bluuebunny/crossref_metadata_2025 huggingface dataset:
Endogenous expression of E1A in human cells enhances the effect of adenovirus E3 on class I major histocompatibility complex antigen expression
Authors: Routes J M , Metz B A , Cook J L
Published: January 2020

Group C human adenovirus (Ad) serotypes (e.g., Ad type 2 [Ad2] and Ad5) cause persistent infections in humans. One explanation for Ad persistence is an ineffective cytotoxic T-lymphocyte response due to diminished cell surface expression of class I major histocompatibility antigen (MHC Ag) on Ad-infected cells, an effect mediated by the Ad E3 19-kDa glycoprotein (E3 effect). However, we previously reported that, except for the Ad5 E1-transformed human cell line 293, a variety of human lymphoid, epithelial, and fibroblastic cells are resistant to the E3 effect during Ad5 infection (J. M. Routes and J. L. Cook, J. Immunol. 144:2763-2770, 1990). The present study tested the hypothesis that endogenous expression of E1A proteins in 293 cells sensitizes cells to this E3 effect, resulting in an enhanced downregulation of surface class I MHC Ag expression following Ad5 infection. Human epithelial and fibroblastic cells expressing E1A gene products for at least 72 h exhibited an enhanced E3 effect following Ad5 infection that was independent of baseline levels of surface class I MHC Ag expression and of E1A induction of E3 19-kDa glycoprotein expression. There was a direct correlation between the level of endogenous E1A expressed and the magnitude of the E3 effect. We postulate that the in vivo existence of cells stably expressing either E1A proteins or E1A-like activities in the microenvironment of Ad5 infection provides a reservoir of Ad-infected cells that is relatively protected from the virus-specific cytotoxic T-lymphocyte response, thereby favoring Ad persistence in humans.
Interchangeability of the adsorption proteins of bacteriophages Ff and IKe
Authors: Endemann H , Gailus V , Rasched I
Published: January 2020

The wild-type adsorption protein (g3p) of filamentous phage IKe cannot be exchanged with its analogous protein in the related Ff (M13, fd, and f1) phage particles. Deletion mutants of the protein, however, are assembled into Ff phage particles. These hybrid Ff phage particles bearing deleted IKe g3p attach to N pili, thus conserving the host attachment property of the protein but not its infection-initiating function. This means that the attachment specificity is determined by IKe g3p independently of other phage components in contact with it. Infection initiation function, the process in which phage DNA is released into the host, in contrast seems to require either more complex structural features of the protein (for example, a certain oligomeric structure) provided only in the original particle, or a concerted action of g3p with another particle component, not replaceable by its homologous counterpart in the related phage.
Experimental transmission of human hepatitis delta virus to the laboratory mouse
Authors: Netter H J , Kajino K , Taylor J M
Published: January 2020

Human hepatitis delta virus (HDV), obtained from the serum of an experimentally infected woodchuck, was injected into either the peritoneal cavity or the tail vein of both adult CB17 mice and mice with a severe combined immunodeficiency (CB17-scid mice). Three lines of evidence indicated that the virus was able to reach the liver and infect hepatocytes: (i) the amount of HDV genomic RNA detected in the liver by Northern (RNA) analysis increased during the first 5 to 10 days postinoculation, reaching a peak that was about threefold the amount in the original inoculum; (ii) also detected in the liver was the viral antigenomic RNA, which is complementary to the genomic RNA found in virions, and is diagnostic for virus replication; and (iii) by immunoperoxidase staining of liver sections, the delta antigen was detected in the nuclei of scattered cells identifiable as hepatocytes. In all of the mice, clearance of the infection occurred between 10 and 20 days after inoculation. The half-life for clearance was about 3 days in CB17-scid mice, indicating that clearance of infection did not involve a T- and B-cell-dependent immune response. Cell-to-cell spread of the initial infection was not detected. One possible interpretation of our results is that HDV infection of hepatocytes is directly cytopathic. Also, the results imply that chronic infection of the liver in humans may require continuous spread of virus within the liver. Alternatively, HDV in the absence of helper virus may be unable to cause a chronic infection of hepatocytes in vivo.
cis-acting requirements for the replication of flock house virus RNA 2
Authors: Ball L A , Li Y
Published: January 2020

To examine the cis-acting requirements for RNA replication, a cDNA clone of flock house virus (FHV) RNA 2 was transfected into baby hamster kidney cells and transcribed to yield RNAs that had terminal extensions of different lengths or that lacked internal regions of the molecule. These RNAs were tested for their ability to be replicated by FHV replicase that was provided by cotransfection of purified FHV RNA 1. The results showed that RNA replication was inhibited by terminal extensions, particularly those at the 5' end of the RNA, despite the fact that these extensions were corrected during RNA replication. A negative-sense transcript with a 12-nucleotide 3' extension was replicated to produce a positive-sense RNA that had the correct 5' end, showing that the replicase could select its correct initiation site from within a longer template. A uridine residue at the second position of the positive strand was an important determinant of template activity. RNA molecules with large internal deletions that amounted to almost 50% of the 1,400 nucleotides of RNA 2 replicated as efficiently as full-length molecules, but only if they contained an internal region that lay between nucleotides 538 and 616. Thirty-six spontaneous deletions of RNA 2 that arose during sequential replicative passages all conserved the same internal region of the molecule. These results establish that both terminal and internal regions of FHV RNA 2 play essential roles in making the molecule a competent template for replication.
Secondary structure of gp160 and gp120 envelope glycoproteins of human immunodeficiency virus type 1: a Fourier transform infrared spectroscopic study
Authors: Decroly E , Cornet B , Martin I , Ruysschaert J M , Vandenbranden M
Published: January 2020

