bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro susceptibility of T lymphocytes from chimpanzees (Pan troglodytes) to human herpesvirus 6 (HHV-6): a potential animal model to study the interaction between HHV-6 and human immunodeficiency virus type 1 in vivo
Authors: Lusso P , Markham P D , DeRocco S E , Gallo R C
Published: January 2020

The in vitro susceptibility of several nonhuman primate species to human herpesvirus 6 (HHV-6) was investigated. Only peripheral blood mononuclear cells from chimpanzees (Pan troglodytes) were found permissive to productive infection by HHV-6, indicating that the host range of HHV-6, albeit limited, may not be restricted to Homo sapiens. However, natural HHV-6 infection in chimpanzees, as well as in the other species tested, could not be documented by serological analysis. As previously observed with human cells, HHV-6 infection of chimpanzee peripheral blood mononuclear cells was highly cytopathic and the infected cells exhibited phenotypic features of activated T lymphocytes. Although in humans the majority of HHV-6-infected lymphocytes displayed the CD4 antigen, in chimpanzees a mixed CD4+ and CD8+ phenotype was observed. HHV-6 was also shown to productively coinfect individual chimpanzee T cells with human immunodeficiency virus type 1, resulting in an accelerated induction of cytopathicity. In light of these findings, we propose the utilization of chimpanzees as a potential animal model system to investigate the in vivo interaction between HHV-6 and human immunodeficiency virus type 1 and its relevance to the development of acquired immune deficiency syndrome.
Induction of protective immunity in animals vaccinated with recombinant vaccinia viruses that express PreM and E glycoproteins of Japanese encephalitis virus
Authors: Yasuda A , Kimura-Kuroda J , Ogimoto M , Miyamoto M , Sata T , Sato T , Takamura C , Kurata T , Kojima A , Yasui K
Published: January 2020

A cDNA clone representing the genome of structural proteins of Japanese encephalitis virus (JEV) was inserted into the thymidine kinase gene of vaccinia virus strains LC16mO and WR under the control of a strong early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. Indirect immunofluorescence and fluorescence-activated flow cytometric analysis revealed that the recombinant vaccinia viruses expressed JEV E protein on the membrane surface, as well as in the cytoplasm, of recombinant-infected cells. In addition, the E protein expressed from the JEV recombinants reacted to nine different characteristic monoclonal antibodies, some of which have hemagglutination-inhibiting and JEV-neutralizing activities. Radioimmunoprecipitation analysis demonstrated that two major proteins expressed in recombinant-infected cells were processed and glycosylated as the authentic PreM and E glycoproteins of JEV. Inoculation of rabbits with the infectious recombinant vaccinia virus resulted in rapid production of antiserum specific for the PreM and E glycoproteins of JEV. This antiserum had both hemagglutination-inhibiting and virus-neutralizing activities against JEV. Furthermore, mice vaccinated with the recombinant also produced JEV-neutralizing antibodies and were resistant to challenge with JEV.
The Epstein-Barr virus (EBV) ORI1yt enhancer is not B-cell specific and does not respond synergistically to the EBV transcription factors R and Z
Authors: Gruffat H , Moreno N , Sergeant A
Published: January 2020

The Epstein-Barr virus DR promoter is located upstream of the PstI repeats, and in addition to the TATA box, it contains an upstream region (positions -69 to -220) responsive to EB1 (Z) (the BZLF1-encoded transcription factor) and an enhancer with two functionally distinct domains, A and B. Domain B has been described as a B-cell-specific EB1-responsive element (P. M. Lieberman, J. M. Hardwick, and S. D. Hayward, J. Virol. 63:3040-3050, 1989) activated synergistically by EB1 and R, an EBV early product encoded by the open reading frame BRLF1 (M. A. Cox, J. Leahy, and J. M. Hardwick, J. Virol. 64:313-321, 1990). We show here that domain B is an R-responsive element in HeLa cells and is therefore not an EB1-responsive B-cell-specific element. However, there is an EB1-binding site (ZRE-B) located within the R-responsive enhancer region. ZRE-B can be deleted without affecting the R-dependent enhancer activity. Moreover, there is no cooperation or synergy between R and EB1 when activating the B domain (ZRE-B plus the R-responsive element) positioned as an enhancer. ZRE-B is therefore not part of the R-inducible enhancer. We have tested several subregions of the DR enhancer B domain, either alone or in combination, for their capacity to transmit the R-activating signal to the rabbit beta-globin promoter. We found that the R-responsive element is composed of four protoenhancers that span the whole B domain. These protoenhancers alone are weakly or not responsive to R. One of the protoenhancers contains the overlapping palindromes 5'-TTGTCCcgtGGACAAaTGTCC-3'. However, one palindrome, either alone or duplicated, or the overlapping palindromes did not respond to R.
Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies: peripheral glycosylation of HIV envelope glycoprotein gp120 may be a target for virus neutralization
Authors: Hansen J E , Clausen H , Nielsen C , Teglbjaerg L S , Hansen L L , Nielsen C M , Dabelsteen E , Mathiesen L , Hakomori S I , Nielsen J O
Published: January 2020

