bluuebunny/crossref_metadata_2025 huggingface dataset:
Secondary structure of the human T-cell leukemia virus type 1 rex-responsive element is essential for rex regulation of RNA processing and transport of unspliced RNAs
Authors: Toyoshima H , Itoh M , Inoue J , Seiki M , Takaku F , Yoshida M
Published: January 2020

Rex protein of human T-cell leukemia virus type 1 (HTLV-1) induces cytoplasmic expression of unspliced gag/pol mRNA and singly spliced env mRNA and thus is essential for replication of the virus. This regulation requires a cis-acting rex-responsive element (RXE), located in the 3' region of the viral RNA. By external deletion, we have identified RXE composed of 205 nucleotides. The secondary structure of RXE was confirmed by studies on its susceptibility to nuclease digestions to consist of four stem-loops and a long stretch of stem structure. Substitution and deletion mutations revealed that two regions of the stem-loops and their secondary structures are essential for rex regulation. Similar secondary structures were found in the corresponding regions of HTLV-2, bovine leukemia virus and human immunodeficiency virus. Furthermore, a sequence of 11 nucleotides in the RXE was found to be conserved in the secondary structures of HTLV-1, HTLV-2, and bovine leukemia virus. These observations suggest that the secondary structure as well as the conserved sequence may be important in expression of unspliced RNA even with diverged sequences as observed in these viruses.
cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication
Authors: Hartl M , Willnow T , Fanning E
Published: January 2020

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.
A DNA replication-positive mutant of simian virus 40 that is defective for transformation and the production of infectious virions
Authors: Peden K W , Spence S L , Tack L C , Cartwright C A , Srinivasan A , Pipas J M
Published: January 2020

Simian virus 40 (SV40) mutant 5002 carries base pair substitutions of C-5109----T and C-5082----T. These mutations lie in a region of the genome that encodes amino acids common to the large and small viral tumor antigens (T and t antigens, respectively) and result in amino acid substitutions of Leu-19----Phe and Pro-28----Ser. In contrast to wild-type SV40, which produces large plaques that are clearly visible 8 days postinfection, mutant 5002 is defective for productive infection, producing tiny plaques that arise at around 21 days postinfection. However, 5002 is capable of replicating viral DNA and producing normal amounts of capsid proteins, indicating that the mutations alter an activity of T antigen that is required subsequent to DNA synthesis, such as maturation, viral assembly, or release of virions. The mutant T antigen has normal ATPase activity, is phosphorylated in a manner that is indistinguishable from that of the wild-type T antigen, and retains the ability to oligomerize. 5002 complements mutants defective in T antigen host range-adenovirus helper function for productive infection. Thus, T antigen encodes two activities that affect at least two different steps in viral infection other than DNA replication, one inactivated by mutations in the host range-adenovirus helper domain and one inactivated by the mutations present in 5002. The 5002-encoded T antigen is also defective for transformation of REF52 cells when expressed from the normal SV40 early promoter, although this defect can be partially overcome by expressing the protein from stronger promoters.
Molecular structure of the cell-attachment protein of reovirus: correlation of computer-processed electron micrographs with sequence-based predictions
Authors: Fraser R D , Furlong D B , Trus B L , Nibert M L , Fields B N , Steven A C
Published: January 2020

The receptor-recognition interaction that initiates reovirus infection is mediated by the sigma 1 protein, located at the vertices of the icosahedral virion. We have applied computer-based image-averaging techniques to electron micrographs of negatively stained preparations of sigma 1 purified from virions (serotype 2 Jones). Combining these results with inferences based on the amino acid sequence has led to a molecular model in which the overall folding of the chains is described; its conformation embodies motifs, coiled-coil alpha-helices and nodular multichain elements rich in beta-sheets, previously detected in the corresponding proteins of other viruses, but with some novel variations. Sigma 1 is a filamentous lollipop-shaped molecule with an overall length of approximately 48 nm; it has a flexible "tail," approximately 40 nm long by 4 to 6 nm wide, terminating at its distal end in a globular "head," approximately 9.5 nm in diameter. The purified protein is a tetramer (4 by 50 kilodaltons) consisting of two similarly oriented dimers bonded side by side and in register. For each chain, a cluster of hydrophobic residues at its amino terminus resides at the proximal end of the tail; next, an alpha-helical domain (residues 25 to 172) participates in a two-chained coiled coil, 22 nm long, with two such coiled coils pairing laterally to form the proximal half of the tail. The remainder of the tail (residues 173 to approximately 316) is less uniform in width and is expected to be rich in beta-sheet; the interdimer bonding is evidently sustained through this portion of the molecule. Finally, the globular head consists of the carboxy-terminal domains (which contain the receptor-binding sites) folded into compact globular conformations; in appropriate side views, the head is resolved into two subunits, presumably contributed by the respective dimers. This model for how the four sigma 1 polypeptide chains are threaded in parallel through the fiber is supported by the observed match between an empirical curvature profile, which identifies the locations of relatively flexible sites along the tail, and the flexibility profile predicted on the basis of the model. Appraisal of the interactions that stabilize the coiled coils suggests that (i) the alpha-helices are individually only marginally stable, a property that may be of significance with regard to the retracted conformation in which sigma 1 is accommodated in the intact virion, and (ii) the predominant interactions between the two coiled coils are likely to involve hydrogen bonding between patches of uncharged residues.
In vitro infection of human hepatoma (HepG2) cells with hepatitis B virus
Authors: Bchini R , Capel F , Dauguet C , Dubanchet S , Petit M A
Published: January 2020

