bluuebunny/crossref_metadata_2025 huggingface dataset:
Fine mapping of transcripts expressed from the US6 gene family of human cytomegalovirus strain AD169
Authors: Jones T R , Muzithras V P
Published: January 2020

By sequence analysis (K. Weston and B. G. Barrell, J. Mol. Biol. 192:177-208, 1986), the human cytomegalovirus (HCMV) strain AD169 HindIII X DNA fragment contains six open reading frames (US6 through US11; called the US6 family) which may encode glycoproteins. Sense transcripts from the US6 family were mapped. The kinetics of appearance of steady-state cytoplasmic RNA was different for each transcription unit. The 1.5-kb US11-US10 and the 1.7-kb US9-US8 transcripts belonged to the early kinetic class. The former reached peak abundance by 8 h postinfection, while the latter peaked at 24 h postinfection. These RNAs greatly decreased in abundance by 48 to 72 h after infection, unlike transcripts from other HCMV early transcription units reported previously. US6 and US7 messages were most abundant at late times postinfection. US6 transcripts utilized different initiation sites at early or late times postinfection. There was evidence for both spliced and unspliced messages from this family. In a transient expression assay, chimeric plasmids containing the regions upstream of the mapped transcription initiation sites were active in promoting indicator gene expression in HCMV-infected, but not uninfected, human foreskin fibroblast cells.
Antigenic and genetic variation in cytopathic hepatitis A virus variants arising during persistent infection: evidence for genetic recombination
Authors: Lemon S M , Murphy P C , Shields P A , Ping L H , Feinstone S M , Cromeans T , Jansen R W
Published: January 2020

Variants of hepatitis A virus (pHM175 virus) recovered from persistently infected green monkey kidney (BS-C-1) cells induced a cytopathic effect during serial passage in BS-C-1 or fetal rhesus kidney (FRhK-4) cells. Epitope-specific radioimmunofocus assays showed that this virus comprised two virion populations, one with altered antigenicity including neutralization resistance to monoclonal antibody K24F2, and the other with normal antigenic characteristics. Replication of the antigenic variant was favored over that of virus with the normal antigenic phenotype during persistent infection, while virus with the normal antigenic phenotype was selected during serial passage. Viruses of each type were clonally isolated; both were cytopathic in cell cultures and displayed a rapid replication phenotype when compared with the noncytopathic passage 16 (p16) HM175 virus which was used to establish the original persistent infection. The two cytopathic virus clones contained 31 and 34 nucleotide changes from the sequence of p16 HM175. Both shared a common 5' sequence (bases 30 to 1677), as well as sequence identity in the P2-P3 region (bases 3249 to 5303 and 6462 to 6781) and 3' terminus (bases 7272 to 7478). VP3, VP1, and 3Cpro contained different mutations in the two virus clones, with amino acid substitutions at residues 70 of VP3 and 197 and 276 of VP1 of the antigenic variant. These capsid mutations did not affect virion thermal stability. A comparison of the nearly complete genomic sequences of three clonally isolated cytopathic variants was suggestive of genetic recombination between these viruses during persistent infection and indicated that mutations in both 5' and 3' nontranslated regions and in the nonstructural proteins 2A, 2B, 2C, 3A, and 3Dpol may be related to the cytopathic phenotype.
Protein phosphatase 2A dephosphorylates simian virus 40 large T antigen specifically at residues involved in regulation of DNA-binding activity
Authors: Scheidtmann K H , Virshup D M , Kelly T J
Published: January 2020

Treatment of purified simian virus 40 large T antigen (LT) with protein phosphatase 2A stimulates LT-dependent DNA unwinding and replication (D. M. Virshup, M. G. Kauffman, and T. J. Kelly, EMBO J. 8: 3891-3898, 1989). The specificity of the catalytic subunit of protein phosphatase 2A toward LT was investigated by two-dimensional peptide mapping. Increasing amounts of phosphatase sequentially removed the phosphates from serine residues 120, 123, 677, and perhaps 679, residues which have been implicated in regulating the DNA-binding activity of LT.
Defective retroviruses can disperse in the human genome by intracellular transposition
Authors: Tchenio T , Heidmann T
Published: January 2020

Using an assay for retrotransposition detection (T. Heidmann, O. Heidmann, and J. F. Nicolas, Proc. Natl. Acad. Sci. USA 85:2219-2223, 1988), we demonstrated that a defective retrovirus deleted for the gag, pol, and env open reading frames can disperse in the genome of human HeLa cells by intracellular transposition, at a frequency close to 10(-6) events per cell per generation. Transposition requires cooperation in trans for the gag and pol gene products and may be associated with the release of low amounts of noninfectious retroviruslike particles which are the hallmarks but not the intermediates of this transposition process. Similar events could account for the dispersion at high copy number of some of the human endogenous sequences related to retroviruses and for the occurrence of noninfectious retroviruslike particles in human placenta and several tumor cell lines (reviewed by E. Larsson, N. Kato, and M. Cohen, Curr. Top. Microbiol, Immunol, 148:115-132, 1989).
Human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein
Authors: Helseth E , Olshevsky U , Furman C , Sodroski J
Published: January 2020

