bluuebunny/crossref_metadata_2025 huggingface dataset:
Divergent transcription of early 35- and 94-kilodalton protein genes encoded by the HindIII K genome fragment of the baculovirus Autographa californica nuclear polyhedrosis virus
Authors: Friesen P D , Miller L K
Published: January 2020

The organization of viral genes within the 3.7-kilobase-pair HindIII-K/EcoRI-S region of the Autographa californica nuclear polyhedrosis virus genome (85 to 88 map units) was determined by using a combination of nucleotide sequencing, transcriptional mapping, and in vitro translation of hybrid selected RNA. Two nonoverlapping genes, extending in opposite directions and encoding polypeptides with molecular weights of 35,000 and 94,000 (35K and 94K polypeptides), were identified. Unspliced, messenger-active RNAs were transcribed from both genes early (2 h) after infection. Indicative of immediate-early genes, transcription of the divergent RNAs was unaffected by the protein synthesis inhibitor, cycloheximide. Late in infection, abundant RNAs were transcribed from promoters located at least 2.5 kilobase pairs upstream from the gene encoding the 35K polypeptide. These transcripts completely overlapped both the 35K and 94K polypeptide genes but apparently lacked protein-coding potential, suggesting that the transcripts may play a role in the suppression of early viral gene expression.
The A+T-rich sequence of the simian virus 40 origin is essential for replication and is involved in bending of the viral DNA
Authors: Hertz G Z , Young M R , Mertz J E
Published: January 2020

The origin-promoter region of simian virus 40 contains a 17-base-pair sequence composed exclusively of adenine (A) and thymine (T). We constructed a linker replacement mutant in which this stretch of A's and T's was reduced to 11 base pairs. While not affecting the level of early gene transcription, this mutation reduced the accumulation of viral DNA in COS cells at least 10(4) fold. In addition, a restriction fragment containing the wild-type A + T-rich region migrated in nondenaturing polyacrylamide gels with an anomalous mobility characteristic of bent DNA; however, the corresponding fragment from the mutant migrated less anomalously. Therefore, bending of the DNA in this region may play a role in some step in viral DNA replication.
pp60c-src has less affinity for the detergent-insoluble cellular matrix than do pp60v-src and other viral protein-tyrosine kinases
Authors: Loeb D M , Woolford J , Beemon K
Published: January 2020

A difference in affinity for a Nonidet P-40-insoluble cellular matrix was observed between the products of the viral and cellular src genes. It has previously been demonstrated that pp60v-src is associated with a detergent-insoluble matrix containing the cellular cytoskeleton (J. G. Burr, G. Dreyfuss, S. Penman, and J. M. Buchanan, Proc. Natl. Acad. Sci. USA 77:3484-3488, 1980). We observed a similar association of the transforming proteins of Fujinami sarcoma virus (P130gag-fps) and Yamaguchi 73 avian sarcoma virus (P90gag-yes), both of which are tyrosine-specific protein kinases. However, we found that the endogenous c-src product, pp60c-src, was not tightly bound to the detergent-insoluble matrix. This does not appear to have been due to differences in the cytoskeleton between transformed and nontransformed cells since pp60c-src was also solubilized by nonionic detergent in cells transformed by Rous sarcoma virus. This difference in the affinities of the v-src and c-src products for cytoskeletal proteins may contribute to the inability of pp60c-src to transform cells.
5' long terminal repeats of myc-associated proviruses appear structurally intact but are functionally impaired in tumors induced by avian leukosis viruses
Authors: Goodenow M M , Hayward W S
Published: January 2020