Carbohydrate structures are often involved in the initial adhesion of pathogens to target cells. In the present study, a panel of anticarbohydrate monoclonal antibodies (MAbs) was tested for their ability to inhibit in vitro human immunodeficiency virus infectivity. MAbs against three different N- and O-linked carbohydrate epitopes (LeY, A1, and sialyl-Tn) were able to block infection by cell-free virus as well as inhibit syncytium formation. Inhibition of virus infectivity was independent of virus strain (HTLVIIIB or patient isolate SSI-002), the cell line used for virus propagation (H9 or MT4), and the cell type used as the infection target (MT4, PMC, or selected T4 lymphocytes). Inhibition was observed when viruses were preincubated with MAbs but not when cells were preincubated with MAbs before inoculation, and the MAbs were shown to precipitate 125I-labeled gp120. The MAbs therefore define carbohydrate structures expressed by the viral envelope glycoprotein gp120, indicating that glycans of the viral envelope are possible targets for immunotherapy or vaccine development or both.
Mutational analysis of simian virus 40 small-t antigen
Authors: Jog P , Joshi B , Dhamankar V , Imperiale M J , Rutila J , Rundell K
Published: January 2020

Several point mutations in the simian virus 40 (SV40) small-t antigen have been analyzed for their effects on protein stability, transformation, transactivation, and binding of two cellular proteins. All mutations which affected cysteine residues in two cysteine clusters produced highly unstable small-t antigens. Four point mutations outside these clusters and one in-frame deletion mutant, dl890, produced stable proteins but reduced transformation efficiency. These were able to transactivate the EII promoter and bind the cellular proteins, suggesting that these activities are not sufficient for small-t-mediated enhancement of transformation.
Intrachromosomal recombination mediated by papovavirus large T antigens
Authors: St-Onge L , Bouchard L , Laurent S , Bastin M
Published: January 2020

To investigate the mechanism by which the large T antigen (T-Ag) of polyomavirus and simian virus 40 can promote recombination in mammalian cells, we analyzed homologous recombination events occurring between two defective copies of the polyomavirus middle T (pmt) oncogene lying in close proximity on the same chromosome in a rat cell line. Reconstitution of a functional pmt gene by spontaneous recombination occurred at a rate of about 2 x 10(-7) per cell generation. Introduction of the polyomavirus large T (plt) oncogene into the cell line by DNA transfection promoted recombination very efficiently, with rates in the range of 10(-1) to 10(-2) per cell generation. Recombination was independent of any amplification of viral sequences and could even be promoted by the large T-Ag from simian virus 40, which cannot activate polyomavirus DNA replication. To explain the role of large T-Ag, we propose a novel mechanism of nonconservative recombination involving slipped-strand mispairing between the two viral repeats followed by gap repair synthesis.
Conformational change of the coronavirus peplomer glycoprotein at pH 8.0 and 37 degrees C correlates with virus aggregation and virus-induced cell fusion
Authors: Sturman L S , Ricard C S , Holmes K V
Published: January 2020

We have obtained biochemical and electron microscopic evidence of conformational changes at pH 8.0 and 37 degrees C in the coronavirus spike glycoprotein E2 (S). The importance of these changes is reflected in the loss of virus infectivity, the aggregation of virions, and increased virus-induced cell fusion at the same pH. Coronavirus (MHV-A59) infectivity is exquisitely sensitive to pH. The virus was quite stable at pH 6.0 and 37 degrees C (half-life, approximately 24 h) but was rapidly and irreversibly inactivated by brief treatment at pH 8.0 and 37 degrees C (half-life, approximately 30 min). Virions treated at pH 8.0 and 37 degrees C formed clumps and large aggregates. With virions treated at pH 8.0 and 37 degrees C, the amino-terminal peptide E2N (or S1) was released from virions and the remaining peptide, E2C (S2), was aggregated. Viral spikes isolated from detergent-treated virions also aggregated at pH 8.0 and 37 degrees C. Loss of virus infectivity and E2 (S) aggregation at pH 8.0 and 37 degrees C were markedly enhanced in the presence of dithiothreitol. On the basis of the effects of dithiothreitol on the reactions of the peplomer, we propose that release of E2N (S1) and aggregation of E2C (S2) may be triggered by rearrangement of intramolecular disulfide bonds. The aggregation of virions and the isolated E2 (S) glycoprotein at pH 8.0 and 37 degrees C or following treatment with guanidine and urea at pH 6.0 and 37 degrees C indicate that an irreversible conformational change has been induced in the peplomer glycoprotein by these conditions. It is interesting that coronavirus-induced cell fusion also occurred under mildly alkaline conditions and at 37 degrees C. Some enveloped viruses, including influenza viruses and alphaviruses, show conformational changes of spike glycoproteins at a low pH, which correlates with fusion and penetration of those viruses in acidified endocytic vesicles. For coronavirus MHV-A59, comparable conformational change of the spike glycoprotein E2 (S) and cell fusion occurred at a mildly alkaline condition, suggesting that coronavirus infection-penetration, like that of paramyxoviruses and lentiviruses, may occur at the plasma membrane, rather than within endocytic vesicles.
Human immunodeficiency virus vpr product is a virion-associated regulatory protein
Authors: Cohen E A , Dehni G , Sodroski J G , Haseltine W A
Published: January 2020

The vpr product of the human immunodeficiency virus type 1 (HIV-1) acts in trans to accelerate virus replication and cytopathic effect in T cells. Here it is shown that the HIV-1 viral particle contains multiple copies of the vpr protein. The vpr product is the first regulatory protein of HIV-1 to be found in the virus particle. This observation raises the possibility that vpr acts to facilitate the early steps of infection before de novo viral protein synthesis occurs.
Transient dominant selection of recombinant vaccinia viruses
Authors: Falkner F G , Moss B
Published: January 2020