The secondary structure of the precursor (gp160) of the envelope protein of human immunodeficiency virus type 1 (BH10) and its receptor-binding subunit (gp120) was studied by Fourier-transformed attenuated total reflection spectroscopy. A higher alpha-helix/beta-sheet ratio in the gp120 subunit than in the precursor indicates a structural heterogeneity between the two subunits (gp120 and gp41), in agreement with classical secondary-structure predictions. The secondary structure of gp41 was estimated and compared with existing models. The high alpha-helical content in gp41 and the dominant beta-sheet content in gp120 resemble the distribution in influenza virus hemagglutinin subunits.
Genetic analysis of the cofactor requirement for human immunodeficiency virus type 1 Tat function
Authors: Madore S J , Cullen B R
Published: January 2020

The Tat protein of human immunodeficiency virus type 1 is a potent transcriptional trans activator of the viral long terminal repeat promoter element. Tat function requires the direct interaction of Tat with a cis-acting viral RNA target sequence termed the trans-activation response (TAR) element and has also been proposed to require at least one cellular cofactor. We have used a genetic approach to attempt to experimentally define the role of the cellular cofactor in Tat function and TAR binding. Our data suggest that neither Tat nor the cellular cofactor binds to TAR alone in vivo and indicate, instead, that the interaction of Tat with its cellular cofactor is a prerequisite for TAR binding. The known species tropism of lentivirus Tat proteins appears to arise from the fact that not only Tat but also the cellular cofactor can markedly influence the RNA sequence specificity of the resultant protein complex. These data also suggest that the Tat cofactor is likely a cellular transcription factor that has been highly conserved during vertebrate evolution. We hypothesize that the primary function of Tat is to redirect this cellular factor to a novel viral RNA target site and to thereby induce activation of viral gene expression.
Analysis of deletions and thermosensitive mutations in Rous sarcoma virus gag protein p10
Authors: Dupraz P , Spahr P F
Published: January 2020

Rous sarcoma virus protein p10 is a gag component of the virion present in stoichiometric amount but of unknown function. To characterize this protein, a series of mutants of p10 with linker insertions or deletions was generated by site-directed mutagenesis of a cloned proviral DNA. The deletions and two of the linkers insertions, which disrupted proline pairs, reduced the yield of virus particles upon transfection. These two linker insertion mutants were moreover thermosensitive for this phenotype, producing fewer virus particles at 41 degrees C than at 36 degrees C. Examination of the intracellular viral proteins demonstrated that for all mutants, the amount of gag precursor was similar to the wild-type level. Moreover, the amount of mature gag CA that could be detected by this analysis was similar between each of the mutants and the wild type. This finding suggests that the transport of gag to the membrane and the initial stages of maturation were not affected by the mutations. The virus particles contained normal amounts of active reverse transcriptase, showing that the gag-pol polyprotein was incorporated and cleaved properly. Viral RNA was quantitatively and qualitatively similar in mutant and wild-type virions. However, the infectivity of the mutants virions differed; one of the thermosensitive linker insertions that had no effect on particle production at 36 degrees C was nevertheless noninfectious at that temperature. Together, these data suggest that the p10 protein is involved in a late steps of virus maturation, possibly budding, and perhaps also in an early event of viral infection.
The two zinc fingers in the human immunodeficiency virus type 1 nucleocapsid protein are not functionally equivalent
Authors: Gorelick R J , Chabot D J , Rein A , Henderson L E , Arthur L O
Published: January 2020

The highly conserved zinc fingers in retroviral nucleocapsid (NC) proteins have the general structure Cys-(X)2-Cys-(X)4-His-(X)4-Cys. Human immunodeficiency virus type 1 (HIV-1) contains two Zn2+ fingers, and mutants were constructed in which the native sequence of each Zn2+ finger was maintained but their positions in the NC protein were changed. Mutants had either two first-finger sequences (pNC1/1), two second-finger sequences (pNC2/2), or reversed first- and second-finger sequences (pNC2/1). Cells transfected with mutant or wild-type clones produced similar levels of Tat, Gag, Pol, and Env proteins, formed syncytia, and shed viruslike particles that were indistinguishable by electron microscopy. However, the pNC2/1 and pNC2/2 mutants were inefficient in packaging genomic RNA (less than 15% of wild-type levels), whereas the pNC1/1 mutant packaged approximately 70% of wild-type levels of RNA. No infectious virus could be detected with either the pNC2/1 or pNC2/2 mutants, whereas the pNC1/1 mutant appeared to sustain a low level of replication and reverted to a competent wild-type-like viral species after a 2- to 4-week lag period. The data strongly suggest that the two Zn2+ fingers of HIV-1 are not functionally equivalent and that the first Zn2+ finger in the Gag precursor plays a more prominent role in RNA selection and packaging. The data also indicate that both Zn2+ fingers in the mature NC protein play as yet unknown roles in viral assembly or the early stages of the viral infection process.
trans processing of vaccinia virus core proteins
Authors: Lee P , Hruby D E
Published: January 2020