B-cell lymphomas induced in chickens infected with avian leukosis viruses are characterized by integration of the virus within the cellular myc locus and alteration of c-myc expression. Although avian leukosis viruses are intact, replication-competent retroviruses, the structures of many myc-associated proviruses are altered by deletions, raising the possibility that proviral defectiveness plays an essential role in oncogenesis. We found that all myc-associated proviruses in 21 independent tumors had deletions, which were confined to the viral genome and did not extend into adjacent cellular sequences. Deletions were not random but, in at least 85% of the myc-associated proviruses, involved a region near the 5' end of the proviral genome where elements implicated in control of viral gene expression have been localized. A second class of deletions involved sequences in the 3' half of the viral genome and included the splice acceptor site used in generating viral env mRNA. Both the 5' and 3' long terminal repeats of myc-associated proviruses appeared to be structurally intact in most tumors, although the 5' long terminal repeats were not involved in expression of either U5-myc transcripts or detectable steady-state viral RNAs. A complex array of repeated sequence elements surrounded the junctions of the internal deletions in two myc-associated proviruses. The organization of the deleted proviruses was similar to that of deleted unintegrated viral molecules, consistent with a model in which deletions occurred prior to integration.
Immunoglobulin A monoclonal antibodies protect against Sendai virus
Authors: Mazanec M B , Nedrud J G , Lamm M E
Published: January 2020

Immunoglobulin A anti-Sendai virus HN protein monoclonal antibodies, generated via a mucosal immunization protocol, were shown to neutralize virus in vitro and, when passively administered to the mouse respiratory tract, to protect against Sendai virus in vivo. Thus, immunoglobulin A antibodies by themselves can protect against respiratory virus infection.
Activated Ha-ras can cooperate with defective simian virus 40 in the transformation of nonestablished rat embryo fibroblasts
Authors: Michalovitz D , Fischer-Fantuzzi L , Vesco C , Pipas J M , Oren M
Published: January 2020

We studied the ability of activated Ha-ras to cooperate with simian virus 40 (SV40) in the transformation of nonestablished rat embryo fibroblasts. Cotransfection with Ha-ras greatly accelerated the rate of focus induction by wild-type SV40. Moreover, a series of transformation-defective SV40 mutants could be partially complemented by Ha-ras. This was true not only for mutants retaining an intact N-terminal immortalization-competent domain, but also for a nonkaryophilic SV40 mutant. In the latter case, all detectable T antigen was cytoplasmic, indicating that efficient transformation can be achieved through the interaction of two nonnuclear proteins. By employing cell lines derived with various SV40 mutants, it was determined that the ability to complex with p53 depends on the integrity of a relatively large region in the C-terminal half of large T. Finally, we report that nonkaryophilic SV40 large T forms a complex with the major heat shock protein HSP70, and we discuss its possible implications.
Generation of altered transcripts by retroviral insertion within the c-myb gene in two murine monocytic leukemias
Authors: Gonda T J , Cory S , Sobieszczuk P , Holtzman D , Adams J M
Published: January 2020

Two murine monocytic leukemia cell lines, WEHI-265 and WEHI-274, were found to carry a rearranged c-myb gene. The rearrangements are due to insertion of a deleted Moloney murine leukemia virus (Mo-MLV) provirus in the 5' region of the c-myb gene and thus are similar to rearrangements in the ABPL tumors (G. L. C. Shen-Ong, M. Potter, J. F. Mushinski, S. Lavu, and E. P. Reddy, Science 226:1077-1080, 1984). In each cell line, the retroviral insertion has induced high levels of two aberrant RNA species, which, as in the ABPL tumors (G. L. C. Shen-Ong, H. C. Morse, M. Potter, and J. F. Mushinski, Mol. Cell. Biol. 6:380-392, 1986), contain both viral (Mo-MLV) and cellular (myb) sequences. Both species lack the sequences encoding the amino terminus of the c-myb protein and thus could encode a protein which, like the v-myb gene products (and the predicted ABPL myb proteins), is truncated at the amino terminus. We have found that the larger (5.3 kilobase [kb]) and more abundant of the tumor-specific myb RNAs was predominantly nuclear, while the smaller species (3.9 kb) was cytoplasmic. Furthermore, our data imply that the 3.9-kb RNA was derived from the 5.3-kb RNA by an additional splice which utilized a cryptic splice acceptor site within the viral gag sequences. On the basis of subcellular distribution and predicted translational potential, we conclude that the 3.9-kb RNA is probably the mRNA which encodes a truncated myb protein. We also show that, due to different insertion points in W265 and W274, the W274 myb RNAs contained sequences from a c-myb exon upstream of the exons represented in the W265 (and ABPL) RNAs. The significance of our findings with regard to transformation by myb in these tumors is discussed.
The membrane glycoprotein of Friend spleen focus-forming virus: evidence that the cell surface component is required for pathogenesis and that it binds to a receptor
Authors: Li J P , Bestwick R K , Spiro C , Kabat D
Published: January 2020