An in vitro system for production of hepatitis B virus (HBV) was established by infection of human hepatoma (HepG2) cells. HBV particles obtained from the serum of a chronic hepatitis B surface antigen (subtype ad) carrier were used to inoculate HepG2 cells. HBV envelope and core proteins were synthesized de novo by the infected cells and secreted into the medium 3 to 6 days postinfection. Viral covalently closed circular DNA, the putative template for viral RNA transcription, accumulated in the cells with increasing time postinfection. The HBV-infected HepG2 cells were maintained for several months (HepG2-BV cell line) and continued producing viral antigens. Both HBV DNA replicative intermediates and major HBV transcripts were identified in HepG2-BV cells. Complete HBV particles, which contain HBV DNA and DNA polymerase activity and express the three antigenic specificities of the envelope (hepatitis B surface antigen, pre-S2, and pre-S1), were released into the culture supernatant. Thus, successful in vitro infection of transformed human hepatocytes raising stable HBV-producing cells was achieved for the first time. This strongly suggests that HepG2 cells have a receptor(s) for virus attachment and penetration. Such a system represents a significant advance for the study of HBV-target cell interactions as the early events of HBV infection.
Constitutive expression of human immunodeficiency virus (HIV) nef protein in human astrocytes does not influence basal or induced HIV long terminal repeat activity
Authors: Bachelerie F , Alcami J , Hazan U , Israël N , Goud B , Arenzana-Seisdedos F , Virelizier J L
Published: January 2020

Since human immunodeficiency virus (HIV) nef has been suggested to exert regulatory effects on HIV long terminal repeat (LTR) activity, we transiently transfected HIV LTR chloramphenicol acetyltransferase or luciferase expression vectors into a human astrocytoma clone (U-373nef) that constitutively expresses the HIV nef gene. In these cells, basal HIV LTR activity, as well as tumor necrosis factor-induced or tat-driven activity, was similar to that in control cells. Lack of any detectable effect of HIV nef on LTR activity was not the result of mutations in integrated nef DNA, as was shown by polymerase chain reaction. These data suggest that the role of nef in HIV genome transcription does not necessarily involve a direct influence on HIV LTR activity.
Tandemly repeated hexamer sequences within the beta interferon promoter can function as an inducible regulatory element in activation by the adenovirus E1B 19-kilodalton protein
Authors: Shiroki K , Kato H , Kawai S
Published: January 2020

The expression of the human beta interferon (IFN-beta) gene was activated by the adenovirus E1B-19K protein. The sequence within the IFN-beta promoter which is related to the activation was analyzed by chloramphenicol acetyltransferase (CAT) assay. The repeated hexamer units, present within the region between -109 and -65 relative to the cap site, were required for the activation of the IFN-beta gene by the E1B-19K protein. The (AAGTGA)8 region, as a typical hexamer of the consensus sequences, was tested for function in the activation by the E1B-19K protein. When the hexamer (AAGTGA)4-8 was inserted upstream of several reporter genes (such as p55cat, pdlE1A-CAT, and pE1B-CAT) which were inefficiently stimulated, the CAT activities of these fusion genes were efficiently stimulated by the E1B-19K protein. These results show that the tandemly repeated hexamer sequences within the IFN-beta promoter can function as an inducible regulatory element in the activation by the adenovirus E1B 19K protein.
Human papillomavirus type 1 produces redundant as well as polycistronic mRNAs in plantar warts
Authors: Palermo-Dilts D A , Broker T R , Chow L T
Published: January 2020

Human papillomavirus type 1 (HPV-1) causes plantar warts. On the basis of previously mapped mRNAs and sequence homologies of HPV-1 to other papillomaviruses, we designed oligonucleotide primers and employed the polymerase chain reaction to recover HPV-1 cDNAs from plantar warts. Seven spliced RNA species were characterized, including three not previously detected, and the coding potentials of each were deduced. The most abundant viral mRNA encodes an E1i--E4 protein. One new species is predicted to encode the full-length E2 protein, and another can, theoretically, encode the E2-C or E1-M proteins, three products that regulate mRNA transcription and DNA replication. One RNA species originating from a novel HPV promoter in the upstream regulatory region has the potential to encode the minor capsid protein L2. A newly recognized E5a open reading frame (ORF) is contained in all mRNAs that are polyadenylated at the E-region poly(A) site and also in a putative L2 mRNA. Three distinct species, two of which are derived from the upstream regulatory region promoter, have the potential to encode the L1 protein; the third species also contains the entire coding region of the E1i--E4 protein 5' to the L1 ORF. Both the E1i--E4 mRNA and the potentially bicistronic L1 mRNA are derived from a promoter located in the E7 ORF. We uncovered no evidence of alternatively spliced mRNAs that could account for the multiple, abundant E4 proteins in plantar warts, suggesting that posttranslational modification is mainly responsible for the observed protein heterogeneity.
The mammalian upstream element factor recognizes two sites in the adenovirus type 2 IVa2-major late promoter intergenic region and stimulates both promoters
Authors: Moncollin V , Kempf A , Egly J M
Published: January 2020