The three major vaccinia virus (VV) virion proteins (4a, 4b, and 25K) are proteolytically matured from larger precursors (P4a, P4b, and P25K) during virus assembly. Within the precursors, Ala-Gly-X motifs have been noted at the putative processing sites, with cleavage apparently taking place between the Gly and X residues. To identify the sequence and/or structural parameters which are required to define an efficient cleavage site, a trans-processing assay system has been developed by tagging the carboxy terminus of the P25K polypeptide (precursor of 25K) with an octapeptide FLAG epitope, which can be specifically recognized by a monoclonal antibody. By using transient expression assays with cells coinfected with VV, the proteolytic processing of the chimeric gene product (P25K:FLAG) was monitored by immunoblotting procedures. The relationship between the P25K:FLAG precursor and the 25K:FLAG cleavage product was established by pulse-chase experiments. The in vivo cleavage of P25K:FLAG was inhibited by the drug rifampin, implying that the reaction was utilizing the same pathway as authentic VV core proteins. Moreover, the 25K:FLAG protein was found in association with mature virions in accord with the notion that cleavage occurs concomitantly with virion assembly. Site-directed mutagenesis of the Ala-Gly-Ala motif at residues 31 to 33 of the P25K:FLAG precursor to Ile-Asp-Ile blocked production of the 25K:FLAG product. The efficiency of 25K:FLAG production (33.71%) is, however, approximately only half of the production of 25K (63.98%) within VV-infected cells transfected with pL4R:FLAG. One explanation for the lower efficiency of 25K:FLAG production was suggested by the observation in the immunofluorescent-staining experiment that 25K:FLAG-related proteins were not specifically localized to the virus assembly factories (virosomes) within VV-infected cells, although virosome localization was prominent for P25K-related polypeptides. Since VV core protein proteolytic processing is believed to take place during virion maturation, only the P25K:FLAG which was assembled into immature virions could undergo proteolytic maturation. Furthermore during these experiments, a potential cleavage intermediate (25K') of P25K was identified. Amino acid residues 17 to 19 (Ala-Gly-Ser) of the P25K precursor were implicated as the intermediate cleavage site, since no 25K':FLAG product was produced from a mutant precursor in which the sequence was altered to Ile-Asp-Ile. Taken together, these results provide biochemical and genetic evidence to support the hypothesis that the Ala-Gly-X cleavage motif plays a critical role in VV virion protein proteolytic maturation.
Assembly, processing, and infectivity of human immunodeficiency virus type 1 gag mutants
Authors: Wang C T , Barklis E
Published: January 2020

We studied the effects of gag mutations on human immunodeficiency virus type 1 (HIV-1) assembly, processing, and infectivity by using a replication-defective HIV expression system. HIV mutants were screened for infectivity by transduction of a selectable marker and were examined for assembly by monitoring particle release from transfected cells. Gag protein processing and reverse transcriptase activities of mutant particles were also assayed. Surprisingly, most Gag protein mutants were assembled and processed. The two exceptions to this rule were a myristylation-minus mutant, and one gag matrix domain mutant which expressed proteins that were trapped intracellularly. Interestingly, a mutant with a 56-amino-acid deletion within the HIV gag capsid domain still could assemble and process virus particles, exhibited a wild-type retrovirus particle density, and had wild-type reverse transcriptase activity. Indeed, although most HIV-1 gag mutants were noninfectious or poorly infectious, they produced apparently normal particles which possessed significant reverse transcriptase activities. These results strongly support the notion that the HIV-1 Gag proteins are functionally involved in post-assembly, postprocessing stages of virus infectivity.
Identification of transactivation-response sequences in the long terminal repeat of bovine immunodeficiency-like virus
Authors: Carpenter S , Nadin-Davis S A , Wannemuehler Y , Roth J A
Published: January 2020

Transient expression assays using the reporter gene that encodes chloramphenicol acetyltransferase were used to identify cis-acting sequences necessary for bovine immunodeficiency-like virus (BIV) transactivation. Computer analyses identified two RNA stem-loop structures located immediately downstream of the transcription start site in the long terminal repeat. Deletion analysis of the long terminal repeat indicated that sequences containing the proximal stem-loop structure located between +4 and +31 are required for virus-specific transactivation. Therefore, BIV likely utilizes a mechanism of transactivation similar to that of the human and simian lentiviruses.
A poliovirus minireplicon containing an inactive 2A proteinase is expressed in vaccinia virus-infected cells
Authors: Pal-Ghosh R , Morrow C D
Published: January 2020