The leukemogenic membrane glycoprotein of Friend spleen focus-forming virus (SFFV) has an apparent Mr of 55,000 (gp55), is encoded by a recombinant env gene, and occurs on cell surfaces and in intracellular organelles. There is evidence that the amino-terminal region of gp55 forms a dualtropic-specific domain that is connected to the remainder of the glycoprotein by a proline-rich linker (C. Machida, R. Bestwick, B. Boswell, and D. Kabat, Virology 144:158-172, 1985). Using the colinear form of a cloned polycythemic strain of SFFV proviral DNA, we constructed seven in-phase env mutants by insertion of linkers and by a deletion. The mutagenized SFFVs were transfected into fibroblasts and were rescued by superinfection with a helper murine leukemia virus. Four of the mutants cause erythroblastosis. These include one with a 6-base-pair (bp) insert in the ecotropic-related sequence near the 3' end of the gene, two with a 12- or 18-bp insert in the region that encodes the proline-rich linker, and one with a 6-bp insert in the dualtropic-specific region. The other mutants (RI, Sm1, and Sm2) are nonpathogenic and contain lesions in dualtropic-specific region. The other mutants (RI, Sm1, and Sm2) are nonpathogenic and contain lesions in dualtropic-specific sequences that are highly conserved among strains of SFFV. A pathogenic revertant (RI-rev) was isolated from one mouse that developed erythroblastosis 3 weeks after infection with RI. RI-rev contains a second-site env mutation that affects the same domain as the primary mutation does and that increases the size of the encoded glycoprotein. All pathogenic SFFVs encode glycoproteins that are expressed on cell surfaces, whereas the nonpathogenic glycoproteins are exclusively intracellular. The pathogenic SFFVs also specifically cause a weak interference to superinfection by dualtropic MuLVs. These results are compatible with the multidomain model for the structure of gp55 and suggest that processing of gp55 to plasma membranes is required for pathogenesis. The amino-terminal region of gp55 binds to dualtropic murine leukemia virus receptors, and this interaction is preserved in the SFFV mutants that cause erythroblastosis.
Replication of human hepatitis delta virus in primary cultures of woodchuck hepatocytes
Authors: Taylor J , Mason W , Summers J , Goldberg J , Aldrich C , Coates L , Gerin J , Gowans E
Published: January 2020

We obtained two lines of evidence that monolayer cultures of primary woodchuck hepatocytes support replication of the genome of human hepatitis delta virus (HDV). (i) From a Northern (RNA blot) analysis of the HDV-related RNA in infected cultures, both genomic and antigenomic 1.7-kilobase RNA species were detected at 11 days after infection. The ratio of genomic RNA to antigenomic RNA was 2:1 to 10:1, comparable to that previously reported in studies of experimentally infected chimpanzees and woodchucks. (ii) Replication in culture was also demonstrated by in situ hybridization with a strand-specific probe. Such studies showed that only a small fraction of the cultured cells supported replication and that in such cells the relative and absolute levels of the HDV RNAs were comparable to those in liver cells infected in vivo. Furthermore, as with the in vivo studies, the HDV RNAs were predominantly localized to the nucleus. In summary, we demonstrated that cultured cells supported both the early events of HDV adsorption and penetration and the intermediate events of genome replication.
Herpes simplex virus 1 protein kinase is encoded by open reading frame US3 which is not essential for virus growth in cell culture
Authors: Purves F C , Longnecker R M , Leader D P , Roizman B
Published: January 2020