A general method for constructing and selecting recombinant vaccinia viruses with insertions, deletions, or mutations in any gene that is similar in principle to one originally devised for Saccharomyces cerevisiae (S. Scherer and R. W. Davis, Proc. Natl. Acad. Sci. USA 76:4951-4955, 1979) is described. The selectable marker used, Escherichia coli guanine phosphoribosyltransferase, is not retained within the final recombinant virus, and hence, this procedure may be used serially to introduce several foreign genes or to make multiple site-directed mutations.
Enhancer functions in U3 of Akv virus: a role for cooperativity of a tandem repeat unit and its flanking DNA sequences
Authors: Lovmand S , Kjeldgaard N O , Jørgensen P , Pedersen F S
Published: January 2020

cis-Acting transcriptional control elements in the U3 region of the murine retrovirus Akv were analyzed in mouse NIH 3T3 fibroblast cells by using a transient expression vector system based upon a complete long terminal repeat with linked flanking sequences. Deletion analysis pointed to the essential role of sequences within the 99-base-pair direct repeats, and a fragment encompassing the two repeats was found to possess orientation-independent enhancer activity when positioned either upstream or downstream of the transcriptional unit. Removal of one copy of the 99-base-pair repeat led to a reduction in activity of about 2.5-fold when located in an intact U3 environment but to reductions of up to 2 orders of magnitude when placed in other sequence contexts. Our studies of enhancer functions in the presence of one versus two copies of the tandem repeat point to duplicate functions of repeat sequences and sequences flanking the repeat region and emphasize the complex overall organization of this U3 region.
Efficient duck hepatitis B virus production by an avian liver tumor cell line
Authors: Condreay L D , Aldrich C E , Coates L , Mason W S , Wu T T
Published: January 2020

Duck hepatitis B virus (DHBV) is produced in small amounts following transfection of human hepatoma or hepatoblastoma cell lines with cloned viral DNA. In a search for better hosts for DHBV replication, two avian liver cell lines were investigated. One of these cell lines, LMH, produced 5 to 10 times more DNA replicative intermediates and 10 to 20 times more infectious DHBV than did either of the two human cell lines, HuH-7 and Hep G2. Utilization of cell lines in genetic analyses of virus replication is often dependent upon obtaining efficient complementation between cotransfected viral genomes. We assayed transcomplementation of a viral polymerase (pol) gene mutant, which is rather inefficient in transfected human cells, and found that viral DNA synthesis was at least 20 times more efficient following cotransfection of LMH cells than in similarly transfected HuH-7 cells. Recombination, a potential interpretation problem in complementation assays, occurred at low levels in the cotransfected cultures but was substantially reduced or eliminated by creation of an LMH subline stably expressing the viral polymerase. This cell line, pol-7, supported the replication of DHBV pol mutants at ca. 10 to 15% of the level of virus replication obtained following transfection with wild-type viral DNA. By transcomplementation of a pol gene mutant in LMH cells, we were able to produce sufficient virus with the mutant genome to investigate the role of polymerase in covalently closed circular DNA amplification. Our results substantiate the hypothesis that covalently closed circular DNA is synthesized by the viral reverse transcriptase.
Characterization of envelope proteins of alcelaphine herpesvirus 1
Authors: Adams S W , Hutt-Fletcher L M
Published: January 2020

Alcelaphine herpesvirus 1 is a gammaherpesvirus which causes malignant catarrhal fever, an acute lymphoproliferative disorder of cattle and other susceptible Bovidae, which is almost invariably fatal. A preliminary analysis of proteins induced by the virus indicated that as many as six glycoproteins and one nonglycosylated molecule might be present in the virus envelope. Monoclonal antibodies selected for recognition of virion envelope proteins included two that recognized a complex of infected cell proteins, designated the gp115 complex, and neutralized virus infectivity in the absence of complement. The gp115 complex consisted of five glycoproteins of 115, 110, 105, 78, and 48 kilodaltons (kDa), and all except the 48-kDa species reacted with antibody in Western blots (immunoblots). Pulse-chase experiments analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions suggested that the 110-kDa protein was the precursor molecule which was processed by addition of sugars to 115 kDa. The 115-kDa protein was cleaved to form a disulfide-linked heterodimer of 78 and 48 kDa, which was the mature form of the molecule incorporated into the virion envelope. The glycoprotein contained N-linked sugars, but little or no O-linked sugar was present. The relative abundance of the mature protein and its ability to induce neutralizing antibodies suggest that it will prove useful to studies aimed at elucidating the biology and pathogenesis of alcelaphine herpesvirus 1.
Expression of collagenlike sequences by a tumor virus, herpesvirus saimiri
Authors: Geck P , Whitaker S A , Medveczky M M , Medveczky P G
Published: January 2020

Sequencing demonstrates that the oncogenic regions of a group A strain and a group C strain of herpesvirus saimiri are nonhomologous. A bicistronic viral mRNA from this region is transcribed in tumor cells transformed by a highly oncogenic group C virus. The first open reading frame is homologous to collagen; no such sequences were found in group A or B strains. This is the first report that a virus encodes for sequences similar to those of a connective tissue protein.
The putative cytoplasmic domain of the pseudorabies virus envelope protein gIII, the herpes simplex virus type 1 glycoprotein C homolog, is not required for normal export and localization
Authors: Solomon K A , Robbins A K , Whealy M E , Enquist L W
Published: January 2020