Insertion of four amino acids into various locations within the amino-terminal halves of the human immunodeficiency virus type 1 gp120 or gp41 envelope glycoprotein disrupts the noncovalent association of these two envelope subunits (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. A. Haseltine, and J. Sodroski, Science 237:1351-1355, 1987). To localize the determinants on the gp120 envelope glycoprotein important for subunit association, amino acids conserved among primate immunodeficiency viruses were changed. Substitution mutations affecting either of two highly conserved regions located at the amino (residues 36 to 45) and carboxyl (residues 491 to 501) ends of the mature gp120 molecule resulted in nearly complete dissociation of the envelope glycoprotein subunits. Partial dissociation phenotypes were observed for some changes affecting residues in the third and fourth conserved gp120 regions. These results suggest that hydrophobic regions at both ends of the gp120 glycoprotein contribute to noncovalent association with the gp41 transmembrane glycoprotein.
Isolation from African Sykes' monkeys (Cercopithecus mitis) of a lentivirus related to human and simian immunodeficiency viruses
Authors: Emau P , McClure H M , Isahakia M , Else J G , Fultz P N
Published: January 2020

Analysis of serum samples from 100 wild-caught or colony-born Sykes' monkeys (Cercopithecus mitis) in Kenya revealed that 59 animals had antibodies cross-reactive to human immunodeficiency virus type 2 (HIV-2) and to simian immunodeficiency viruses (SIVs). A lentivirus, designated SIVsyk, was isolated from five of six seropositive asymptomatic Sykes' monkeys, but in four cases isolation was possible only after depletion of CD8+ lymphocytes and cocultivation of the CD4(+)-enriched cell population with peripheral blood mononuclear cells from seronegative Sykes' monkeys. SIVsyk resembled other SIVs and HIVs morphologically, had an Mg2(+)-dependent reverse transcriptase enzyme, and replicated in and was cytopathic for CEMx174 and Sup-T1 cells. SIVsyk differred substantially from other SIVs, however, in that it failed to replicate in normal human, mangabey, and macaque peripheral blood mononuclear cells and serum from seropositive Sykes' monkeys immunoprecipitated env antigens from HIV-1 as well as from HIV-2, SIVsmm, and SIVagm. These data demonstrate a high prevalence of natural infection in Sykes' monkeys in Kenya with a lentivirus that appears to be unique with respect to its host range and antigenic cross-reactivity.
Functional similarities between human immunodeficiency virus type 1 and simian virus 40 kappa B proto-enhancers
Authors: Phares W , Herr W
Published: January 2020

To search for broadly active enhancer elements within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we have used a proto-enhancer amplification assay. In this assay, the enhancer region of simian virus 40 (SV40) is replaced by heterologous regulatory sequences. Upon passage in African green monkey kidney cells. SV40 growth revertants can arise by amplification (usually duplication) of active protoenhancers within the heterologous sequences. Most of the HIV-1 U3 regulatory sequences were assayed; only amplification of one or both of the HIV-1 enhancer core kappa B motifs consistently resulted in viable SV40 virus. Examination of the cell-specific enhancer activity of the individual HIV-1 kappa B proto-enhancers showed that, like the broadly active SV40 kappa B proto-enhancer (C proto-enhancer), they are all active in noninduced cell lines of either lymphoid (H9 and Jurkat) or nonlymphoid (HeLa and CV-1) origin. Unexpectedly, one of three kappa B point mutants that exhibit little or no activity in unstimulated cells is as highly induced in stimulated Jurkat cells as are the wild-type kappa B proto-enhancers. This point mutation shows that kappa B-related proto-enhancers can display markedly different activation properties in unstimulated cells yet still activate transcription to similar levels in stimulated cells.
Human immunodeficiency virus (HIV)-infected tumor xenografts as an in vivo model for antiviral therapy: role of alpha/beta interferon in restriction of tumor growth in nude mice injected with HIV-infected U937 tumor cells
Authors: Puddu P , Locardi C , Sestili P , Varano F , Petrini C , Modesti A , Masuelli L , Gresser I , Belardelli F
Published: January 2020

The host factors involved in the restriction of tumor growth were studied in nude mice transplanted with a cloned line of chronically human immunodeficiency virus (HIV)-infected U937 cells. HIV-infected and uninfected U937 cells exhibited the same growth patterns in culture. However, HIV-infected cells were not tumorigenic when injected subcutaneously in nude mice, whereas large solid tumors were observed in mice injected with uninfected U937 cells. Injection of nude mice with antibody to alpha/beta interferon (IFN-alpha/beta) enabled HIV-infected U937 cells to grow progressively in approximately 90 to 100% of mice. HIV-infected U937 cells formed solid tumors in the majority (60 to 90%) of either immunosuppressed (splenectomized, irradiated, and anti-asialo-GM1-treated) or genetically immunodeficient (bg/nu/xid) nude mice. In mice treated with antibodies to IFN-alpha/beta with established HIV-positive tumors, a direct correlation was found between p24 antigenemia and tumor size. Treatment of established HIV-positive U937 cell tumors with human IFN-alpha or mouse IFN-alpha/beta resulted in a clear-cut inhibition of both tumor growth and p24 HIV antigenemia. In contrast, treatment with tumor necrosis factor alpha markedly inhibited tumor growth but did not significantly decrease serum p24 levels. 3'-Azido-3'-deoxythymidine treatment did not affect either tumor growth or the levels of serum p24 antigen. These data indicate that endogenous IFN-alpha/beta is a crucial factor in the restriction of both tumor growth and p24 antigenemia in mice injected with HIV-infected tumor cells. Moreover, the results suggest that the development of HIV-1 p24 antigenemia in athymic immunosuppressed mice may represent an interesting in vivo model for anti-HIV therapy.
Human T-cell leukemia virus (HTLV) type II Rex protein binds specifically to RNA sequences of the HTLV long terminal repeat but poorly to the human immunodeficiency virus type 1 Rev-responsive element
Authors: Yip M T , Dynan W S , Green P L , Black A C , Arrigo S J , Torbati A , Heaphy S , Ruland C , Rosenblatt J D , Chen I S
Published: January 2020