Earlier reports have described a novel protein kinase in cells infected with herpes simplex or pseudorabies viruses. These novel enzymes were characterized by their acceptance of protamine as a substrate and by their differential chromatographic behavior in anion-exchange chromatography. We report that this activity was not present in extracts of uninfected cells or of cells infected with a mutant constructed so as to contain a deletion in the US3 open reading frame mapping in the small component of herpes simplex virus 1 DNA. The activity was present in extracts of cells infected with wild-type virus and with a recombinant in which the US3 open reading frame had been rescued. Our results are consistent with the observation reported earlier that the coding sequences predict an amino acid motif common to protein kinases and lead to the conclusion that the US3 open reading frame encodes a virus-specific protein kinase that is not required for virus growth in cells in culture.
Association of Epstein-Barr virus early antigen diffuse component and virus-specified DNA polymerase activity
Authors: Li J S , Zhou B S , Dutschman G E , Grill S P , Tan R S , Cheng Y C
Published: January 2020

The role of Epstein-Barr virus (EBV) early antigen diffuse component (EA-D) and its relationship with EBV DNA polymerase in EBV genome-carrying cells are unclear, EBV-specified DNA polymerase was purified in a sequential manner from Raji cells treated with phorbol-12,13-dibutyrate and n-butyrate by phosphocellulose, DEAE-cellulose, double-stranded DNA-cellulose, and blue Sepharose column chromatography. Four polypeptides with molecular masses of 110,000, 100,000, 55,000, and 49,000 daltons were found to be associated with EBV-specified DNA polymerase activity. A monoclonal antibody which could neutralize the EBV DNA polymerase activity was prepared and found to recognize 55,000- and 49,000-dalton polypeptides. An EA-D monoclonal antibody, R3 (G. R. Pearson, V. Vorman, B. Chase, T. Sculley, M. Hummel, and E. Kieff, J. Virol. 47:183-201, 1983), was also able to recognize these same two polypeptides associated with EBV DNA polymerase activity. It was concluded that EBV EA-D polypeptides, as identified by R3 monoclonal antibody, are critical components of EBV DNA polymerase.
Transcription of interferon-stimulated genes is induced by adenovirus particles but is suppressed by E1A gene products
Authors: Reich N , Pine R , Levy D , Darnell J E
Published: January 2020

Interferon treatment of cell cultures results in the rapid transcriptional induction of a specific set of genes. In this paper we explore the effect of cellular infection by several adenoviruses, both wild type and mutant, on the expression of these genes. Infection with adenovirus induces the transcription of the interferon-stimulated genes in the absence of any protein synthesis. In fact, the inhibition of protein synthesis during a wild-type infection produces enhanced stimulation of transcription of these genes. Experiments with viral mutants indicate the ability to specifically suppress this transcription maps to the E1A gene. In addition, the E1A gene products are capable of suppressing the specific transcriptional induction of interferon-stimulated promoters during cotransfection experiments and therefore presumably during viral infection. The dual effect of adenovirus on the expression of interferon-stimulated genes may represent an example of action and evolutionary reaction between virus and host.
Progression of the phenotype of transformed cells after growth stimulation of cells by a human papillomavirus type 16 gene function
Authors: Noda T , Yajima H , Ito Y
Published: January 2020

Alteration of the growth properties of the established murine fibroblast cell lines NIH 3T3 and 3Y1 was studied in monolayer cultures and in cells suspended in semisolid medium after introduction of a cloned human papillomavirus type 16 (HPV16) DNA. HPV 16 DNA stimulated both cell lines to grow beyond their saturation densities in monolayer cultures without any apparent morphological changes or tendency to pile up. These cells were also stimulated to grow in soft agar. Since essentially all the cells that received the viral gene were stimulated to grow, the growth-stimulatory activity of HPV16 appeared to be due to the direct effect of a viral gene function. The NIH 3T3 cells showed an additional change in growth properties upon prolonged incubation of dense monolayers of cells containing the HPV16 DNA; morphologically recognizable dense foci appeared at a frequency of about 10(-3). These cells, when cloned from the foci, grew more rapidly in soft agar than the parental cells and were morphologically transformed. In other words, there were two sequential steps in cell transformation induced by HPV16. Practically all the viral DNAs were present in the cells as large rearranged multimers and were integrated into host chromosomal DNA. There was no obvious difference in the state of viral DNA in the cells of the original clone or the three subclones derived from it as dense foci. There was no difference in the amount or the number of viral RNA species expressed in the cells at these two stages. The secondary changes in the growth properties of NIH 3T3 cells appear to be due to some cellular alterations.
Suppression of RNA synthesis by a specific antiviral activity in Sindbis virus-infected Aedes albopictus cells
Authors: Condreay L D , Brown D T
Published: January 2020