The adenovirus type 2 major late upstream element factor (UEF) recognizes two similar elements that lie between the major late promoter (MLP) and IVa2 promoter cap sites (the previously characterized MLP-UE from nucleotides -49 to -67 and the IVa2-UE from nucleotides -98 to -122). DNase I footprinting and gel retention assays showed that the UEF has a lower affinity for the IVa2-UE than for the MLP-UE. In vitro transcription experiments demonstrated first that the IVa2 promoter, which lacks a consensus TATA box, may work, as does the MLP, in the absence of its proximal upstream element and second that the IVa2-UE stimulated IVa2 transcription two- to threefold, as MLP-UE did for the MLP. In addition, we demonstrated that the more distal upstream element has a weak stimulatory effect on transcription of both promoters.
Virus-lymphocyte interactions: inductive signals necessary to render B lymphocytes susceptible to vesicular stomatitis virus infection
Authors: Schmidt M R , Woodland R T
Published: January 2020

We examined the inductive signals necessary to render B lymphocytes capable of supporting a productive vesicular stomatitis virus infection. Small murine splenic B cells in the G0 phase of the cell cycle were cultured with stimulators which allow progression through various stages in the activation and/or differentiation pathway leading to antibody secretion. We found that vesicular stomatitis virus expression is dependent on the state of B-cell activation and that three distinct phases can be defined. A nonsupportive state, which is defined by the failure to produce infection centers, viral proteins, or PFUs, is characteristic of freshly isolated small B cells, B cells cultured 48 h without further stimulation, or B cells in the G1 phase of the cell cycle induced by culture with T-cell-derived lymphokines. This refractory state was not due to a failure of virus uptake. Activation of G0 B cells with anti-immunoglobulin at doses which allow entry into the S phase rendered them capable of synthesizing viral proteins and increased the number of B cells producing infection centers, without enhancing PFU production on a per cell basis. In contrast, B cells stimulated with multiple inductive signals provided by anti-immunoglobulin and lymphokines showed increased infectious particle production (7 PFU per infection center). Lipopolysaccharide stimulation, acting through another induction pathway, caused the maximum increase in the number of infected B cells and production of infectious particles (25 PFU per infection center).
Organ tropism of Sendai virus in mice: proteolytic activation of the fusion glycoprotein in mouse organs and budding site at the bronchial epithelium
Authors: Tashiro M , Yamakawa M , Tobita K , Klenk H D , Rott R , Seto J T
Published: January 2020

Wild-type Sendai virus is exclusively pneumotropic in mice, while a host range mutant, F1-R, is pantropic. The latter was attributed to structural changes in the fusion (F) glycoprotein, which was cleaved by ubiquitous proteases present in many organs (M. Tashiro, E. Pritzer, M. A. Khoshnan, M. Yamakawa, K. Kuroda, H.-D. Klenk, R. Rott, and J. T. Seto, Virology 165:577-583, 1988). These studies were extended by investigating, by use of an organ block culture system of mice, whether differences exist in the susceptibility of the lung and the other organs to the viruses and in proteolytic activation of the F protein of the viruses. Block cultures of mouse organs were shown to synthesize the viral polypeptides and to support productive infections by the viruses. These findings ruled out the possibility that pneumotropism of wild-type virus results because only the respiratory organs are susceptible to the virus. Progeny virus of F1-R was produced in the activated form as shown by infectivity assays and proteolytic cleavage of the F protein in the infected organ cultures. On the other hand, much of wild-type virus produced in cultures of organs other than lung remained nonactivated. The findings indicate that the F protein of wild-type virus was poorly activated by ubiquitous proteases which efficiently activated the F protein of F1-R. Thus, the activating protease for wild-type F protein is present only in the respiratory organs. These results, taken together with a comparison of the predicted amino acid substitutions between the viruses, strongly suggest that the different efficiencies among mouse organs in the proteolytic activation of F protein must be the primary determinant for organ tropism of Sendai virus. Additionally, immunoelectron microscopic examination of the mouse bronchus indicated that the budding site of wild-type virus was restricted to the apical domain of the epithelium, whereas budding by F1-R occurred at the apical and basal domains. Bipolar budding was also observed in MDCK monolayers infected with F1-R. The differential budding site at the primary target of infection may be an additional determinant for organ tropism of Sendai virus in mice.
Emergence of viruses resistant to neutralization by V3-specific antibodies in experimental human immunodeficiency virus type 1 IIIB infection of chimpanzees
Authors: Nara P L , Smit L , Dunlop N , Hatch W , Merges M , Waters D , Kelliher J , Gallo R C , Fischinger P J , Goudsmit J
Published: January 2020