The human T-cell leukemia viruses (HTLVs) encode a trans-regulatory protein, Rex, which differentially regulates viral gene expression by controlling the cytoplasmic accumulation of viral mRNAs. Because of insufficient amounts of purified protein, biochemical characterization of Rex activity has not previously been performed. Here, utilizing the baculovirus expression system, we purified HTLV type II (HTLV-II) Rex from the cytoplasmic fraction of recombinant baculovirus-infected insect cells by heparin-agarose chromatography. We directly demonstrated that Rex specifically bound HTLV-II 5' long terminal repeat RNA in both gel mobility shift and immunobinding assays. Sequences sufficient for Rex binding were localized to the R-U5 region of the HTLV-II 5' long terminal repeat and correlate with the region required for Rex function. The human immunodeficiency virus type 1 (HIV-1), has an analogous regulatory protein, Rev, which directly binds to and mediates its action through the Rev-responsive element located within the HIV-1 env gene. We demonstrated that HTLV-II Rex rescued an HIV-1JR-CSF Rev-deficient mutant, although inefficiently. This result is consistent with a weak binding activity to the HIV-1 Rev-responsive element under conditions in which it efficiently bound the HTLV-II long terminal repeat RNA.
African swine fever virus attachment protein
Authors: Carrascosa A L , Sastre I , Viñuela E
Published: January 2020

Treatment of African swine fever virus particles with nonionic detergents released proteins p35, p17, p14, and p12 from the virion. Of these proteins, only p12 bound to virus-sensitive Vero cells but not to virus-resistant L or IBRS2 cells. The binding of p12 was abolished by whole African swine fever virus and not by similar concentrations of subviral particles that lacked the external proteins. A monoclonal antibody (24BB7) specific for p12 precipitated a protein that, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol, showed a molecular mass of 17 kDa (p17*) instead of 12 kDa as found in the presence of 2-mercaptoethanol. The relationship between these two proteins was confirmed by the conversion of p17* to p12 when the former was isolated from polyacrylamide gels in the absence of 2-mercaptoethanol and subsequently treated with the reducing agent. The supernatant obtained after immunoprecipitation with the p12-specific antibody lacked the virus-binding protein.
In vivo transcription of the bovine leukemia virus tax/rex region in normal and neoplastic lymphocytes of cattle and sheep
Authors: Jensen W A , Rovnak J , Cockerell G L
Published: January 2020

Expression of bovine leukemia virus (BLV) has been considered to be blocked at the transcriptional level in vivo, since viral RNA species are not readily detected in freshly isolated leukocytes from BLV-infected animals. However, the presence of a persistent antiviral antibody response in infected animals suggests that some degree of virus expression must occur in vivo. The purpose of this study was to determine whether BLV RNA species could be detected by using the polymerase chain reaction in normal or neoplastic lymphoid cells freshly isolated from naturally or experimentally BLV-infected cattle and sheep, respectively. Primers designed to detect a 2.1-kb doubly spliced BLV tax/rex-specific mRNA were used to amplify cDNA copies of RNA derived from infected animals. The amplified viral product was then detected with a radiolabeled BLV tax/rex-specific probe. BLV-specific RNA was detected readily in freshly isolated peripheral blood leukocytes derived from BLV-seropositive cattle or sheep with persistent lymphocytosis and less readily in peripheral blood leukocytes from BLV-seropositive but hematologically normal animals. BLV-specific RNA was also detected in fresh samples of BLV-induced lymphosarcomas. Normal and neoplastic lymphoid cells from BLV-seronegative animals were uniformly negative under similar conditions. These primers also amplified the same viral product from genomic DNA derived from BLV-seropositive animals, providing further evidence for in vivo transcription and suggesting that BLV RNA-dependent DNA polymerase is capable of reverse transcribing the 2.1-kb mRNA in vivo. The demonstration of transcriptional products of BLV in vivo proves that viral latency in BLV infection is incomplete.
A domain of the hepadnavirus capsid protein is specifically required for DNA maturation and virus assembly
Authors: Yu M , Summers J
Published: January 2020

Mutations introduced into the capsid gene of duck hepatitis B virus (DHBV) were tested for their effects on viral DNA synthesis and assembly of enveloped viruses. Four classes of mutant phenotypes were observed among a series of deletions of covering the 3' end of the capsid open reading frame. Class I mutant capsids were able to support normal single-stranded and relaxed circular viral DNA synthesis; class II mutant capsids supported normal single-stranded DNA synthesis but not relaxed circular DNA synthesis; class III mutant capsids resembled class II capsids, but viral DNA synthesis was inhibited 5- to 10-fold; and class IV capsids were severely restricted in their ability to support viral DNA synthesis. Class I capsids were assembled into enveloped virions, but class II, III, and IV capsids were not. Viral DNA synthesized inside class II capsids was normal with respect to minus-strand DNA initiation, plus-strand DNA initiation, and circularization of the DNA, but plus strands failed to be elongated to mature 3-kb DNA. The results suggest that a function of the capsid protein specifically required for viral DNA maturation is also required for assembly of nucleocapsids into envelopes. Thus, class II mutants appear to be defective in the appearance of the "packaging signal" for virus assembly (J. Summers and W. Mason, Cell 29:403-415, 1982).
Intracellular distribution of input vesicular stomatitis virus proteins after uncoating
Authors: Rigaut K D , Birk D E , Lenard J
Published: January 2020