It has been difficult to evaluate the role of individual viral proteins in poliovirus replication because a suitable complementation system has not yet been developed. To approach this problem, we constructed a chimeric human immunodeficiency virus type 2 (HIV-2)-gag-poliovirus minireplicon in which regions of the gag gene of HIV-2 were inserted in the poliovirus genome between nucleotides 1174 and 2470. Transfection of this chimeric RNA into HeLa cells results in the replication of the minireplicon and expression of an HIV-2-gag-P1 fusion protein which can be immunoprecipitated with antibodies to HIV-2-gag. Expression of the HIV-2-gag-P1 fusion protein was dependent on replication of the chimeric RNA genome. Although the chimeric HIV-2-gag-poliovirus RNA genome replicated in poliovirus-infected cells, transfection of the chimeric HIV-2-gag-poliovirus genome into vaccinia virus-infected cells resulted in increased replication as measured by analysis of chimeric RNA. The increase in replication correlated with an increase in the expression of the HIV-2-gag-P1 fusion protein in vaccinia virus-infected cells. To characterize this system, we constructed a mutation in the 2A gene to change a cysteine at amino acid 109 to a serine. Expression of the HIV-2-gag-P1 fusion protein was not detected when the HIV-2-gag-poliovirus genome containing the 2A mutation was transfected into HeLa cells, demonstrating the mutation was lethal for replication. When the chimeric genome was transfected into poliovirus-infected cells, no RNA replication or expression of the HIV-2-gag-P1 fusion protein was observed. In contrast, transfection of this genome into vaccinia virus-infected cells resulted in replication of the chimeric RNA and expression of two proteins with larger molecular masses than the HIV-2-gag-P1 proteins, possibly representing HIV-2-gag-P1-2A and HIV-2-gag-P1-2ABC fusion proteins. The transfection of the chimeric HIV-2-gag-poliovirus genome containing the 2A mutation into poliovirus-vaccinia virus coinfected cells resulted in the expression and partial processing of the two larger HIV-2-gag-P1 fusion proteins to give the correct molecular mass for the HIV-2-gag-P1 fusion protein. The 2A mutation was reconstructed back into the full-length infectious cDNA of poliovirus. Transfection of this cDNA into vaccinia virus-infected cells followed by immunoprecipitation with anticapsid antibodies demonstrated the presence of two proteins with molecular masses larger than P1, possibly P1-2A and P1-2ABC fusion proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
Analysis of the functional and host range-determining regions of the murine ectropic and amphotropic retrovirus envelope proteins
Authors: Morgan R A , Nussbaum O , Muenchau D D , Shu L , Couture L , Anderson W F
Published: January 2020

A series of Moloney murine leukemia virus (Mo-MuLV) envelope gene constructs were analyzed for biological activity. Three classes of recombinant envelopes were examined: insertions, deletions, and chimeras. Insertion (4 to 5 amino acids) and deletion (31 to 62 amino acids) mutants spanned most of the SU (gp70)-coding region and were all biologically inactive. Radioimmunoprecipitation demonstrated that the mutant envelope proteins were incorrectly processed. The Pr80env envelope precursor proteins failed to obtain the proper posttranslational modifications and were not cleaved into SU (gp70) and TM (p15E), suggesting that disruption of Pr80env structure prevents intracellular transport and processing. To analyze the functional domains of the SU portion of the Env protein, we assembled several chimeric constructs. In these constructs, portions of the ecotropic Mo-MuLV envelope gene were replaced with corresponding sequences from the 4070A amphotropic MuLV envelope. Using a retroviral vector pseudotyping assay, 5 of 12 chimeric envelope proteins were shown to be biologically active. Host range was determined by retroviral vector transduction of the appropriate cell, by viral interference studies, and by the productive infection of Chinese hamster ovary cells expressing the murine ecotropic receptor. These results permit assignment of the amino acids responsible for host range determination. Ecotropic host range is determined by the first 88 amino acids of the Mo-MuLV SU, while the amphotropic host range-determining region spans the first 157 amino acids of the 4070A SU.
Primary rat cells expressing a hybrid polyomavirus-simian virus 40 large T antigen have altered growth properties
Authors: Manfredi J J , Prives C
Published: January 2020

Expression of simian virus 40 (SV40) large T antigen efficiently immortalizes and transforms primary cells. We previously reported that a hybrid polyomavirus-SV40 large T antigen, PyT1-521-SVT336-708, binds to both p53 and pRb but does not transform an established rat cell line (J. J. Manfredi and C. Prives, J. Virol. 64:5250-5259, 1990). Here we show that this hybrid large T antigen is capable of immortalizing primary rat cells. Plasmids that express resistance to G418 sulfate and either SV40 large T antigen or PyT1-521-SVT336-708 were transfected into primary rat embryo fibroblasts, and cell lines were established. The cell lines that expressed PyT1-521-SVT336-708 were not fully transformed but did exhibit altered growth properties. Although these PyT1-521-SVT336-708-expressing lines did not form foci, they did grow in low serum and grew to a high saturation density; these cell lines also formed colonies in soft agar, but their colonies were much smaller than those seen with an SV40 large-T-antigen-expressing line. PyT1-521-SVT336-708 also demonstrated the ability to cooperate with activated Ha-ras to form foci on primary rat embryo fibroblasts. Surprisingly, two types of morphologies in such lines were observed: refractile and spindle shaped. Although there was no correlation between T-antigen level and morphology, all lines that displayed refractile morphology expressed high levels of p21ras. Since the p53 binding activity of PyT1-521-SVT336-708 appears to be intact, these results suggest that there are functions residing in the amino end of SV40 large T antigen which are necessary for full transformation that are missing from the amino end of polyomavirus large T antigen. Conversely, conferring the ability to bind to p53 on an amino-terminal fragment of polyomavirus large T antigen, although not enough to allow full transformation function, does increase its oncogenic activity in saturation density and soft agar growth assays.
Basis for selective incorporation of glycoproteins into the influenza virus envelope
Authors: Naim H Y , Roth M G
Published: January 2020