Emergence in two chimpanzees of human immunodeficiency virus type 1 (HIV-1) IIIB variants resistant to neutralization by the preexisting antibody is described. Viruses isolated from the HIV-1 IIIB gp120-vaccinated and -challenged animal were more resistant to neutralization by the chimpanzee's own serum than viruses isolated from the naive infected animal, indicating immune pressure as the selective mechanism. However, all reisolated viruses were 16- to 256-fold more neutralization resistant than the inoculum virus to antibodies binding to the third variable domain (V3) of the HIV-1 external envelope. Early chimpanzee serum samples that neutralized the inoculum strain but not the reisolated viruses were found to bind an HIV-1 IIIB common nonapeptide (IQRGPGRAF) derived from the gp120 isolate-specific V3 domain shown to induce isolate-specific neutralization in other animals. Amplification of the V3 coding sequence by polymerase chain reaction and subsequent sequence analysis of the neutralization-resistant variants obtained from in vivo-infected animals indicated that early resistance to neutralization by an HIV-1 IIIB monoclonal antibody (0.5 beta) was conferred by changes outside the direct binding site for the selective neutralizing antibody. The reisolated neutralization-resistant isolates consisted of the lower-replication-competent virus subpopulations of the HIV-1 IIIB stock, as confirmed by biological and sequence analyses. In vitro passage of the HIV-1 IIIB stock through chimpanzee and human peripheral blood mononuclear cell cultures void of HIV-specific antibody resulted in homogenic amplification of the more-replication-competent subpopulation preexisting in the original viral stock, suggesting a role for the immune system in suppressing the more-replication-competent viruses.
At least two types of control regions can be found among naturally occurring BK virus strains
Authors: Sundsfjord A , Johansen T , Flaegstad T , Moens U , Villand P , Subramani S , Traavik T
Published: January 2020

The various strains of BK virus (BKV) exhibit a remarkable degree of heterogeneity in the transcriptional control region, which may affect the biological characteristics of a BKV strain. We describe the detection and sequencing of BKV control regions directly from urine samples and after propagation in cell culture. A BKV strain [BKV (TU)] with a control region anatomy not described earlier, as well as a BKV (WW)-like strain [BKV (WWT)], was detected in urine samples by direct sequencing of polymerase chain reaction products. Urine inocula containing BKV (WWT) yielded BKV (TU) upon one passage in cell culture, while BKV (TU) did not change its control region during propagation in cell culture. Analysis of the nucleotide sequence of the transcriptional control regions revealed a partial deletion and duplication in BKV (TU) compared with BKV (WWT). In addition, the control region of BKV (TU) contains two point mutations relative to BKV (WWT). This indicates that both virus strains were probably present in the BKV (WWT)-dominated urine inocula, rather than that BKV (WWT) genomes were rearranged into BKV (TU) genomes during cell propagation. The heterogeneity of the control region of BKV strains is discussed in relation to both confirmed and putative transcription factor-binding sites.
trans-dominant inhibition of herpes simplex virus transcriptional regulatory protein ICP4 by heterodimer formation
Authors: Shepard A A , Tolentino P , DeLuca N A
Published: January 2020

Herpes simplex virus encodes a 175-kilodalton immediate-early transactivating protein referred to as ICP4. A mutant ICP4 molecule expressed from a stable transformed cell line lacks the sequences required for transactivation yet retains the ability to specifically associate with DNA and to form homodimers. Expression of the mutant ICP4 peptide from this cell line, designated X25, resulted in the inhibition of herpes simplex virus growth. Wild-type ICP4 homodimers were depleted in X25-infected cells by the formation of heterodimers containing the wild-type ICP4 molecule and the mutant peptide. While the ICP4 heterodimer retained DNA-binding activity, immunological studies suggest that the wild-type subunit of the heterodimer is conformationally altered in a region that serves as the antigenic epitope. Physical studies that determined the composition of the heterodimer and its native size and approximate shape support this observation. The structural change is in a region of ICP4 genetically implicated as important for transactivation and may result in an alteration in an interaction between ICP4 and a target protein essential to promote transcriptional activation. Sequestering wild-type monomers of a viral regulatory protein into heterodimers which are less proficient in transactivation may explain the dominant inhibitory activity of the X25 cells, resulting in attenuation of viral growth.
The E2 transactivator of bovine papillomavirus type 1 is expressed from multiple promoters
Authors: Vaillancourt P , Nottoli T , Choe J , Botchan M R
Published: January 2020