We have examined the fate of input viral proteins following the uncoating of vesicular stomatitis virus (VSV) by immunofluorescence microscopy, immunoelectron microscopy, and cell fractionation. VSV was adsorbed to BHK cells and allowed to become internalized in the presence of 100 mM NH4Cl; the NH4Cl was then removed to initiate synchronized uncoating. The three major structural proteins of VSV, the matrix protein (M), the nucleocapsid protein (N), and the glycoprotein (G), were each distributed uniquely after uncoating. Immunofluorescence microscopy showed that both G and N proteins retained a punctate distribution, whereas M protein was diffusely distributed throughout the cytoplasm, suggesting that it had become soluble. Immunoelectron microscopy showed that N protein was found in clusters (presumably in intact nucleocapsids) associated with the cell cytoskeleton and in unfused virions in endosomes and lysosomes. M protein was found diffusely distributed throughout the cytoplasm and also in endosomes and lysosomes. G protein was found only in association with endosomes and lysosomes after uncoating. Electrophoretic analysis of the high-speed cytosol fraction from infected cells showed that it contained chiefly M protein. The amount of M protein in the cytosol increased continuously during 90 min of uncoating, confirming its solubilization during uncoating. M protein was not covalently modified by phosphorylation upon uncoating, as evidenced by its mobility on nonequilibrium pH gradient gel electrophoresis. We suggest that those nucleocapsids associating with the cytoskeleton after uncoating may represent the sites of primary viral transcription.
The N-terminal 31 amino acids of human immunodeficiency virus type 1 envelope protein gp120 contain a potential gp41 contact site
Authors: Ivey-Hoyle M , Clark R K , Rosenberg M
Published: January 2020

We have compared the expression of full-length gp160 envelope protein from human immunodeficiency virus type 1 with that of a deletion mutant lacking the N-terminal 31 amino acids of the mature protein (gp160 delta 32). The gp160 and gp160 delta 32 proteins are processed to yield gp41 and gp120 or gp120 delta 32, respectively. In contrast to full-length gp120, gp120 delta 32 failed to associate with gp41 at the cell surface, despite conformational integrity as judged by soluble CD4 binding. Thus, the N-terminal 31 amino acids of gp120, which contain hyperconserved sequences, are likely involved in forming a contact site for gp41.
Encephalomyocarditis virus RNA polymerase preparations, with and without RNA helicase activity
Authors: Dmitrieva T M , Norkina K B , Agol V I
Published: January 2020

RNA template- and primer-dependent preparations of RNA polymerase were purified from encephalomyocarditis virus-infected Krebs-2 cells, using a three-step chromatographic procedure. The RNA duplex-unwinding activity of these preparations was investigated by two assays, using a partially double-stranded RNA template (encephalomyocarditis virus RNA annealed with a long segment of antisense transcript). Less purified preparations of the polymerase appeared to be able to efficiently displace, in an ATP-dependent and RNA elongation-dependent reaction, the antisense segment from the template. However, upon a more extensive purification, the unwinding activity of the RNA polymerase preparations was lost.
The 5'-terminal nucleotides of hepatitis A virus RNA, but not poliovirus RNA, are required for infectivity
Authors: Harmon S A , Richards O C , Summers D F , Ehrenfeld E
Published: January 2020

A series of plasmids containing hepatitis A virus (HAV) cDNA was constructed such that positive-strand HAV RNA could be transcribed with T7 RNA polymerase. The plasmids differed in the number of 5'-terminal nucleotides representing the junctions between vectors and HAV sequences that were present in the transcripts. When these transcripts were used to transfect cultured BS-C-1 cells, it was found that only those transcripts that contained all of the 5'-terminal HAV nucleotides, in addition to one or more nucleotides from the vector, were capable of initiating an infectious cycle leading to production of progeny virus. Transcripts that contained one 5'-terminal nucleotide from the vector sequence but were missing two uridylate residues corresponding to the first two nucleotides of HAV sequences, or were missing U and C residues corresponding to nucleotides 2 and 3 of the HAV sequence, were not infectious. A similar plasmid containing poliovirus cDNA was engineered to produce transcripts similarly lacking the first two uridylate residues of the poliovirus RNA sequence. These transcripts were infectious.
Deletion analysis of brome mosaic virus 2a protein: effects on RNA replication and systemic spread
Authors: Traynor P , Young B M , Ahlquist P
Published: January 2020