Glycoprotein gIII of pseudorabies virus is a member of a conserved gene family found in at least seven diverse herpesviruses. We report here that the putative cytoplasmic domain of gIII is not required for transport to the cell surface and, unlike the prototype domain from herpes simplex virus type 1 glycoprotein C, is not required for stable membrane anchoring. Furthermore, this domain does not appear to be essential for incorporation of the glycoprotein into virions.
Differences in the basal activity of the long terminal repeat determine different replicative capacities of two closely related human immunodeficiency virus type 1 isolates
Authors: Golub E I , Li G G , Volsky D J
Published: January 2020

Two human immunodeficiency virus type 1 (HIV-1) variants derived from a single parental isolate were found to differ substantially in their ability to replicate in CD4-positive cells. Using transient chloramphenicol acetyltransferase expression assays, we show that the long terminal repeat (LTR) of the better-replicating virus has significantly higher capacity than that of the companion virus to direct gene expression in T cells. Sequence data and site-specific mutagenesis experiments demonstrate that the higher LTR activity of the better-replicating HIV-1 is due to a combined effect of two mutations: (i) a point mutation in position -94 (relative to the transcriptional start site), which is located between the two subunits of the HIV-1 enhancer, and (ii) a duplication of 24 base pairs in positions -128 to -151, which was not previously known to be involved in any regulatory function. The presence of these mutations increases the basal level of the LTR-driven gene expression and does not influence the degree of induction caused by the viral tat gene product or by cell activation. Reciprocal exchange of LTRs between the respective viral DNAs results in a change of a recombinant virus replication pattern consistent with the activity of the particular LTR. These experiments suggest that the HIV-1 LTR is one of the sites which determines the functional heterogeneity of HIV-1.
Cloning and characterization of human immunodeficiency virus type 1 variants diminished in the ability to induce syncytium-independent cytolysis
Authors: Stevenson M , Haggerty S , Lamonica C , Mann A M , Meier C , Wasiak A
Published: January 2020

The phenomenon of interference was exploited to isolate low-abundance noncytopathic human immunodeficiency virus type 1 (HIV-1) variants from a primary HIV-1 isolate from an asymptomatic HIV-1-seropositive hemophiliac. Successive rounds of virus infection of a cytolysis-susceptible CD4+ cell line and isolation of surviving cells resulted in selective amplification of an HIV-1 variant reduced in the ability to induce cytolysis. The presence of a PvuII polymorphism facilitated subsequent amplification and cloning of cytopathic and noncytopathic HIV-1 variants from the primary isolate. Cloned virus stocks from cytopathic and noncytopathic variants exhibited similar replication kinetics, infectivity, and syncytium induction in susceptible host cells. The noncytopathic HIV-1 variant was unable, however, to induce single-cell killing in susceptible host cells. Construction of viral hybrids in which regions of cytopathic and noncytopathic variants were exchanged indicated that determinants for the noncytopathic phenotype map to the envelope glycoprotein. Sequence analysis of the envelope coding regions indicated the absence of two highly conserved N-linked glycosylation sites in the noncytopathic HIV-1 variant, which accompanied differences in processing of precursor gp160 envelope glycoprotein. These results demonstrate that determinants for syncytium-independent single-cell killing are located within the envelope glycoprotein and suggest that single-cell killing is profoundly influenced by alterations in envelope sequence which affect posttranslational processing of HIV-1 envelope glycoprotein within the infected cell.
T-cell lymphoma lines derived from rat thymomas induced by Moloney murine leukemia virus: phenotypic diversity and its implications
Authors: Lazo P A , Klein-Szanto A J , Tsichlis P N
Published: January 2020

The phenotype of 27 Moloney murine leukemia virus-induced rat thymic lymphomas and 36 cell lines derived from these tumors was determined by using 18 monoclonal antibodies directed against hematopoietic cell surface determinants. The cell lines and the primary tumors from which they were derived were clonally related as determined by the pattern of provirus integration and the pattern of rearrangement of the T-cell receptor beta and delta and Igh loci. The differentiation phenotype of the primary tumors and the cell lines derived from them were related. The differences observed between the primary tumors and the cell lines could be explained either by the selection of subpopulations of tumor cells during establishment in culture or by the phenotypic instability of the tumor cells. One cell line (LE3Sp) underwent the transition from a CD4+ CD8+ to a CD4+ CD8- phenotype following exposure to interleukin-2 in culture. Both the primary tumors and the cell lines derived from them express a wide range of phenotypes which correspond to multiple stages in T-cell development. This observation suggests that the pleiomorphism of retrovirus-induced lymphomas, which had been suggested previously from the analysis of mouse tumors, is an intrinsic property of the process of oncogenesis and is not due to the transformation of different types of cells by spontaneously arising leukemogenic variants of the inoculated virus. The wide spectrum of phenotypes expressed by these tumors suggests that Moloney murine leukemia virus may infect and transform T cells at various stages of development. Alternatively, the target cells may be immature T-cell precursors which, following transformation, continue to differentiate. A host of early findings, suggesting that the repertoire of target cells is restricted to poorly differentiated hematopoietic progenitors, and the ability of the LE3Sp cell line to differentiate in culture indicate that the latter possibility may be more likely. The data in this report address the extent and mechanism of the phenotypic variability of retrovirus-induced rodent T-cell lymphomas. In addition, they demonstrate the potential usefulness of the T-cell lymphoma lines we have established in studies of oncogenesis and T-cell differentiation.
Envelope glycoprotein of avian hemangioma retrovirus induces a thrombogenic surface on human and bovine endothelial cells
Authors: Resnick-Roguel N , Eldor A , Burstein H , Hy-Am E , Vlodavsky I , Panet A , Blajchman M A , Kotler M
Published: January 2020