An antiviral protein is released by mosquito cells persistently infected with Sindbis virus. Differences in both sensitivity to and production of this virus-specific activity were apparent in three independently produced Aedes albopictus cell lines. This activity inhibits total viral RNA synthesis in a time-dependent manner. The antiviral effect is maximally realized when cells are treated with the activity 48 h before infections. These data suggest that the antiviral activity induces an antiviral state in treated cells which prevents the formation or efficient function of viral RNA-synthesizing complexes.
Identification of point mutations in the envelope gene of Moloney murine leukemia virus TB temperature-sensitive paralytogenic mutant ts1: molecular determinants for neurovirulence
Authors: Szurek P F , Yuen P H , Jerzy R , Wong P K
Published: January 2020

ts1, a temperature-sensitive mutant of Moloney murine leukemia virus TB, induces hind-limb paralysis in mice. The DNA of both the ts1 and Moloney murine leukemia virus TB env genes has been sequenced, and the encoded amino acid sequences have been deduced from the DNA sequences. Four amino acids in the ts1 envelope protein have been identified which may be responsible for the ts1 phenotype, which includes temperature sensitivity, nonprocessing of Pr80env, and neurovirulence.
Organization of the transcriptional control region of the E1b gene of adenovirus type 5
Authors: Parks C L , Banerjee S , Spector D J
Published: January 2020

Genetic analysis of the transcriptional control sequences of the E1b gene of adenovirus type 5 identified two regions that stimulated specific transcription by whole cell extracts from uninfected cells. The first region, located within 50 nucleotides (position -50) 5' to the transcription initiation (cap) site, contains a G+C-rich consensus-binding site (GC box) for the transcription factor Sp1 and a TATA box. Unambiguous stimulatory activity of the second region, between positions -358 and -127, was observed only in the absence of the GC box. DNase I protection experiments (footprinting) with crude nuclear extracts from uninfected cells revealed multiple DNA-protein interactions at the control region. Proximal to the initiation site, both the GC box and the cap site were protected; however, protection of the TATA box was not observed. In the distal region, four protein-binding sites, designated I through IV, were located between positions -250 and -120. Three of the four mapped in protein-coding sequences of the adjacent E1a gene. Sites I and II were 5' to position -218 whereas sites III and IV were 3' to position -218. This finding was consistent with results of the transcriptional analysis indicating that subsets of the distal region were sufficient for stimulation of transcription in vitro in the absence of the GC box. Within the boundaries of site I, a 10-base-pair protected sequence was similar to one located 5' to the adenovirus E1a, E2a, E3, E4, E2 late, and polypeptide IX transcription initiation sites. Sequences within the boundaries of the other three sites were similar to those within other viral and cellular enhancers.
Bovine papillomavirus E2 gene regulates expression of the viral E5 transforming gene
Authors: Prakash S S , Horwitz B H , Zibello T , Settleman J , DiMaio D
Published: January 2020

We have performed transient-expression experiments with CV1 monkey kidney cells to investigate the role of the bovine papillomavirus type 1 (BPV1) E2 gene in the regulation of the E5 transforming gene. Direct analysis of the 7-kilodalton open reading frame (ORF) E5 protein and measurements of the expression of an E5-chloramphenicol acetyltransferase fusion protein indicate that the efficient expression of ORF E5 requires the full-length E2 gene, which can be supplied in trans. The viral long control region is required in cis for this response to ORF E2, and it acts in a position- and orientation-independent fashion characteristic of a transcriptional enhancer. Deletion analysis suggests that the P2443 promoter is required for efficient expression of the E5 gene. The E2 repressor activity encoded in the 3' end of the E2 gene inhibits the expression of ORF E5. These effects define a major BPV1 regulatory circuit and appear to explain the transformation behavior of a variety of BPV1 mutants.
Influenza virus regulates protein synthesis during infection by repressing autophosphorylation and activity of the cellular 68,000-Mr protein kinase
Authors: Katze M G , Tomita J , Black T , Krug R M , Safer B , Hovanessian A
Published: January 2020