Brome mosaic virus (BMV) genomic RNA2 encodes the 94-kDa 2a protein, which is one of two BMV nonstructural proteins required for RNA replication and subgenomic mRNA transcription. 2a contains a central polymeraselike region, which has extensive sequence similarity with the Sindbis virus nsP4 and tobacco mosaic virus (TMV) 183-kDa replication proteins, and also contains N- and C-terminal flanking segments without counterparts in the Sindbis virus and TMV nonstructural proteins. To further investigate the roles of the central and flanking segments in 2a, we have constructed a series of deletion and frameshift mutants in a biologically active BMV RNA2 cDNA clone and tested their ability to support viral RNA replication in barley protoplasts and systemic infection in whole barley plants. The entire 125-amino-acid C-terminal segment following the polymeraselike region was dispensable for RNA replication and transcription. Within the 200-amino-acid N-terminal flanking segment, deletion of the first 50 residues dramatically reduced genomic and subgenomic RNA accumulation, and deletion of 100 or more residues abolished detectable RNA synthesis. All mutations removing residues from the central polymeraselike domain also blocked RNA replication in trans. Sequences required in cis for RNA2 replication or stability were found to occur within the first 300 nucleotides of the 2a coding region. In whole barley plants, systemic infection was inhibited even by 2a deletions that supported strong RNA replication in protoplasts. Some replication-competent 2a variants failed to spread to uninoculated leaves, while other showed 10- to 500-fold-reduced virus yield in both inoculated and uninoculated leaves. These reductions were not due to any defects in RNA2 encapsidation.
Expression of human immunodeficiency virus type 1 (HIV-1) gag, pol, and env proteins from chimeric HIV-1-poliovirus minireplicons
Authors: Choi W S , Pal-Ghosh R , Morrow C D
Published: January 2020

Recent studies have demonstrated that genomes of poliovirus with deletions in the P1 (capsid) region contain the necessary viral information for RNA replication. To test the effects of the substitution of foreign genes on RNA replication and protein expression, chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes were constructed in which regions of the gag, pol, or env gene of HIV-1 were substituted for regions of the P1 gene in the infectious cDNA clone of type 1 Mahoney poliovirus. The HIV-1 genes were inserted between nucleotides 1174 and 2956 of the poliovirus cDNA so that the translational reading frame was maintained between the HIV-1 genes and the remaining poliovirus genes. The chimeric genomes were positioned downstream from a T7 RNA polymerase promoter and transcribed in vitro by using T7 RNA polymerase, and the RNA was transfected into HeLa cells. A Northern (RNA blot) analysis of the RNA from transfected cells demonstrated the appropriate-size RNA, corresponding to the full-length chimeric genomes, which increased over time. Immunoprecipitation with antibodies specific for poliovirus RNA polymerase or sera from AIDS patients demonstrated the expression of the poliovirus RNA polymerase and HIV-1 proteins as fusions with the poliovirus P1 protein. The expression of the HIV-1-poliovirus P1 fusion protein was dependent upon an intact RNA polymerase gene, indicating that RNA replication was required for efficient expression. A pulse-chase analysis of the protein expression from the chimeric genomes demonstrated the initial rapid proteolytic processing of the polyprotein from the chimeric genomes to give HIV-1-poliovirus P1 fusion protein in transfected cells; the HIV-1 gag-P1 and HIV-1 pol-P1 fusion proteins exhibited a greater intracellular stability than the HIV-1 env-P1 fusion protein. Finally, superinfection with wild-type poliovirus of HeLa cells which had been transfected with the chimeric genomes did not significantly affect the expression of chimeric fusion protein. The results are discussed in the context of poliovirus RNA replication and demonstrate the feasibility of using poliovirus genomes (minireplicons) as novel vectors for expression of foreign proteins.
E1A represses wild-type and F9-selected polyomavirus DNA replication by a mechanism not requiring depression of large tumor antigen transcription
Authors: DePolo N J , Villarreal L P
Published: January 2020

Polyomavirus (Py) DNA replication may be regulated to a low-level replication state in specific target cells in mice as well as in certain undifferentiated murine cell lines, such as embryocarcinoma (EC) cells. To investigate possible mechanisms by which such control may occur, we have examined the effects of E1A on Py DNA replication. Adenovirus E1A proteins repress transcriptional activation of various enhancers, including those of Py, and can stimulate DNA replication in quiescent cells, but E1A effects on Py DNA replication were unknown. We found that constitutive E1A expression in NIH 3T3 cells depressed Py DNA replication very strongly. Two F9 EC cell-selected Py enhancer variants, PyF441 and PyF101, were also examined because undifferentiated EC cells are hypothesized to have an E1A-like activity responsible for the Py restriction, and these variants activate Py DNA replication in cis in undifferentiated F9 cells. Both variants were repressed by E1A, indicating that E1A activity in 3T3 cells is not equivalent to undifferentiated F9 cell E1A-like activity. We also examined transient inducible E1A expression in cells supplying Py large tumor antigen (T-Ag). Py DNA replication was again repressed, and the inhibition increased with E1A induction. Analysis of T-Ag mRNA levels indicated that E1A repression of Py DNA replication was not an indirect result of depression of T-Ag transcription. This suggests that E1A may repress Py DNA replication by a more direct mechanism, possibly by blocking enhancer activation of DNA replication in a manner uncoupled with enhancer transcriptional control.
Expression of the herpes simplex virus 1 alpha transinducing factor (VP16) does not induce reactivation of latent virus or prevent the establishment of latency in mice
Authors: Sears A E , Hukkanen V , Labow M A , Levine A J , Roizman B
Published: January 2020