Vascular endothelial cells are a target for blood-borne pathogens which may affect their integrity and thromboresistant properties. Here, we report that cultured bovine and human endothelial cells lose their thromboresistance following interaction with the avian hemangioma-inducing retrovirus. We show that the envelope (env) gene product, glycoprotein 85, is responsible for this effect, which appears soon after infection without viral replication or cell transformation. Induction of thrombogenicity is associated with a reduction in prostacyclin release and increased expression of tissue factor. These observations may explain the occurrence of thrombosis frequently observed in association with the hemangiosarcomas induced by avian hemangioma-inducing retrovirus. These unique endothelial cell-virus interactions may also be a model for the pathogenesis of various vascular diseases.
Involvement of nuclear factor I-binding sites in control of Akv virus gene expression
Authors: Olsen H S , Lovmand S , Lovmand J , Jørgensen P , Kjeldgaard N O , Pedersen F S
Published: January 2020

The U3 region of Akv murine leukemia virus carries a 99-base-pair repeat that is associated with transcriptional enhancement in murine NIH 3T3 cells. Deletion analysis points to a critical function of a region within the repeat unit related to the recognition sequences for nuclear factor I proteins but distinct from the sites previously analyzed in related viruses. Nuclear proteins binding to the critical site were detected in NIH 3T3 cells and in mouse livers. A protein fraction binding to this site was purified from mouse livers by ion-exchange and DNA affinity chromatography and shown to have nuclear factor I properties. Mutations that caused a partial or complete reduction of the in vitro binding were introduced into an Akv long terminal repeat with one 99-base-pair repeat copy driving a reporter gene, and the expression activities of the mutants in NIH 3T3 cells were found to correspond to their in vitro binding activities. This correlation strongly supports the role of nuclear factor I proteins in Akv expression. Residual expression activity was, however, detected in mutants devoid of in vitro binding. This residual activity may relate to the presence of additional sequences with homology to nuclear factor I binding sites both within and outside the repeat region. The ability of these sites to bind crude and purified protein fractions with nuclear factor I activity was analyzed, and the role of the sites within and outside the repeat region for control of gene expression of Akv and related viruses is discussed.
Incorporation of chimeric gag protein into retroviral particles
Authors: Weldon R A , Erdie C R , Oliver M G , Wills J W
Published: January 2020

The product of the Rous sarcoma virus (RSV) gag gene, Pr76gag, is a polyprotein precursor which is cleaved by the viral protease to yield the major structural proteins of the virion during particle assembly in avian host cells. We have recently shown that myristylated forms of the RSV Gag protein can induce particle formation with very high efficiency when expressed in mammalian cells (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). We made use of this mammalian system to examine the abilities of foreign antigens to be incorporated into particles when fused directly to the myristylated Gag protein. Our initial experiments showed that removal of various portions of the viral protease located at the carboxy terminus of the RSV Gag protein did not disrupt particle formation. We therefore chose this region for coupling of iso-1-cytochrome c from Saccharomyces cerevisiae to Gag. This was accomplished by constructing an in-frame fusion of the CYC1 and gag coding sequences at a common restriction endonuclease site. Expression of the chimeric gene resulted in synthesis of the Gag-cytochrome fusion protein and its release into the cell culture medium. The chimeric particles were readily purified by simple centrifugation, and transmission electron microscopy of cells that produced them revealed a morphology similar to that of immature type C retrovirions.
Antibodies with specificities against a dispase-produced 15-kilodalton hexon fragment neutralize adenovirus type 2 infectivity
Authors: Varga M J , Bergman T , Everitt E
Published: January 2020

During the entrance of adenovirus type 2 into cells, it has been suggested that the virion undergoes a conformational change. In this investigation, we have further characterized the hypothetical conformational change, which the structural protein hexon undergoes in response to low pH. From pH 5.0 to pH 6.0, the proteolytic enzyme dispase cleaved the hexon into a few distinct fragments with a dominating low-molecular-weight fragment with a molecular weight of 15,000 (15K peptide), whereas between pH 6.5 and pH 8.0, the cleavage of the hexon was negligible. The degradation of the hexon with dispase at low pH was not due to an increased activity or alteration of the active site of dispase at low pH. The 15K fragment was identified as a segment of the N-terminal part of the hexon polypeptide beginning at amino acid residue 5. An immune serum produced in response to acid-treated and glutaraldehyde-fixed hexons contained a small amount of antibodies directed towards the 15K fragment, as judged by Western immunoblotting. An anti-15K antibody fraction was isolated by affinity chromatography by removing antibodies recognizing the hexon in the alkaline configuration. Such antibodies displayed a higher relative titer at pH 5.0 than at pH 7.5 in an enzyme-linked immunosorbent assay. The isolated antibodies showed a specific neutralizing capacity five times higher than that of the corresponding unfractionated polyclonal anti-hexon serum; however, the neutralizing ability was independent of pH. The neutralization of adenovirus type 2 infection by the isolated anti-15K antibodies implies that the N-terminal end of the hexon may play a critical role in the early steps of the virion-cell interaction.
Function of Semliki Forest virus E3 peptide in virus assembly: replacement of E3 with an artificial signal peptide abolishes spike heterodimerization and surface expression of E1
Authors: Lobigs M , Zhao H X , Garoff H
Published: January 2020

