-kb transcript that contained all IR2 sequences. In vitro translation of the 4.2-kb RNA yielded a major protein of approximately 130 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. This protein corresponds to the predicted IR2 product of 1,165 amino acids that would be in frame with the major IE polypeptide (IE1 = 200 kDa; 1,487 amino acids) and thus would be a 5'-truncated form of the IE1 polypeptide. The presence and potential role of the IR2 gene embedded within the IR1 gene increase the complexity of the regulation of the IE gene region during various stages of a productive infection.
The sequence of human immunodeficiency virus type 2 circle junction suggests that integration protein cleaves the ends of linear DNA asymmetrically
Authors: Whitcomb J M , Hughes S H
Published: January 2020

The sequence of the human immunodeficiency virus type 2 circle junction was determined. The most common sequence found between the conserved CA and TG dinucleotides at the ends of the integrated provirus was five bases long (GGTAC). This suggests that the integration of human immunodeficiency virus type 2 DNA is accompanied by the asymmetric loss of two and three bases, respectively, from the U3 and U5 ends of the linear double-stranded DNA prior to integration.
Posttranscriptional regulation of beta interferon expression in erythroid Friend cells treated with gamma interferon
Authors: Marziali G , Fiorucci G , Coccia E M , Percario Z , Raber J , Battistini A , Rossi G B , Affabris E , Romeo G
Published: January 2020

Treatment of Friend erythroleukemia cells (FLC) with gamma interferon (IFN-gamma) in the presence of anti-IFN-beta antibodies reduces the effectiveness of the antiviral state and the induction of 2'-5'-oligoadenylate synthetase activity, indicating that the antiviral activity of IFN-gamma in FLC is in part mediated by the production of IFN-beta. Accordingly, IFN-gamma induces a less pronounced antiviral state in FLC resistant to IFN-alpha/beta than in wild-type cells. Moreover, while results of run-on assays indicate that both IFN-alpha and -beta genes are constitutively transcribed in these cells, FLC treatment with IFN-gamma induces only IFN-beta mRNA accumulation. These results indicate that posttranscriptional mechanisms are involved in the regulation of IFN-beta and -alpha expression by IFN-gamma. The low amounts of the induced IFN-beta synergize with IFN-gamma in mounting the potent antiviral effect.