A feature of the cascade regulation of herpes simplex virus 1 gene expression in productive infection is that the first genes to be expressed, the alpha genes, are transactivated by a structural component of the virion designated as the alpha transinducing factor (alpha TIF). In this study, we have tested the hypothesis that latent infection of sensory neurons results from the failure of alpha TIF, a tegument protein, to be transported from the nerve endings to the nucleus of the sensory neuron. Two viruses were constructed. The first recombinant virus (R6003) contained a second copy of the alpha TIF gene placed under the control of a metallothionein promoter. The second recombinant virus (R6004) is identical to R6003 except for the presence of a stop codon inserted at amino acid 70 of the second alpha TIF gene. The metallothionein promoter inserted into the viral genome was shown to be expressed, and alpha TIF mRNA was detected by in situ hybridization of sections of trigeminal ganglia of mice infected with R6003, both untreated and those given cadmium injections. In all experiments, there were no significant differences in the recovery of latent virus from mice infected with R6003 or R6004, whether injected with cadmium or not. Cadmium administration at the time of infection and at intervals thereafter did not preclude establishment of latency. In another series of experiments, transgenic mice expressing the metallothionein-driven alpha TIF did not differ from nontransgenic siblings with respect to the incidence of latent virus in trigeminal ganglia. We conclude that the absence of alpha TIF cannot alone account for the establishment of latency.
Antiviral antibodies attenuate T-cell-mediated immunopathology following acute lymphocytic choriomeningitis virus infection
Authors: Wright K E , Buchmeier M J
Published: January 2020

The role of antiviral antibody in resistance to acute lymphocytic choriomeningitis virus infection has been examined by passive transfer of monoclonal antibodies and intracerebral challenge infection. Protection of mice from lethal T-cell-mediated acute disease was observed following passive administration of antibodies either 1 day before or up to 2 days after infection. Viral replication was suppressed in protected mice, and the cytotoxic T-cell response to virus was also diminished. Virus was cleared from the brain and other tissues of protected mice without development of lethal immunopathology, suggesting that preexisting antibody may play a significant role in modulating potentially destructive effects of T-cell-mediated immune responses to pathogens.
Expression of a human cytomegalovirus receptor correlates with infectibility of cells
Authors: Nowlin D M , Cooper N R , Compton T
Published: January 2020

Previous studies have demonstrated that human cytomegalovirus (HCMV) specifically binds to a fibroblast membrane glycoprotein(s) with a molecular mass from 30 to 34 kDa. In this study, the distribution of the putative receptor proteins was analyzed in a variety of cell types, including cell types representative of those that are infected in vivo. Using a sensitive microbinding assay (to score virus attachment) and an indirect detection method (to score HCMV-binding proteins), we found that the 34- and 32-kDa HCMV binding proteins are ubiquitous molecules, broadly distributed among diverse cell types. In addition, the level of virus attachment was found to correlate with the abundance of the 34- and 32-kDa cellular proteins, while the ability of the virus to penetrate cells and initiate infection did not. The results support the hypothesis that the 34- and 32-kDa cellular proteins represent the HCMV (attachment) receptor. The data also support the notion that additional cellular components are required for virus entry and fusion.
Indirect role of T cells in development of polioencephalitis and encephalomyelitis induced by encephalomyocarditis virus
Authors: Topham D J , Adesina A , Shenoy M , Craighead J E , Sriram S
Published: January 2020

Infection of female BALB/c mice with encephalomyocarditis virus results in the development of a paralytic syndrome in 7 to 10 days postinoculation. Previous studies had suggested the involvement of an immune component in the development of central nervous system pathology. We have examined the effects of T-cell depletion on the development of polioencephalitis (neuronal necrosis and inflammation of the brain and brain stem) and the relative contribution of the CD4+ and CD8+ subsets following the establishment of viremia. We show that monoclonal antibody depletion of T cells is effective in the reduction of polioencephalitis when given prior to viral inoculation. However, administration of the antibodies 12 h or more after viral inoculation failed to alter the development of polioencephalitis or encephalomyelitis. We conclude that T cells are involved in the development of central nervous system disease during the initial stages of infection but are not responsible for the later progression of disease.
Transformation by the oncogenic latent membrane protein correlates with its rapid turnover, membrane localization, and cytoskeletal association
Authors: Martin J , Sugden B
Published: January 2020

The latent membrane protein (LMP) of Epstein-Barr virus (EBV) has a short half-life (V. R. Baichwal and B. Sugden, J. Virol, 61:866-875, 1987; K.P. Mann and D. Thorley-Lawson, J. Virol, 61:2100-2108, 1987), is localized in patches in the membrane (D. Liebowitz, D. Wang, and E, Kieff, J. Virol, 58:233-237, 1986), and associates with the cytoskeleton in EBV-immortalized B lymphocytes (D. Liebowitz, R. Kopan, E. Fuchs, J. Sample, and E. Kieff, Mol. Cell. Biol. 7:2299-2308, 1987; K. P. Mann and D. Thorley-Lawson, J. Virol. 61:2100-2108, 1987). Deletion mutants of LMP that are either positive or negative in the induction both of anchorage-independent growth of BALB/c 3T3 cells (V. R. Baichwal and B. Sugden, Oncogene 4:67-74, 1989) and of cytotoxicity in a variety of cells (W. Hammerschmidt, B. Sugden, and V. R. Baichwal, J. Virol. 63:2469-2475, 1989) have been studied to identify the biochemical properties of this protein that correlate with its effects on cell growth. Mutant LMP proteins that are metabolically stable, do not associate with the cytoskeleton, and exhibit a diffuse plasma membrane localization also do not induce anchorage-independent growth in rodent cells or cytotoxicity in B lymphoblastoid cells. In contrast, a mutant of LMP that is functionally identical to the wild-type protein has a half-life, membrane localization, and cytoskeletal association similar or identical to those of LMP. These results are consistent with the hypothesis that LMP's rapid turnover, association with the cytoskeleton, and patching in the membrane are required for it to affect cell growth.
Identification and characterization of the hamster polyomavirus middle T antigen
Authors: Courtneidge S A , Goutebroze L , Cartwright A , Heber A , Scherneck S , Feunteun J
Published: January 2020