The ability of mutant or chimeric A/Japan hemagglutinins (HAs) to compete for space in the envelope of A/WSN influenza viruses was investigated with monkey kidney fibroblasts that were infected with recombinant simian virus 40 vectors expressing the Japan proteins and superinfected with A/WSN influenza virus. Wild-type Japan HA assembled into virions as well as WSN HA did. Japan HA lacking its cytoplasmic sequences, HAtail-, was incorporated into influenza virions at half the efficiency of wild-type Japan HA. Chimeric HAs containing the 11 cytoplasmic amino acids of the herpes simplex virus type 1gC glycoprotein or the 29 cytoplasmic amino acids of the vesicular stomatitis virus G protein were incorporated into virions at less than 1% the efficiency of HAtail-. Thus, the cytoplasmic domain of HA was not required for the selection process; however, foreign cytoplasmic sequences, even short ones, were excluded. A chimeric HA having the gC transmembrane domain and the HA cytoplasmic domain (HgCH) was incorporated at 4% the efficiency of HAtail-. When expressed from simian virus 40 recombinants in this system, vesicular stomatitis virus G protein with or without (Gtail-) its cytoplasmic domain was essentially excluded from influenza virions. Taken together, these data indicate that the HA transmembrane domain is required for incorporation of HA into influenza virions. The slightly more efficient incorporation of HgCH than G or Gtail- could indicate that the region important for assembling HA into virions extends into part of the cytoplasmic domain.
Molecular cloning of infectious ecotropic murine leukemia virus AK7 from an emv-14-positive AKXL-5 mouse and the resistance of AK7 to recognition by cytotoxic T lymphocytes
Authors: White H D , Green W R , Giné N R
Published: January 2020

The AKXL-5 recombinant inbred mouse strain is positive for the endogenous ecotropic murine leukemia virus emv-14, the only emv present in its germ line. emv-14 is of particular interest because spleen cells expressing emv-14 virus escape recognition by anti-AKR/Gross virus-specific cytotoxic T lymphocytes. We report here the isolation and characterization of a replication-competent emv clone, pAK7, derived from an AKXL-5 mouse. This clone is novel in that it encodes a variant ecotropic murine leukemia virus that, when expressed in SC.Kb target cells, fails to be recognized efficiently by anti-AKR/Gross virus cytotoxic T lymphocytes. The pAK7 clone can therefore be used to further probe mechanisms of escape from cell-mediated immunity.
Repression of bovine papillomavirus type 1 transcription by the E1 replication protein
Authors: Sandler A B , Vande Pol S B , Spalholz B A
Published: January 2020

Bovine papillomavirus type 1 (BPV-1) is the prototype virus for the study of papillomavirus gene regulation. The functions of the BPV-1 E2 proteins in transcriptional regulation have been well characterized. The BPV-1 E1 protein is required for viral DNA replication and can bind to the origin of replication alone or in a complex with the E2 transactivator protein. In this study, we demonstrated that the BPV-1 E1 protein is also involved in transcriptional regulation. The E1 protein significantly repressed E2-transactivated transcription from the major early promoter P89. This activity is consistent with the elevated level of P89 transcription observed in BPV-1 E1 open reading frame mutants. Transcriptional repression by E1 correlated with the ability of an E1-E2 protein complex to bind the replication origin but was not dependent on viral DNA replication. These studies identify a new mechanism involved in the regulation of papillomavirus transcription which has implications regarding expression of the viral transforming functions.
Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells
Authors: Bai M , Harfe B , Freimuth P
Published: January 2020

The adenovirus penton base protein has a cell rounding activity and may lyse endosomes during virus entry into the cytoplasm. We found that penton base that was expressed in Escherichia coli also caused cell rounding and that cells adhered to polystyrene wells that were coated with the protein. Mutant analysis showed that both properties required an Arg-Gly-Asp (RGD) sequence at residues 340 to 342 of penton base. In flat adherent cells, virus mutants with amino acid substitutions in the RGD sequence were delayed in virus reproduction and in the onset of viral DNA synthesis. In nonadherent or poorly spread cells, the kinetics of mutant virus reproduction were similar to those of wild-type adenovirus type 2. Expression of the mutant phenotype exclusively in the flat cells that we tested supports a model in which penton base interacts with an RGD-directed cell adhesion molecule during adenovirus uptake or uncoating.
cdc2 phosphorylation of threonine 124 activates the origin-unwinding functions of simian virus 40 T antigen
Authors: McVey D , Ray S , Gluzman Y , Berger L , Wildeman A G , Marshak D R , Tegtmeyer P
Published: January 2020

Phosphorylation of simian virus 40 (SV40) T antigen on threonine 124 activates viral DNA replication in vivo and in vitro. We have manipulated the modification of T-antigen residue 124 both genetically and biochemically and have investigated individual replication functions of T antigen under conditions suitable for in vitro DNA replication. We find that the hexamer assembly, helicase, DNA polymerase alpha-binding, and transcriptional-autoregulation functions are independent of phosphorylation of threonine 124. In contrast, neither T antigen with an alanine mutation of threonine 124 made in human cells nor unphosphorylated T antigen made in Escherichia coli binds the SV40 replication origin as stably as phosphorylated wild-type T antigen does. Furthermore, modification of threonine 124 is essential for complete unwinding of the SV40 replication origin. We conclude that phosphorylation of threonine 124 enhances specific interactions of T antigen with SV40 origin DNA. Our findings do not exclude the possibility that phosphorylation of threonine 124 may affect additional undefined steps in DNA replication. We also show that DNase footprinting and KMnO4 modification assays are not as stringent as immunoprecipitation and origin-dependent strand displacement assays for detecting defects in the origin-binding and -unwinding functions of T antigen. Differences in the assays may explain discrepancies in previous reports on the role of T-antigen phosphorylation in DNA binding.
Gibbon ape leukemia virus and the amphotropic murine leukemia virus 4070A exhibit an unusual interference pattern on E36 Chinese hamster cells
Authors: Eglitis M A , Eiden M V , Wilson C A
Published: January 2020