The E2 proteins of bovine papillomavirus type 1 (BPV-1) are a family of site-specific DNA-binding proteins which regulate viral transcription by repression and activation. Repressors E2-TR and E8/E2 are expressed from promoters P5 (P3080) and P3 (P890), respectively. Previous reports have provided evidence that the transcript for the 48-kilodalton transactivator is initiated from a promoter proximal to the open reading frame encoding this protein (P2440 or P4). Our studies extend these findings and show that the E2 transactivation gene is expressed from multiple promoters. We have described the isolation of a cDNA (N15-2) which represents an RNA species expressed from the P3 promoter. The major exon of this species was produced by splicing to an acceptor located at nucleotide 2558 and contained the complete E2 open reading frame. The acceptor is probably utilized by yet another more abundant mRNA expressed from the P2 promoter (A. Stenlund, J. Zabielski, H. Ahola, J. Moreno-Lopez, and U. Pettersson, J. Mol. Biol. 182:541-554, 1985). Linked to a surrogate promoter, the N15-2 cDNA can transactivate an E2-responsive reporter gene. BPV-1 plasmids containing mutations either in the 2558 splice acceptor or in the P4 promoter showed significantly reduced transforming ability and reduced ability to transactivate an E2-responsive reporter, while a double mutant was inactive in both assays. The transformation defect was complemented by an E2 expression vector, and the BPV genome absolutely required the E2 protein to transactivate in the second assay. Thus, these genetic experiments show that alternate modes of E2 expression contribute to the E2 mRNA pool. Direct analysis of cytoplasmic RNA from transformed cultured cells proves that transcripts containing the 2558 acceptor exon are approximately as abundant as the P4 type E2 mRNAs. Furthermore, analysis of the E2 proteins present in various cell lines harboring specific BPV-1 mutants, including the 2558 acceptor mutant, proves that alternate modes of E2 expression exist. The ability of the E2 activator and repressors to each be independently expressed from multiple E2-responsive promoters probably adds to the resiliency of the latent virus as a plasmid and may be important for its homeostasis within the cell in different environmental or developmental situations.
Mutational analysis of a native substrate of the human immunodeficiency virus type 1 proteinase
Authors: Partin K , Kräusslich H G , Ehrlich L , Wimmer E , Carter C
Published: January 2020

Proteolytic processing of the gag/pol precursor by the human immunodeficiency virus type 1 proteinase is essential for the production of infectious viral particles. Although the sites of virus-specific cleavages have been determined, the primary amino acid sequences surrounding these sites are heterogeneous and the determinants that direct the cleavage specificity exhibited by human immunodeficiency virus type 1 proteinase remain largely undefined. We performed mutational analysis of the Tyr/Pro site, which produces the amino terminus of the viral capsid protein, and the Phe/Pro site, which produces the amino terminus of the proteinase. Mutations were made in a clone encoding a frameshift mutation that results in the expression of equimolar amounts of the substrate and proteinase in the form of a truncated gag/pol precursor. After single-amino-acid substitutions were made, their effects on proteolytic processing were examined by in vitro transcription and in vitro translation of the synthetic mRNA; translation products were then processed by exogenously added purified proteinase. Single-amino-acid substitutions yielded both substrates which were processed with wild-type efficiency and substrates on which processing was impaired. At the Tyr/Pro site in gag, processing was severely inhibited by substitutions within the P4, P2, P1, and P2' positions. The Phe/Pro site in pol, however, demonstrated far greater tolerance to amino acid substitution. These data suggest that the primary amino acid sequence around a scissile bond is more critical for cleavage of the Tyr/Pro site than the Phe/Pro site.
Infection of the human monocytic cell line Mono Mac6 with human immunodeficiency virus types 1 and 2 results in long-term production of virus variants with increased cytopathogenicity for CD4+ T cells
Authors: L'age-Stehr J , Niedrig M , Gelderblom H R , Sim-Brandenburg J W , Urban-Schriefer M , Rieber E P , Haas J G , Riethmüller G , Ziegler-Heitbrock H W
Published: January 2020

The recently established human monocytic cell line Mono Mac6 expressing distinct characteristics of mature monocytes/macrophages was tested for its susceptibility to infection with human immunodeficiency virus. Inoculation of the cells with the T-cell-tropic human immunodeficiency virus strains human T-lymphotropic virus type IIIB and lymphadenopathy-associated virus type 2 led to a noncytopathic productive infection becoming apparent only after a latency period of up to 56 days. The infectibility of the Mono Mac6 cells was dependent on low levels of CD4 expression, as demonstrated by blocking experiments with various CD4-specific antibodies. Increasing with time after infection (greater than 200 days), the cultured Mono Mac6 cells released virus variants which showed shortened latency periods when passaged onto uninfected Mono Mac6 cells. Also, cytopathogenicity for several CD4+ T cells of the Mono Mac6-derived virus was drastically increased; thus, the infection of the H9 cell line with low doses of virus (less than 0.1 50% tissue culture infective dose per cell) led to giant syncytium formation within 1 day and subsequent death of all fused cells. We propose Mono Mac6 cells as a new model for the study of human immunodeficiency virus infecting the monocyte/macrophage lineage, particularly with regard to virus-host cell interaction and the influence of cell differentiation and activation on latency and development of virulence. The human immunodeficiency virus-infected Mono Mac6 cell may also serve as a valuable tool for in vitro testing of antiviral therapies.
Human T-cell leukemia virus type I trans activator induces class I major histocompatibility complex antigen expression in glial cells
Authors: Sawada M , Suzumura A , Yoshida M , Marunouchi T
Published: January 2020