We investigated the mechanisms by which influenza virus prevents shutoff of protein synthesis by a cellular protein kinase normally activated during infection. Earlier work has shown that influenza virus superinfection of cells previously infected by the adenovirus VAI RNA-negative mutant dl331 resulted in selective translation of influenza virus mRNAs and suppression of the elevated protein kinase levels normally found in cells infected by the mutant alone (M. G. Katze, B. M. Detjen, B. Safer, and R. M. Krug, Mol. Cell. Biol. 6:1741-1750, 1986). We elucidated the mechanisms of this kinase repression and can now report that influenza virus encodes a gene product which functions to directly block the autophosphorylation and activity of the interferon-induced, double-stranded-RNA-activated protein kinase, P68. Suppressed P68 activity was found not only in doubly infected cells but also in cells infected by influenza virus alone. Moreover, the decrease in P68 activity correlated with a decrease in the endogenous levels of phosphorylation of the alpha subunit of the eucaryotic initiation factor eIF-2, the natural substrate of the protein kinase. Suppression of P68 activity occurred as early as 2 h after influenza virus infection and required viral gene expression beyond the level of primary mRNA transcription to take place. We confirmed our in vivo observations with in vitro mixing experiments which showed that the influenza virus inhibitor can act in trans to block P68 activity. Combined repression of P68 function and eIF-2 alpha phosphorylation during influenza virus infection is essential for continued catalytic recycling of eIF-2 and efficient mRNA translation.
Vaccinia virus gene D8 encodes a virion transmembrane protein
Authors: Niles E G , Seto J
Published: January 2020

Transcription mapping studies and DNA sequence analysis of the vaccinia virus HindIII D fragment predict that gene D8 encodes a protein 304 amino acids in length, with a molecular mass of 35,426 daltons, that is expressed at late times in infection. In order to determine whether the native D8 protein is required for virus propagation, we constructed a frameshift mutation in the D8 coding sequence. Virus containing this mutation were isolated and shown to replicate in a single-step growth experiment with wild type virus growth kinetics, demonstrating that the normal-length D8 protein is not essential for virus propagation in tissue culture. In order to investigate the synthesis of the wild-type and the mutant D8 proteins in virus-infected cells, we raised polyclonal antisera to a fusion protein consisting of a portion of the D8 coding sequence linked to the Escherichia coli trpE gene. Western blot (immunoblot) analysis of the time course of D8 protein synthesis in cells infected with either wild-type or mutant virus demonstrated that D8 protein was synthesized late in infection in each case and accumulated throughout the experiment. To determine whether the D8 protein was incorporated into the mutant or wild-type virus, purified virions were fractionated into Nonidet P-40-soluble, deoxycholate-soluble, and detergent-insoluble fractions. In both the wild-type and the mutant viruses, the D8 protein was an integral viral protein. The wild-type protein partitioned into the Nonidet P-40-soluble fraction, suggesting that it was a viral membrane protein. The mutant protein fractionated into the detergent-insoluble component, demonstrating that although the altered protein was incorporated into the virus, it was found in a abnormal location. In order to determine whether the D8 protein was present on the virion surface, the susceptibility of the D8 protein to proteolysis was tested by analyzing the products of incubation of the wild-type and mutant viruses with either chymotrypsin or trypsin. These studies demonstrated that the wild-type D8 protein was a transmembrane protein with a major extraviral domain that was released largely intact from the virus by trypsin. The mutant D8 protein was relatively refractory to proteolysis, confirming the hypothesis that although it is associated with the virus, it is in a conformation different from that of the wild-type protein. Tryptic digestion of the wild-type virus increased plaque formation severalfold, concomitant with the removal of the extraviral domain of the D8 protein.(ABSTRACT TRUNCATED AT 400 WORDS)
Development of a sensitive quantitative focal assay for human immunodeficiency virus infectivity
Authors: Chesebro B , Wehrly K
Published: January 2020