The gibbon ape leukemia virus (GaLV), the amphotropic mouse leukemia virus (A-MLV) 4070A, and the xenotropic mouse leukemia virus (X-MLV) exhibit wide but not identical species host ranges. However, most Chinese hamster cells resist infection by all three viruses. We have now determined that the Chinese hamster cell line E36 differs from other Chinese hamster cell lines in that it is susceptible to infection by wild-type GaLV, A-MLV, and X-MLV. Surprisingly, analysis of the interference pattern of GaLV and A-MLV in E36 cells indicated that GaLV and A-MLV interfere in a nonreciprocal fashion. E36 cells productively infected with GaLV were resistant to superinfection by both GaLV and amphotropically packaged recombinant retroviral vectors. In contrast, E36 cells infected with A-MLV were resistant to superinfection with an amphotropic vector but could still be infected by a GaLV vector. These results imply the existence of a receptor on E36 cells that interacts with both GaLV and A-MLV.
Translational inhibition mediated by a short upstream open reading frame in the human cytomegalovirus gpUL4 (gp48) transcript
Authors: Degnin C R , Schleiss M R , Cao J , Geballe A P
Published: January 2020

The human cytomegalovirus (CMV) virion glycoprotein gpUL4 (gp48) gene expresses a transcript that contains three AUG codons upstream from the one used to initiate synthesis of the gp48 protein. Previously we reported that the second of these AUG codons, AUG2, was necessary but insufficient for inhibition of downstream translation (M. Schleiss, C. R. Degnin, and A. P. Geballe, J. Virol. 65:6782-6789, 1991). We now demonstrate that the coding information of the upstream open reading frame initiated by AUG2 (uORF2) is critical for the inhibitory signal. Several missense mutations, particularly those involving the carboxy-terminal codons of uORF2, inactivate the inhibitory signal, while mutations that preserve the coding content of uORF2 uniformly retain the inhibitory signal. The uORF2 termination codon is essential for inhibition, but leader sequences further downstream are not critical. Conservation of uORF2 among clinical strains of CMV suggests that uORF2 provides an important function in the CMV infectious cycle. Although these results indicate that the peptide product of uORF2 mediates the inhibitory effect, we demonstrate that the uORF2 signal acts only in cis, and we propose a model of inhibition by the gp48 uORF2 signal.
Role of the His-Cys finger of Moloney murine leukemia virus integrase protein in integration and disintegration
Authors: Jonsson C B , Roth M J
Published: January 2020

Retroviral integrases mediate site-specific endonuclease and transesterification reactions in the absence of exogenous energy. The basis for the sequence specificity in these integrase-viral DNA recognition processes is unknown. Structural analogs of the disintegration substrate were made to analyze the disintegration reaction mechanism for the Moloney murine leukemia virus (M-MuLV) integrase (IN). Modifications in the target DNA portion of the disintegration substrate decreased enzymatic activity, while substitution of the highly conserved CA in the viral long terminal repeat portion had no effect on activity. The role of the His-Cys finger region in catalysis was addressed by N-ethylmaleimide (NEM) modification of the cysteine residues of M-MuLV IN as well as by mutations. Both integration activities, 3' processing, and strand transfer, were completely inhibited by NEM modification of M-MuLV IN, while disintegration activity was only partially sensitive. However, structural analogs of the disintegration substrates that were modified in the target DNA and had the conserved CA removed were not active with NEM-treated M-MuLV IN. In addition, mutants made in the His-Cys region of M-MuLV IN were examined and found to also be completely blocked in integration but not disintegration activity. These data suggest that the domains of M-MuLV IN that are required for the forward integration reaction substrate differ from those required for the reverse disintegration reaction substrate.
Functional analysis of interactions between Tat and the trans-activation response element of human immunodeficiency virus type 1 in cells
Authors: Luo Y , Madore S J , Parslow T G , Cullen B R , Peterlin B M
Published: January 2020

Transcriptional trans-activation of the human immunodeficiency virus type 1 long terminal repeat requires that the virally encoded Tat effector interacts with its target trans-activation response element (TAR) RNA stem-loop. Although the arginine-rich region of Tat from amino acids 49 to 59 is sufficient to bind to TAR RNA in vitro, the RNA-binding domain of Tat has not been defined in vivo. Human immunodeficiency virus type 1 also encodes the Rev protein, which acts through an RNA stem-loop called the Rev-response element to transport unspliced and singly spliced viral RNA species from the nucleus to the cytoplasm. To map the RNA-binding domain of Tat, we performed assays that relied on Rev function using the heterologous RNA-tethering mechanism of Tat and the TAR. By examining the effects of selected targeted mutations of Tat on the abilities of hybrid Tat/Rev proteins to rescue the expression of unspliced mRNA via the TAR, we demonstrated that residues throughout the N-terminal 59 amino acids of Tat are required for binding of Tat and TAR RNA in vivo.
Cooperative DNA binding of the bovine papillomavirus E2 transcriptional activator is antagonized by truncated E2 polypeptides
Authors: Monini P , Blitz I L , Cassai E
Published: January 2020