Transfection of the tax gene encoding the trans activator of human T-cell leukemia virus type I into glial line cells induced class I major histocompatibility complex (MHC) antigens on these cells. This occurred through the interaction of tax protein with the gene encoding class I MHC antigens but not through any soluble factors, such as interferons, or factors from glial cells. Since neural cells do not usually express MHC antigens, this novel mechanism may be an intermediate event between viral infection and subsequent immune-mediated pathology in the central nervous system.
Human immunodeficiency virus type 1 cellular host range, replication, and cytopathicity are linked to the envelope region of the viral genome
Authors: York-Higgins D , Cheng-Mayer C , Bauer D , Levy J A , Dina D
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) isolates vary in their in vitro biologic characteristics such as cellular host range, replication kinetics, and cytopathicity. In this study, we molecularly exchanged equivalent regions between two cloned HIV-1 isolates with differing replicative and cytopathic properties. To facilitate generation of recombinant viruses, we used a method involving cotransfection of human monolayer cells with plasmid constructs containing half of the biologically active viral genome. The two halves of the genome were subsequently ligated by intracellular processes to form the complete proviral genome. This method simplifies plasmid construction, since new infectious virus particles can be produced easily from the individual constructs that are correctly ligated in vivo. Results obtained by using recombinant viruses generated in this manner indicate that the ability of HIV to replicate in specific cell types and cytopathicity segregate with the env region of the viral genome.
Polypeptide specificity of antiviral serum antibodies in children naturally infected with human rotavirus
Authors: Brüssow H , Offit P A , Gerna G , Bruttin A , Sidoti J
Published: January 2020

Reassortants between serotype 3 SA11 and serotype 6 NCDV rotaviruses were used to determine the relative amounts of serum-neutralizing antibody to VP4 and VP7 of serotype 3 SA11 rotavirus in children after natural rotavirus exposure. Sera from Ecuadorian children of a population-based study and sera from children of a hospital-based study in Germany (excluding diarrhea patients) demonstrated high titers of VP7-specific but only low titers of VP4-specific antibodies. In contrast, paired sera from German children hospitalized with a symptomatic primary rotavirus gastroenteritis demonstrated a titer increase to VP4 more frequently than to VP7 protein by neutralization test and immunoblotting. For these rotavirus patients, we provided, previously, direct evidence for the development of cross-neutralizing antibodies.
RNA editing by G-nucleotide insertion in mumps virus P-gene mRNA transcripts
Authors: Paterson R G , Lamb R A
Published: January 2020

A guanine nucleotide insertion event has been shown to occur at a specific site within mumps virus P-gene mRNA transcripts. The region of the mRNA containing the site expected to be used for RNA editing and the complementary portion of the genomic RNA were cloned, and their nucleotide sequences were obtained. The genomic RNA was found to possess six C residues at the insertion site, whereas 63% of the P-gene-specific mRNA transcripts were found to have from two to five G residues inserted at this position in the RNA. An unedited mRNA was shown to encode the mumps virus cysteine-rich protein V, and mRNA transcripts containing two and four inserted G residues were translated to yield the mumps virus P and I proteins, respectively.
Defined mutations in the 5' nontranslated sequence of Sindbis virus RNA
Authors: Niesters H G , Strauss J H
Published: January 2020

We have constructed 24 deletion mutants which contain deletions of from 1 to 15 nucleotides in the 5' nontranslated region of Sindbis virus RNA and tested the effect of these mutations on virus replication. The results showed that the first 44 nucleotides, which are capable of forming a hairpin structure, are important for virus replication, as all deletions tested in this region were either lethal or resulted in virus that grew poorly in comparison to the parental virus. Many of these deletions had different effects in mosquito cells than in chicken cells, suggesting that cellular factors, presumably proteins, bind to this region. This domain may function in at least two processes in viral replication. It seems likely that in the minus strand, this sequence element is bound by the viral replicase and promotes RNA replication. In the plus strand, this element may modulate initiation of translation of the nonstructural proteins. The results suggest that the hairpin structure itself is important. All deletions within it had deleterious effects on virus replication, and in particular, deletion of one of the G residues at nucleotide 7 or 8 or of one of the C residues at nucleotide 36 or 37 which are theoretically base-paired with these G's resulted in temperature-sensitive viruses that behaved very similarly. In contrast, large deletions between the 44-nucleotide hairpin and the translation start site at nucleotides 60 to 62 resulted in virus that grew as well as or better than the parental virus in both chicken and mosquito cells. The A residue at position 5 of the HRSP strain used was examined in more detail. Deletion of this A was lethal, whereas substitution by G resulted in a virus that grew poorly, despite the fact that G is present at position 5 in the AR339 parent of HRSP. U at position 5 resulted in a virus that grew less well than the A5 strain but better than the G5 mutant.
Induction of a neutralizing immune response to human respiratory syncytial virus with anti-idiotypic antibodies
Authors: Palomo C , Albar J P , García-Barreno B , Melero J A
Published: January 2020