The Semliki Forest virus spike glycoproteins E1 and p62 form a heterodimeric complex in the endoplasmic reticulum (ER) and are transported as such to the cell surface. In the mature virus particle, the heterodimeric association of E1 and E2 (the cleavage product of p62) is maintained, but as a more labile and acid-sensitive oligomer than the E1-p62 complex. The E3 peptide forms the N-terminal part of the p62 precursor and carries the signal for the translocation of p62 into the lumen of the ER. The question of whether E3 is also important in the formation and stabilization of the E1-p62 heterodimer has been addressed here with the aid of an E3 deletion mutant cDNA. In this construct, the entire E3 was replaced with a cleavable, artificial signal sequence which preserved the membrane topology of an authentic E2. The E3 deletion, when expressed via a recombinant vaccinia virus, abolished heterodimerization of the spike proteins. It also resulted in the complete retention of E1 in the ER and almost total inhibition of E2 transport to the plasma membrane. The oligomerization and transport defect of E1 expressed from the E3 deletion mutant could be complemented with a wild-type p62 provided from a separate coding unit in double infections. These results point to a central role of E3 in complex formation and transport of the viral structural components to the site of budding. In conjunction with earlier work (M. Lobigs and H. Garoff, J. Virol. 64:1233-1240, 1990; J. Wahlberg, W. A. M. Boere, and H. Garoff, J. Virol. 63:4991-4997, 1989), the data support a model of spike protein oligomerization control of Semliki Forest virus assembly and disassembly which may be mediated by the presence of E3 in the uncleaved p62 precursor and release of E3 after cleavage.
Differential expression in human and mouse cells of human immunodeficiency virus pseudotyped by murine retroviruses
Authors: Chesebro B , Wehrly K , Maury W
Published: January 2020

Expression of cell surface CD4 influences susceptibility of cells to human immunodeficiency virus (HIV) infection; however, some CD4-positive human and mouse cells are still resistant to HIV infection. To search for mechanisms of resistance to HIV independent of CD4 expression, HIV expression was studied in human and mouse cells normally resistant to HIV infection by introducing infectious virus by transfection of HIV DNA or infection with HIV pseudotyped with amphotropic or polytropic murine leukemia viruses. The results indicated that even when barriers to viral entry were bypassed, mouse NIH 3T3 cells and Dunni cells still showed a marked reduction in number of cells expressing HIV compared with the human cells studied, although the intensity of immunostaining of individual positive mouse cells was indistinguishable from that seen on permissive human cell lines. CD4 expression in mouse cells or human brain or skin cells did not influence the number of HIV foci observed after transfection with HIV DNA or infection with pseudotyped HIV. These results suggested that in addition to a block in the usual HIV fusion and entry process, CD4-positive mouse cells differed from human cells in exhibiting partial resistance to HIV infection which acted at a postpenetration step in the infection cycle. This resistance was partially overcome when mouse cells were infected by direct exposure to human lymphocytes producing HIV pseudotyped by amphotropic murine leukemia virus.
Novel catalytic activity associated with positive-strand RNA virus infection: nucleic acid-stimulated ATPase activity of the plum pox potyvirus helicaselike protein
Authors: Laín S , Martín M T , Riechmann J L , García J A
Published: January 2020

The cylindrical inclusion protein of potyviruses contains the so-called nucleoside triphosphate binding motif, an amino acid sequence motif present in proteins encoded by most positive-strand RNA viruses, some double-strand RNA viruses, apparently all groups of double-strand DNA viruses, and also several single-strand DNA viruses. Further sequence analysis has allowed to include the cylindrical inclusion protein of potyviruses as a member of a superfamily of helicaselike proteins. In this paper we show that the purified cylindrical inclusion protein of plum pox potyvirus interacts with RNA and ATP and copurifies with a nucleic acid-stimulated ATPase activity. To our knowledge, this is the first time that this kind of enzymatic activity has been experimentally associated with a positive-strand RNA virus-encoded protein.
Expression of yeast L-A double-stranded RNA virus proteins produces derepressed replication: a ski- phenocopy
Authors: Wickner R B , Icho T , Fujimura T , Widner W R
Published: January 2020