Hamster polyomavirus (HaPV) is associated with lymphoid and hair follicle tumors in Syrian hamsters. The early region of HaPV has the potential to encode three polypeptides (which are related to the mouse polyomavirus early proteins) and can transform fibroblasts in vitro. We identified the HaPV middle T antigen (HamT) as a 45-kDa protein. Like its murine counterpart, HamT was associated with serine/threonine phosphatase, phosphatidylinositol-3 kinase, and protein tyrosine kinase activities. However, whereas mouse middle T antigen associates predominantly with pp60c-src and pp62c-yes, HamT was associated with a different tyrosine kinase, p59fyn. The ability of HaPV to cause lymphoid tumors may therefore reside in its ability to associate with p59fyn, a potentially important tyrosine kinase in lymphocytes.
A chimpanzee-passaged human immunodeficiency virus isolate is cytopathic for chimpanzee cells but does not induce disease
Authors: Watanabe M , Ringler D J , Fultz P N , MacKey J J , Boyson J E , Levine C G , Letvin N L
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) readily infects both humans and chimpanzees, but the pathologic outcomes of infection in these two species differ greatly. In attempts to identify virus-cell interactions that might account for this differential pathogenicity, chimpanzee peripheral blood lymphocytes and bone marrow macrophages were assessed in vitro for their ability to support the replication of several HIV-1 isolates. Although the IIIb, RF, and MN isolates did not readily infect chimpanzee peripheral blood lymphocytes, an isolate of HIV-1 passaged in vivo in chimpanzees not only replicated well in both chimpanzee peripheral blood lymphocytes and bone marrow macrophages but also was cytopathic for chimpanzee CD4+ lymphocytes. Because no evidence of HIV-induced disease has been observed in chimpanzees infected with this isolate, in vitro replication to high titers with concomitant loss of CD4+ cells is not, in this instance, a correlate of pathogenicity. These observations, therefore, indicate that caution must be used when making extrapolations from in vitro data to in vivo pathogenesis.
Rhesus papillomavirus type 1 cooperates with activated ras in transforming primary epithelial rat cells independent of dexamethasone
Authors: Schneider J F , McGlennen R C , LaBresh K V , Ostrow R S , Faras A J
Published: January 2020

Rhesus Papillomavirus type 1 (RhPV-1) was recently cloned from a rhesus monkey lymph node metastasis of a penile squamous cell carcinoma. In this paper, we demonstrate that RhPV-1 cooperates with the activated ras oncogene to transform primary cells at a level comparable to human papillomavirus type 16. The viral DNAs were cloned such that their expression was under the control of their natural promoter elements. Unlike human papillomavirus type 16, RhPV-1 DNA cooperated with ras independently of the hormone dexamethasone. However, dexamethasone did have a positive influence on the ability of some RhPV-1 cotransformed cells to grow in soft-agar assays. The transformed cells are highly tumorigenic in vivo in nude mice.
The yeast GAL4 protein transactivates the polyomavirus origin of DNA replication in mouse cells
Authors: Baru M , Shlissel M , Manor H
Published: January 2020

We have replaced the polyomavirus (Py) enhancer, which is an essential component of the Py origin of DNA replication (ori), with five repeats of a 17-bp oligonucleotide including the yeast GAL4 upstream activating sequence (5xGAL4 sites). Plasmids containing this modified Py ori, designated test plasmids, and plasmids encoding either the GAL4 transcriptional activator protein or various derivatives of this protein were cotransfected into mouse cells which constitutively synthesize a temperature-sensitive Py large tumor antigen (T-Ag). Replication of the test plasmids was monitored by Southern blot determinations of the amounts of plasmid DNA that became resistant to cleavage by the enzyme DpnI. These studies showed that in the presence of a functional T-Ag, the GAL4 protein, and hybrid proteins including the GAL4 DNA-binding domain and the activating domain of the adenovirus E1a or herpesvirus VP16 protein transactivated the modified Py ori. A truncated protein including just the GAL4 DNA-binding domain was inactive in these assays. The authentic GAL4 protein was found to be a more efficient replication transactivator than the hybrid proteins. In contrast, chloramphenicol acetyltransferase assays showed that the hybrid proteins were more efficient transcriptional activators than the GAL4 protein. The extent of the GAL4-dependent replication of a plasmid in which the Py early promoter was deleted was 55% lower than that of a plasmid including the promoter. However, the extents of replication of plasmids including two tandem repeats of the remaining Py origin core and 5xGAL4 sites or two origin cores flanking a single cluster of 5xGAL4 sites were 4.8- and 1.6-fold higher than that of the plasmid including a single copy of each element. The replication of a plasmid including two clusters of 5xGAL4 sites flanking a single origin core was below the limit of detection of our assays. These results indicate that the GAL4 and hybrid transactivators do not activate the Py ori by virtue of their interactions with transcription factors that bind promoter elements. Rather, it appears that these activator proteins may interact with the replication initiation complexes, thereby facilitating or inhibiting the initiation of replication.
Human influenza virus hemagglutinin with high sensitivity to proteolytic activation
Authors: Ohuchi R , Ohuchi M , Garten W , Klenk H D
Published: January 2020