Accurate and sensitive quantitation of infectious human immunodeficiency virus (HIV) has been difficult to achieve. In this report, a quantitative focal immunoassay (FIA) for HIV was developed using human HeLa cells rendered susceptible to HIV infection by introduction of the CD4 gene via a retrovirus vector. Infected cells were identified by using human anti-HIV antibodies or mouse monoclonal antibodies specific for HIV together with secondary fluorescein- or peroxidase-conjugated antibody specific for mouse or human immunoglobulins. The assay identified cells infected with either wild-type or culture-adapted HIV isolates and was capable of detecting 1 positive cell in 10(6) cells. The FIA was also effective at detecting cell-free HIV, and in contrast to assays using A3.01, CEM, and other human leukemia cells, the FIA detected most wild-type HIV isolates. HIV neutralization could be determined by using the FIA, and two monoclonal antibodies reactive with HIV gp120 were found to neutralize only the LAV-IIIB strain of HIV. These monoclonal antibodies, as well as antibodies in serum samples from patients with acquired immune deficiency syndrome, were able to inhibit the spread of HIV infection in human lymphocyte suspension cultures but not in CD4-positive HeLa cells growing attached to plastic dishes.
The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors
Authors: Mervis R J , Ahmad N , Lillehoj E P , Raum M G , Salazar F H , Chan H W , Venkatesan S
Published: January 2020

Seven human immunodeficiency virus gag polypeptides were identified in the purified virus and in infected CD4+ lymphocytes by peptide mapping and limited amino acid sequencing of immune-purified proteins. Two gag polyproteins of 55,000 (p55) and 41,000 (p41) daltons were rapidly labeled and readily processed into the major internal gag proteins that were aligned within the gag open reading frame (ORF) as NH2-p16 (MA)-p24 (CA)-p9 (NC)-p7-COOH. The myristoylated p16 (matrix, MA) protein was processed from the myristoylated p55 gag precursor protein. The immunoreactivity of the p16 (MA) protein with region-specific gag antisera and the conservation of the N-terminal myristyl group of the p55 precursor protein in p16 (MA) confirmed its position as the N-terminal-most protein. The p9 (nucleocapsid, NC) protein was localized to residue 378 of the gag ORF, next to the C terminus of the p24/p25 (core antigen, CA) protein. The p9 protein had a repeating Cys residue containing motif which is found in the nucleic acid-binding Cys residue-containing proteins of retroviruses. The p24 (CA) protein, which was localized to residue 133 of the gag ORF, was apparently derived by C-terminal processing of an intermediate polypeptide, p25. Both the mature p24 (CA) and p16 (MA) proteins were phosphorylated at Ser residue(s). We also identified two forms of gag p41 species, one resulting from the C-terminal processing of p55 and the other originating either from N-terminal processing of p55 or from de novo synthesis.
Isolation of poliovirus 2C mutants defective in viral RNA synthesis
Authors: Li J P , Baltimore D
Published: January 2020

Two poliovirus mutants were isolated that contain an oligonucleotide linker insertion in the 2C-coding region of the viral genome. One, 2C-31, has a strongly temperature-sensitive phenotype and the other, 2C-32, forms small plaques on HeLa cell monolayers at all temperatures. Both mutants have a severe temperature-sensitive defect in viral RNA synthesis but little effect on the types of viral protein that are made. Temperature shift experiments showed that the 2C function is continuously required for viral RNA synthesis to proceed. The 2C mutants could be complemented in trans by mutants with mutations in other viral proteins. Protein 2C is also the locus of the guanidine resistance and dependence mutants, a drug whose action also affects viral RNA synthesis. Thus, protein 2C is one that is needed continually for viral RNA synthesis and, at least with these temperature-sensitive alleles, can be provided in trans.
C3b receptor activity on transfected cells expressing glycoprotein C of herpes simplex virus types 1 and 2
Authors: Seidel-Dugan C , Ponce de Leon M , Friedman H M , Fries L F , Frank M M , Cohen G H , Eisenberg R J
Published: January 2020