Cooperative DNA binding of the bovine papillomavirus type 1 (BPV-1) E2 transcriptional activator (E2-TA) is thought to play a role in the transcriptional synergism of multiple E2-responsive DNA elements (J. Ham, N. Dostatni, J.-M. Gauthier, and M. Yaniv, Trends Biochem. Sci. 16:440-444, 1991). Binding-equilibrium considerations show that such involvement is unlikely, thereby suggesting that the E2-TA cooperative capacity may have evolved to play other, different roles. The role of cooperative interactions in the antagonistic activity of BPV-1-positive and BPV-1-negative E2 regulatory proteins was investigated by an in vitro quantitative gel shift assay. Viral repressor E2-TR, a truncated peptide encompassing the activator DNA-binding domain, possesses a small but measurable cooperative capacity. Furthermore, the minimal E2 DNA-binding domain interacts with the activator in a positive, heterocooperative manner. As a result, the in vitro competition of full-length and truncated E2 peptides appears to be (macroscopically) noncooperative. This heterocooperative effect is probably dominant in latently infected G0-G1 cells, in which repressor E2-TR is 10- to 20-fold more abundant than the activator. The data are discussed considering the possible role of homo- and heterocooperative DNA binding in E2-conditional gene expression.
Studies of the major reovirus core protein sigma 2: reversion of the assembly-defective mutant tsC447 is an intragenic process and involves back mutation of Asp-383 to Asn
Authors: Coombs K M , Mak S C , Petrycky-Cox L D
Published: January 2020

The reovirus group C temperature-sensitive mutant tsC447, whose defect maps to the S2 gene, which encodes the major core protein sigma 2, fails to assemble core particles at the nonpermissive temperature. To identify other proteins that may interact with sigma 2 during assembly, we generated and examined 10 independent revertants of the mutant. To determine which gene(s) carried a compensatory suppressor mutation(s), we generated intertypic reassortants between wild-type reovirus serotype 1 Lang and each revertant and determined the temperature sensitivities of the reassortants by efficiency-of-plating assays. Results of the efficiency-of-plating analyses indicated that reversion of the tsC447 defect was an intragenic process in all revertants. To identify the region(s) of sigma 2 that had reverted, we determined the nucleotide sequences of the S2 genes. In all revertant sequences examined, the G at nucleotide position 1166 in tsC447 had reverted to the A present in the wild-type sequence. This reversion leads to the restoration of a wild-type asparagine (in place of a mutant aspartic acid) at amino acid 383 in the sigma 2 sequence. These results collectively indicate that the functional lesion in tsC447 is Asp-383 and that this lesion cannot be corrected by alterations in other core proteins. These observations suggest that this region of sigma 2, which may be important in mediating assembly of the core particle, does not interact significantly with other reovirus proteins.
Inhibition of human immunodeficiency virus type 1 Tat-dependent activation of translation in Xenopus oocytes by the benzodiazepine Ro24-7429 requires trans-activation response element loop sequences
Authors: Braddock M , Cannon P , Muckenthaler M , Kingsman A J , Kingsman S M
Published: January 2020

Two benzodiazepine compounds, [7-chloro-5-(2-pyrryl)-3H-1,4 benzodiazapin-2-(H)-one] (Ro5-3335) and [7-chloro-5-(1H-pyrrol-2-yl)-3H-benzo[e] [1,4] diazepin-2-yl]- methylamine (Ro24-7429), inhibit human immunodeficiency virus type 1 (HIV-1) replication via a specific effect on the function of the transactivator protein, Tat. To gain further insight into the mechanism of action of these compounds, we have tested their effects in an alternative assay for Tat activation in Xenopus oocytes. In this system, translation of trans-activation response element (TAR)-containing RNA is activated by Tat. Both compounds specifically blocked activation of translation in a dose-dependent fashion, with Ro24-7429 showing the greater potency. In the Xenopus oocyte system, as in mammalian cells, mutation of the TAR loop sequences abolishes Tat action. However, it is possible to obtain TAR-specific, Tat-dependent activation of a target RNA with a mutation in the loop provided that this target is in large excess. This result has been interpreted as indicating that a negative factor has been titrated (M. Braddock, R. Powell, A.D. Blanchard, A.J. Kingsman, and S.M. Kingsman, FASEB J. 7:214-222, 1993). Interestingly Ro24-7429 was unable to inhibit the TAR-specific but loop sequence-independent mode of translational activation. This finding suggests that a specific loop-binding cellular factor may mediate the effects of this inhibitor of Tat action. Consistent with this notion, we could not detect any effect of Ro24-7429 on the efficiency of specific Tat binding to TAR in vitro.
The nonstructural glycoprotein of rotavirus affects intracellular calcium levels
Authors: Tian P , Hu Y , Schilling W P , Lindsay D A , Eiden J , Estes M K
Published: January 2020