The plus strand of the L-A double-stranded RNA virus of Saccharomyces cerevisiae has two large open reading frames, ORF1, which encodes the major coat protein, and ORF2, which encodes a single-stranded RNA-binding protein having a sequence diagnostic of viral RNA-dependent RNA polymerases. ORF2 is expressed only as a Gag-Pol-type fusion protein with ORF1. We have constructed a plasmid which expresses these proteins from the yeast PGK1 promoter. We show that this plasmid can support the replication of the killer toxin-encoding M1 satellite virus in the absence of an L-A double-stranded RNA helper virus itself. This requires ORF2 expression, providing a potential in vivo assay for the RNA polymerase and single-stranded RNA-binding activities of the fusion protein determined by ORF2. ORF1 expression, like a host ski- mutation, can suppress the usual requirement of M1 for the MAK11, MAK18, and MAK27 genes and allow a defective L-A (L-A-E) to support M1 replication. These results suggest that expression of ORF1 from the vector makes the cell a ski- phenocopy. Indeed, expression of ORF1 in a wild-type killer makes it a superkiller, suggesting that a target of the SKI antiviral system may be the major coat protein.
Identification of eight determinants in the hemagglutinin molecule of influenza virus A/PR/8/34 (H1N1) which are recognized by class II-restricted T cells from BALB/c mice
Authors: Gerhard W , Haberman A M , Scherle P A , Taylor A H , Palladino G , Caton A J
Published: January 2020

Eight nonoverlapping regions of the hemagglutinin (HA) molecule of influenza virus A/PR/8/34 (PR8), which serve as recognition sites for class II-restricted T cells (TH) from BALB/c mice, have been identified in the form of 10- to 15-amino-acid-long synthetic peptides. These TH determinants are located between residues 110 to 313 of the HA1 polypeptide. From a total of 36 HA-specific TH clones and limiting-dilution cultures of independent clonal origins, 33 (90%) responded to stimulation with one of these peptides. The residual three TH clones appeared to recognize a single additional determinant on the HA1 polypeptide which could not be isolated, however, in the form of a stimulatory peptide. None of the motifs that have been proposed to typify TH determinants were displayed by more than half of these recognition sites. Most unexpected was the finding that none of the TH determinants was located in the ectodomain of the HA2 polypeptide that makes up roughly one-third of the HA molecule. Possible reasons for the preferential recognition of HA1 as opposed to HA2 by TH are discussed.
Characterization of a human cytomegalovirus 1.6-kilobase late mRNA and identification of its putative protein product
Authors: Lahijani R S , Otteson E W , Adlish J D , St Jeor S C
Published: January 2020

In a previous study (J. Martinez, R. S. Lahijani, and S. C. St. Jeor, J. Virol. 63:233-241, 1989), we identified a late, unspliced 1.6-kb mRNA that maps to the HindIII R fragment of human cytomegalovirus (HCMV) AD169. In the present study, the direction of transcription of this mRNA was determined by Northern (RNA) analysis with strand-specific probes. Primer extension was used to precisely map the 5' end of the mRNA. An open reading frame (ORF) designated ORF 2-1, located 176 nucleotides downstream from the cap site of the 1.6-kb mRNA, was identified. A synthetic peptide was made representing a hydrophilic region in the amino terminus of ORF 2-1. Immunoprecipitation and Western immunoblot analysis of infected HEL cell lysates, using affinity-purified antibody to the peptide (anti-P2-1), detected a viral protein with an apparent molecular mass of 58 kDa late in infection. Further support for the presence of this protein in infected-cell lysates was obtained by an enzyme-linked immunosorbent assay. Expression of viral antigens in intact infected HEL cells was assessed by immunofluorescence. General cytoplasmic staining was observed at 62 h postinfection, in contrast to a localized staining observed in the nuclear and perinuclear region at 96 h postinfection.
Rat primary embryo fibroblast cells suppress transformation by the E6 and E7 genes of human papillomavirus type 16 in somatic hybrid cells
Authors: Miyasaka M , Takami Y , Inoue H , Hakura A
Published: January 2020

The E6 and E7 genes of human papillomavirus type 16 (HPV-16) transform established lines of rat cells but not rat cells in primary culture irrespective of the expression of the two genes. The reason for this difference between the susceptibilities of cell lines and primary cells was examined by using hybrid cells obtained by somatic cell fusion of rat cell lines transformed by the E6 and E7 genes of HPV-16 and freshly isolated rat embryo fibroblast cells. In these hybrid cells, transformed phenotypes, including colony formation in soft agar, saturation density, and tumorigenicity in nude mice, were suppressed, whereas hybrid cells between E6/E7-transformed cell lines and normal rat cell lines retained these transformed phenotypes. RNA analysis showed that the E6 and E7 genes were transcribed in both types of hybrid cells. These results suggest that primary rat cells possess intracellular functions that cause posttranscriptional suppression of induction of the transformed phenotypes by the E6 and E7 genes of HPV-16.
Differential rates of processing and transport of herpes simplex virus type 1 glycoproteins gB and gC
Authors: Sommer M , Courtney R J
Published: January 2020