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against monoclonal antibodies that recognized either F glycoprotein 47F or G glycoprotein 63G, 62G, or 74G of the human respiratory syncytial virus Long strain. Anti-Id sera inhibited the virus binding of the immunizing monoclonal antibodies and in some cases the binding of other antibodies reacting with overlapping epitopes. The anti-Id sera also inhibited virus neutralization mediated by the original monoclonal antibodies. Affinity purified anti-Id antibodies were subsequently used to raise a homologous anti-anti-Id response in rabbits. One of the rabbits, inoculated with anti-Id 63G, generated antibodies that reacted with the G protein of respiratory syncytial virus and neutralized the virus to high titers. The antiviral antibodies induced by anti-Id 63G were broadly cross-reactive with strains of the A and B subtypes. However, the specificities of monoclonal antibody 63G and anti-anti-Id 63G were not exactly the same, as indicated by their reaction with escape mutants to antibody 63G. These results demonstrate for the first time the induction of an anti-respiratory syncytial virus response by anti-Id antibodies.
Cytolytic T-lymphocyte responses to respiratory syncytial virus: effector cell phenotype and target proteins
Authors: Nicholas J A , Rubino K L , Levely M E , Adams E G , Collins P L
Published: January 2020

Cytolytic T-lymphocyte (CTL) activity specific for respiratory syncytial (RS) virus was investigated after intranasal infection of mice with RS virus, after intraperitoneal infection of mice with a recombinant vaccinia virus expressing the F glycoprotein, and after intramuscular vaccination of mice with Formalin-inactivated RS virus or a chimeric glycoprotein, FG, expressed from a recombinant baculovirus. Spleen cell cultures from mice previously infected with live RS virus or the F-protein recombinant vaccinia virus had significant CTL activity after one cycle of in vitro restimulation with RS virus, and lytic activity was derived from a major histocompatibility complex-restricted, Lyt2.2+ (CD8+) subset. CTL activity was not restimulated in spleen cells from mice that received either the Formalin-inactivated RS virus or the purified glycoprotein, FG. The protein target structures for recognition by murine CD8+ CTL were identified by using target cells infected with recombinant vaccinia viruses that individually express seven structural proteins of RS virus. Quantitation of cytolytic activity against cells expressing each target structure suggested that 22K was the major target protein for CD8+ CTL, equivalent to recognition of cells infected with RS virus, followed by intermediate recognition of F or N, slight recognition of P, and no recognition of G, SH, or M. Repeated stimulation of murine CTL with RS virus resulted in outgrowth of CD4+ CTL which, over time, became the exclusive subset in culture. Murine CD4+ CTL were highly cytolytic for RS virus-infected cells, but they did not recognize target cells infected with any of the recombinant vaccinia viruses expressing the seven RS virus structural proteins. Finally, the CTL response in peripheral blood mononuclear cells of adult human volunteers was investigated. The detection of significant levels of RS virus-specific cytolytic activity in these cells was dependent on at least two restimulations with RS virus in vitro, and cytolytic activity was derived primarily from the CD4+ subset.
Quantitative polymerase chain reaction analysis of herpes simplex virus DNA in ganglia of mice infected with replication-incompetent mutants
Authors: Katz J P , Bodin E T , Coen D M
Published: January 2020

To study the roles of viral genes in the establishment and maintenance of herpes simplex virus (HSV) latency, we have developed a polymerase chain reaction assay that is both quantitative and sensitive. Using this assay, we analyzed the levels of viral DNA in trigeminal ganglia of mice inoculated corneally with HSV mutants that are defective for virus replication at one or more sites in mice and for reactivation upon ganglionic explant. Ganglia from mice infected with thymidine kinase-negative mutants, which replicate at the site of inoculation and establish latency but do not replicate acutely in ganglia or reactivate upon explant, contained a range of levels of HSV DNA that overlapped with the range found in ganglia latently infected with wild-type virus. On average, these mutant-infected ganglia contained one copy of HSV DNA per 100 cell equivalents (ca. 10(4) molecules), which was 50-fold less than the average for wild-type virus. Ganglia from mice infected with a ribonucleotide reductase deletion mutant, which is defective for acute replication and reactivation upon ganglionic explant, also contained on average one copy of HSV DNA per 100 cell equivalents. We also detected substantial numbers of HSV DNA molecules (up to ca. 10(3] in ganglia of mice infected with an ICP4 deletion mutant and other replication-negative mutants that are severely impaired for viral DNA replication and gene expression. These results raise the possibility that such mutants can establish latency, which could have important implications for mechanisms of latency and for vaccine and antiviral drug development.
Direct and cytokine-mediated activation of protein kinase C induces human immunodeficiency virus expression in chronically infected promonocytic cells
Authors: Kinter A L , Poli G , Maury W , Folks T M , Fauci A S
Published: January 2020