To examine the prerequisites for cleavage activation of the hemagglutinin of human influenza viruses, a cDNA clone obtained from strain A/Port Chalmers/1/73 (serotype H3) was subjected to site-directed mutagenesis and expressed in CV-1 cells by using a simian virus 40 vector. The number of basic residues at the cleavage site, which consists of a single arginine with wild-type hemagglutinin, was increased by inserting two, three, or four additional arginines. Like wild-type hemagglutinin, mutants with up to three additional arginines were not cleaved in CV-1 cells, but insertion of four arginines resulted in activation. When the oligosaccharide at asparagine 22 of the HA1 subunit of the hemagglutinin was removed by site-directed mutagenesis of the respective glycosylation site, only three inserted arginines were required to obtain cleavage. Mutants containing a series of four basic residues were also generated by substituting arginine for uncharged amino acids immediately preceding the cleavage site. The observation that these mutants were not cleaved, even when the carbohydrate at asparagine 22 of HA1 was absent, underscores the fact that the basic peptide had to be generated by insertion to obtain cleavage. The data show that the hemagglutinin of a human influenza virus can acquire high cleavability, a property known to be an important determinant for the pathogenicity of avian influenza viruses. Factors important for cleavability are the number of basic residues at the cleavage site, the oligosaccharide at asparagine 22, and the length of the carboxy terminus of HA1.
Characterization of Pichinde virus infection of cells of the monocytic lineage
Authors: Polyak S J , Rawls W E , Harnish D G
Published: January 2020

To establish a model of viral infection of monocytes, we examined infection of human cells and cell lines of the monocytic series with the arenavirus Pichinde virus. We demonstrate for the first time that human peripheral blood monocytes are susceptible to Pichinde virus infection, as shown by immunoprecipatation of virus-specific polypeptides from infected cells, immunofluorescence analyses, and quantitation of virus production from infected cells. The human promyelocytic leukemia cell line HL60 did not support Pichinde virus replication, even if cells were induced with the phorbol ester phorbol myristate acetate (PMA) to differentiate to monocytes. However, the human promonocytic leukemia cell line THP-1 did support Pichinde virus replication. Replication depended on exposure of the cells to PMA. We examined the nature of the effect of PMA in the induction of THP-1 cells to support Pichinde virus replication. We found that 5 min of exposure of THP-1 cells to PMA is sufficient to support virus growth and that PMA-treated THP-1 cells remain susceptible to infection up to 4 days after the initial PMA treatment. We also showed that infection of PMA-treated THP-1 cells is mediated through protein kinase C (PKC). H7, a PKC inhibitor, was able to block both PMA-induced differentiation and Pichinde virus infection of THP-1 cells. The synthetic diacylglycerol and PKC agonist, diC8, was able to stimulate THP-1 cells to support virus growth, albeit to lower levels than PMA. Dactinomycin abrogated the ability of virus to replicate and suggested a requirement for host cell transcription. The PMA effect did not appear to relate to receptor modulation. These results suggest that PMA-induced susceptibility to Pichinde virus infection occurs at a point later than the initial binding and penetration stages and that infection depends on the activation or differentiation state of the cell.
Pregenomic RNA encapsidation analysis of eleven missense and nonsense polymerase mutants of human hepatitis B virus
Authors: Roychoudhury S , Faruqi A F , Shih C
Published: January 2020

We characterized 11 DNA polymerase mutants of human hepatitis B virus (HBV) which contain single missense or nonsense mutations in the various domains within this gene. Except for mutant 738, a tight association between DNA replication and RNA packaging of these missense pol mutants was observed. Further analysis of HBV core particle-associated RNA indicated that only the 3.5-kb core-specific RNA, but not the precore-specific RNA, is selectively packaged in this tissue culture system. Previously, we have demonstrated that only the 3.5-kb core-specific RNA can serve as an efficient template for pol translation. Taken together, our results suggest that selectivity of HBV RNA packaging occurs as a result of selective translation of pol-containing mRNAs. Furthermore, our data suggest that the RNA encapsidation domain of pol overlaps with all of the domains of pol involved in the synthesis of terminal protein, as well as DNA replication. Finally, on the basis of gradient centrifugation analysis, a pol defect appeared to have no negative effect on the assembly or stability of core particles. A new method to assay RNA encapsidation, as well as potential RNase H activity, is reported.
Host range expansion by recombination of the baculoviruses Bombyx mori nuclear polyhedrosis virus and Autographa californica nuclear polyhedrosis virus
Authors: Kondo A , Maeda S
Published: January 2020

The mechanisms of host specificity of nuclear polyhedrosis viruses (NPVs) (Baculoviridae) were analyzed after coinfection of Bombyx mori NPV (BmNPV) and one of four distinct groups of Spodoptera litura NPV (SlNPV), including an Autographa californica NPV (AcNPV) variant (S. Maeda, Y. Mukohara, and A. Kondo, J. Gen. Virol. 71:2631-2639, 1990), into various lepidopteran cell lines. Replication of BmNPV in nonpermissive cells (TN-386, SF-21, and CLS-79) was induced by coinfection with AcNPV but not with the other three SlNPV groups. These induced progeny NPVs were plaque purified in BmN cells, which are susceptible to only BmNPV, and characterized. Most of these isolates did not replicate in the cell lines in which they were produced, indicating the existence of a helper function of AcNPV for BmNPV replication in nonpermissive cells. Some of these isolates, however, were able to replicate in cell lines nonpermissive to BmNPV, indicating the appearance of a new virus with wider host specificity. DNA restriction endonuclease analysis showed that the isolates exhibiting wider host range were recombinant viruses between the parents, AcNPV and BmNPV, resulting from various types of crossovers of relatively large areas of their genomes. Expansion of host range was also observed in larvae.