The kinetics of processing and transport of herpes simplex virus type 1 (HSV-1) glycoproteins gB and gC was investigated. The conversion of precursor to mature forms and the appearance of the glycoproteins at the infected-cell surface at different times postinfection (p.i.) were studied. gB, synthesized at 4 h p.i., was converted to the mature form with a half-time (t1/2) of 120 min and appeared at the plasma membrane with a t1/2 of 270 min. The gB synthesized at later times p.i. (6, 8, and 10.5 h) was transported less efficiently. Less than 50% of gB synthesized at later times p.i. was processed and transported to the cell surface. gB synthesized in transfected cells was transported to the plasma membrane with kinetics similar to that for gB synthesized at early times p.i. gC was processed efficiently when synthesized at both 8 and 10.5 h p.i., with t1/2 of conversion of pgC to gC of 40 and 60 min, respectively. Approximately 90 to 95% of the gC synthesized was converted to the mature form. The gC synthesized at 8 h p.i. was also transported rapidly to the cell surface, compared with the transport of gB synthesized at the same time, with a t1/2 of 240 min. Greater than 70% of the gC synthesized at 8 h p.i. appeared at the cell surface. The gC synthesized at 10.5 h was transported less efficiently to the cells surface during a 6-h chase.
Gene transfer into human lymphocytes by a defective human immunodeficiency virus type 1 vector
Authors: Poznansky M , Lever A , Bergeron L , Haseltine W , Sodroski J
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) proviruses containing deletions between nucleotides 301 and 319 express viral proteins but exhibit marked attenuation in the packaging of viral RNA into virions (A. Lever, H. Gottlinger, W. Haseltine, and J. Sodroski, J. Virol. 63:4085-4087, 1989; A. Aldovini and R. A. Young, J. Virol. 64:1920-1926, 1990). Here we report that such packaging-defective proviruses can provide trans-acting viral elements required for the transfer of a HIV-1 vector to Jurkat human lymphocytes. The transferred vector was unable to encode HIV-1 genes, indicating that the long terminal repeats and the immediate flanking viral sequences are sufficient for packaging, reverse transcription, and integration. The generation of replication-competent viruses in this system was reduced to undetectable levels by providing the trans-acting viral functions on two separate expression plasmids. This defective retroviral vector provides a means of efficiently introducing desired genetic elements, in the absence of HIV-1 genes, into HIV-1 target cells.
Circular DNA of human immunodeficiency virus: analysis of circle junction nucleotide sequences
Authors: Hong T , Drlica K , Pinter A , Murphy E
Published: January 2020

During infection of cells by retroviruses, some of the nonintegrated viral DNA can be found as a circular form containing two tandem, directly repeated long terminal repeats. The nucleotide sequence at the point where the long terminal repeats join (the circle junction) can be used to deduce the terminal nucleotides of the linear form of the viral DNA. Comparison of the termini of linear viral DNA with sequences at the junctions between the integrated provirus and the host chromosome has revealed that for most retroviruses 2 bp are removed from each end of the linear viral DNA during integration. For human immunodeficiency virus type 1 (HIV-1), however, sequence considerations involving primer-binding sites had suggested that only 1 bp is removed during integration. We obtained the nucleotide sequences at the ends of HIV-1 DNA by using the polymerase chain reaction to amplify fragments corresponding to the HIV-1 circle junction. Of 17 clones containing amplified sequences, 10 had identical circle junctions that contained an additional 4 bp (GTAC) relative to the integrated provirus. This indicates that, as for other retroviruses, 2 bp are removed from each end of the linear HIV-1 viral DNA during integration. The remaining seven isolates contained insertions or deletions at the circle junction.
Role of immune cells in protection against and control of reovirus infection in neonatal mice
Authors: Virgin H W , Tyler K L
Published: January 2020

We studied the role of T cells in resistance to reovirus intestinal and central nervous system infection. Transfer of reovirus-immune adult spleen cells protected neonatal mice from (i) lethal infection with reovirus serotype 3 Dearing (T3D, footpad inoculation) and serotype 3 clone 9 (T3C9, oral inoculation) and (ii) hydrocephalus caused by serotype 1 Lang (T1L, intracranial [i.c.] inoculation). Cell-mediated protection was not serotype specific. While immune cells protected against T1L i.c., they failed to protect against 1/5,000 of the dose of T3D i.c. Two types of experiments showed that both CD4 and CD8 T cells are involved in reovirus resistance. First, immune cell-mediated protection against T3D was abrogated by in vivo treatment with anti-CD4 monoclonal antibody (MAb) and significantly inhibited by in vivo treatment with anti-CD8 MAb. Second, T3C9-infected neonatal mice treated with anti-CD4 and/or anti-CD8 developed a novel disease phenotype, an oily hair syndrome, associated with severe hepatobiliary pathology and increased viral titer in heart and liver. Immune cells and an MAb to the cell attachment protein sigma 1 (MAb G5) protected by different mechanisms. Immune cells were more effective than sigma 1 MAb G5 at controlling primary replication, while sigma 1 MAb G5 was more effective than immune cells at inhibiting neural spread of virus. We conclude that both CD4 and CD8 T cells are important for reovirus resistance, that cells and antibody act preferentially at different stages in pathogenesis in vivo, and that adoptively transferred immune cells can protect both the central nervous system and intestine.
Cellular ras gene activity is required for full neoplastic transformation by polyomavirus
Authors: Raptis L , Marcellus R , Corbley M J , Krook A , Whitfield J , Anderson S K , Haliotis T
Published: January 2020

To investigate the role of ras gene activity in cellular transformation by polyomavirus, murine C3H10T1/2 cells were rendered ras deficient by transfection with an antisense ras gene construct. Ras deficiency resulted in a partial suppression of the polyomavirus-induced transformed phenotype. The production of viral middle T antigen and its association with pp60c-src, increased membrane-associated protein kinase C activity, and morphological transformation were unaffected by the downregulation of c-ras gene expression. On the other hand, stimulated proliferation, focus formation on confluent monolayers of normal cells, and colony formation in soft agar were all greatly reduced in cells containing reduced p21ras levels. It is concluded that ras gene activity is needed for full cell transformation by polyomavirus.