The chronically infected promonocytic clone U1 expresses low-to-undetectable constitutive levels of human immunodeficiency virus (HIV). Virus replication in these cells can be increased up to 25-fold by phorbol esters (phorbol-12-myristate-13-acetate), recombinant cytokines such as tumor necrosis factor-alpha, and cytokine-enriched mononuclear cell supernatants. We have tested specific activators of protein kinases (PK) and PK inhibitors (isoquinolinesulfonamide derivatives), as well as calcium-mobilizing agents, for their effect on constitutive and induced virus expression in U1 cells. Virus expression was measured by reverse transcriptase, Western blot, and nuclear run-on analysis. Activation of PKC by 1-oleyl,2-acetylglycerol, a synthetic analog of the natural ligand 1,2-diacylglycerol, and bryostatin 1 (a recently described specific PKC activator) resulted in a two- to eightfold increase in virus production. In contrast, activators of cyclic-nucleotide-dependent PKs were not effective in inducing virus expression. PK inhibitors were tested for their effect on HIV upregulation by cytokines and other inducing agents. The isoquinolinesulfonamide derivative H7, a potent inhibitor of PKC activation, effectively blocked (70 to 90%) HIV induction by cytokines and phorbol-12-myristate-13-acetate. The derivative HA1004, which is more selective for cyclic-nucleotide-dependent kinases, did not suppress viral induction. In addition, increases in intracellular calcium levels dramatically enhanced HIV production induced by both specific PKC activators and cytokines. These results indicate that activation of PKC is a common pathway involved in the upregulation of HIV expression in chronically infected cells stimulated by cytokines and other inducing agents.
Structural role of the matrix protein of type D retroviruses in gag polyprotein stability and capsid assembly
Authors: Rhee S S , Hunter E
Published: January 2020

To obtain a better understanding of the role of the gag gene-encoded matrix (MA) protein in the assembly and maturation of type D retroviruses, we have made five mutants with specific in-frame deletions within the p10-coding region by the use of oligonucleotide-directed mutagenesis. The changes in the Gag polyprotein made by these mutations resulted in almost identical phenotypes. In cells expressing mutant genomes, the mutant Gag polyproteins were synthesized and modified with myristic acid in a normal manner. However, they were so unstable that the bulk of the newly synthesized polyproteins was degraded within 1 h without being processed into mature structural polypeptides. In contrast, wild-type polyproteins have a processing half-life of 3.0 to 3.5 h. The mutant Gag polyproteins were assembled with very low efficiency into capsids in the cytoplasm of the mutant-infected cells. Moreover, the few capsids that formed were neither released from nor accumulated in the cells. These results suggest that the matrix protein plays an important role in guiding the correct folding of the Gag polyprotein, which is presumably crucial for both stabilizing the molecule and facilitating the intermolecular interactions that occur during assembly of immature capsids.
AGM1+ spleen cells contain gamma interferon (IFN-gamma) gene transcripts in the early, sex-dependent production of IFN-gamma after picornavirus infection
Authors: McFarland H I , Bigley N J
Published: January 2020

Encephalomyocarditis D variant (EMCV-D)-infected spleen cell cultures prepared from diabetes-resistant ICR Swiss female mice produce more gamma interferon (IFN-gamma) activity over a 24-h period than do spleen cell cultures from diabetes-susceptible male mice of this strain. Pretreatment of mice with anti-asialo GM1 eliminates early in vitro IFN-gamma production from 4 to 16 h postinfection (p.i.) and reduces IFN-gamma production from 16 to 24 h p.i. In this study, depletion of spleen cells with anti-Thy-1 by panning greatly reduced IFN-gamma activity in EMCV-D-infected spleen cell cultures throughout a 24-h period. Populations of asialo GM1 (AGM1), L3T4, and Lyt-2-positive cells were isolated from cells harvested at 9 h p.i. from EMCV-D-infected spleen cell cultures by a modified panning technique on polystyrene microscope slides. By in situ hybridization with a [35S]dATP-labeled IFN-gamma cDNA probe, only the AGM1-bearing cells were found to contain detectable IFN-gamma gene transcripts. An AGM1+, Thy-1+ natural killer-like cell is the probable producer of the early, sex-dependent IFN-gamma activity in this system.
Myristylated polyomavirus VP2: role in the life cycle of the virus
Authors: Krauzewicz N , Streuli C H , Stuart-Smith N , Jones M D , Wallace S , Griffin B E
Published: January 2020

The double-stranded genome of the small DNA tumor virus, polyomavirus, is enclosed in a capsid composed of a major protein, VP1, which associates as pentameric capsomeres into an icosahedral structure, and two minor proteins, VP2 and VP3, whose functions and positions within the structure are unknown. The N-terminal glycine of the VP2 coat protein has been shown to be cotranslationally acylated with myristic acid. To study the function of this modification and the role of VP2 in the life cycle of polyomavirus, the N-terminal glycine, critical to the myristylation consensus sequence, has been altered to a glutamic acid or a valine residue by site-directed oligonucleotide mutagenesis. The glycine----glutamic acid mutant DNA has been further studied. When transfected into cells permissive for the polyomavirus full lytic life cycle, this mutant DNA replicated at levels comparable to those of wild-type viral DNA, and small amounts of nonrevertant (mutant) virus could be harvested from the cultures. The virus particles viewed by electron microscopy appeared slightly distorted, but the ratio of full to empty particles was similar to that produced in a wild-type viral infection. Mutant virus was capable of reinfecting permissive cells but with a considerably reduced efficiency.