Rotavirus infection of monkey kidney cells has been reported to result in a significant increase in the concentration of intracellular calcium. This increase in intracellular calcium was associated with viral protein synthesis and cytopathic effects in infected cells. We tested the effect of individual rotavirus proteins on intracellular calcium concentrations in insect Spodoptera frugiperda (Sf9) cells. Insect cells were infected with wild-type baculovirus or baculovirus recombinants that contained an individual rotavirus gene. The cells were harvested at different times postinfection, and the intracellular calcium concentration was measured by using fura-2 as a fluorescent calcium indicator. We found that the concentration of intracellular calcium was increased nearly fivefold in infected Sf9 cells that expressed the nonstructural glycoprotein (NSP4) of group A rotavirus, and this increase in intracellular calcium concentration coincided with NSP4 expression. A similar result was observed in insect cells expressing NSP4 from a group B rotavirus, suggesting the conservation of this function among rotavirus groups. Expression of the other 10 rotavirus proteins or of wild-type baculovirus proteins in Sf9 cells did not significantly increase intracellular calcium levels. These results suggest that the nonstructural glycoprotein NSP4 is responsible for the increase in cytosolic calcium observed in rotavirus-infected cells.
Requirement for double-strand breaks but not for specific DNA sequences in herpes simplex virus type 1 genome isomerization events
Authors: Sarisky R T , Weber P C
Published: January 2020

Herpes simplex virus type 1 (HSV-1) genome isomerization occurs as a result of DNA replication-mediated homologous recombination between several sets of inverted repeat sequences present in the viral DNA. The frequency with which this recombination occurs has been demonstrated to be dependent upon DNA homology length rather than specific sequences. However, the smallest of the viral inverted repeats, the alpha sequence, has been shown to function as a recombinational hot spot, leading to speculation that this sequence may represent a specific element through which genome isomerization is mediated. To investigate this apparent paradox, a quantitative transient recombination assay system was developed and used to examine the recombinogenic properties of a panel of alpha sequence mutants. This analysis revealed that the presence of both the pac1 and pac2 elements was both necessary and sufficient for the induction of high-frequency recombination events by the alpha sequence. However, it was the double-strand break promoted by pac1 and pac2 during cleavage and packaging at the alpha sequence, and not the DNA sequences of the elements themselves, which appeared to be critical for recombination. This was illustrated (i) by the inability of the same pac1 and pac2 sequences to mediate inversion events in cells infected with an HSV-1 mutant which was competent for DNA replication-dependent recombination but defective for the cleavage and packaging process and (ii) by the ability of double-strand breaks generated in non-HSV-1 DNA by an in vivo-expressed restriction endonuclease to significantly stimulate the initiation of recombination events in virus-infected cells. Thus, the alpha sequence appears to act as a hot spot for homologous recombination simply because it happens to coincide with the site of the double-strand break which is generated during the cleavage and packaging process, not because it contains discrete sequences which are required for this activity. However, it was found that this enhanced recombinogenicity disappeared when the element was flanked by regions of extensive sequence homology, particularly that of the large inverted repeats which flank the alpha sequence at its natural site in the HSV-1 genome. These findings are consistent with a model for HSV-1 genome isomerization in which recombination is initiated primarily by multiple random double-strand breaks which arise during DNA replication across the inverted repeats of the genome, rather than by a single specific break which occurs at the alpha sequence during the cleavage and packaging process.
Lower mutation rate of bovine leukemia virus relative to that of spleen necrosis virus
Authors: Mansky L M , Temin H M
Published: January 2020

Genetic variation of the more complex retroviruses in the human T-cell leukemia virus/bovine leukemia virus (HTLV/BLV) group is less than in some other retroviral genera. To test whether reverse transcription of HTLV/BLV group members is less error prone than that of members of other groups, we developed an assay for detecting forward mutations in BLV, similar to that developed for the simpler spleen necrosis virus (SNV). We used this system to study the rates and types of mutations that occur during a single replication cycle. We found that BLV reverse transcription is approximately two and one-half times less error prone than SNV reverse transcription (4.8 x 10(-6) versus 1.2 x 10(-5) mutation per bp per cycle, respectively). The relative numbers of all types of observed mutations (that is, base pair substitutions, frameshifts, deletions, and deletions with insertions) were similar for BLV and SNV.
Use of random systematic mutagenesis to generate viable human rhinovirus 14 chimeras displaying human immunodeficiency virus type 1 V3 loop sequences
Authors: Smith A D , Resnick D A , Zhang A , Geisler S C , Arnold E , Arnold G F
Published: January 2020

Random systematic mutagenesis was used to generate a library of human rhinovirus 14 chimeras that each display a segment from the V3 loop of human immunodeficiency virus type 1. The sequence XXIGPGRAXX, where X could be any of the 20 amino acids, was inserted at the neutralizing immunogenic site II of human rhinovirus 14 between VP2 residues 159 and 160. Twenty-five unique chimeric viruses were isolated, and the identity of their randomized residues was determined. A nonrandom amino acid distribution that may reflect structural requirements for viability was observed at the randomized positions. Fifteen of 25 chimeras were neutralized by one or more of a panel of four anti-human immunodeficiency virus type 1 V3 loop antibody preparations, indicating that antigenicity had been successfully transplanted. Libraries of chimeric viruses produced by using the techniques described may be a source of vaccines and other immunotherapeutic reagents. The random systematic mutagenesis methodology described should be generally useful for the rapid transplantation of foreign sequences into viral coat and other proteins to produce libraries containing members with the desired properties.