Glycoprotein C from herpes simplex virus type 1 (gC-1 from HSV-1) acts as a receptor for the C3b fragment of the third component of complement on HSV-1-infected cell surfaces. Direct binding assays with purified gC-1 and C3b demonstrate that other viral and cellular proteins are not required for this interaction. Although C3b receptor activity is not expressed on HSV-2-infected cell surfaces, purified gC-2 specifically binds C3b in direct binding assays, suggesting that gC-1 and gC-2 are functionally similar. Here, we used a transient transfection system to further characterize the role of gC-1 and gC-2 as C3b receptors and to localize the site(s) on gC involved in C3b binding. The genes for gC-1 and gC-2 were each cloned into a eucaryotic expression vector containing the Rous sarcoma virus long terminal repeat as the promoter and transfected into NIH 3T3 cells. The expressed proteins were similar in molecular size, extent of carbohydrate processing, and antigenic properties to gC-1 and gC-2 purified from infected cells. Using a double-label immunofluorescence assay, we found that both gC-1 and gC-2 were expressed on the surfaces of transfected cells and bound C3b. These results suggest that other proteins expressed during HSV-2 infection prevent receptor activity. We constructed three in-frame deletion mutants of gC-2 to identify domains on the protein important for C3b receptor activity. These mutants lacked amino acids 26 to 73, 219 to 244, or 318 to 346. The mutant protein lacking residues 26 to 73 was reactive with two monoclonal antibodies recognizing distinct epitopes, showed a wild-type pattern of carbohydrate processing, and bound C3b on the transfected cell surface. These results suggest that residues 26 to 73 are not involved in C3b binding. The other two mutant proteins were present on the cell surface, but did not bind C3b. In addition, these mutant proteins showed altered patterns of carbohydrate processing, formed aggregates, and were no longer recognized by the monoclonal antibodies. These properties indicate that removal of residues 219 to 244 or 318 to 346 disrupted the native conformation of gC-2, possibly owing to an alteration in the spacing between critical cysteine residues.
Negative regulation of retrovirus expression in embryonal carcinoma cells mediated by an intragenic domain
Authors: Loh T P , Sievert L L , Scott R W
Published: January 2020

An intragenic region spanning the tRNA primer binding site of a Moloney murine leukemia virus recombinant retrovirus was found to restrict expression specifically in embryonal carcinoma (EC) cells. When the inhibitory domain was present, the levels of steady-state RNA synthesized from integrated recombinant templates in stable cotransformation assays were reduced 20-fold in EC cells but not in C2 myoblast cells. Transient-cotransfection assays showed that repression of a template containing the EC-specific inhibitory component was relieved by an excess of specific competitor DNA. In addition, repression mediated by the inhibitory component was orientation independent. This evidence demonstrates the presence of a saturable, trans-acting negative regulatory factor(s) in EC cells and suggests that the interaction of the factor(s) with the intragenic inhibitory component occurs at the DNA level.
A herpesvirus vector for expression of glycosylated membrane antigens: fusion proteins of pseudorabies virus gIII and human immunodeficiency virus type 1 envelope glycoproteins
Authors: Whealy M E , Baumeister K , Robbins A K , Enquist L W
Published: January 2020

We describe experiments using the swine herpesvirus, pseudorabies virus (PRV), as a vector for expression of hybrid membrane protein genes. In particular, we present the construction and analysis of three infectious PRV mutants expressing chimeric viral membrane proteins composed of portions of the PRV envelope glycoprotein gIII and of the human retrovirus, human immunodeficiency virus type 1 (HIV-1), envelope glycoproteins gp120 and gp41. All of the chimeric genes contain the transcription control sequences and the first 157 codons of PRV gIII (known to contain signals sufficient for efficient export of the encoded peptide out of the cell) fused to different regions of the HIV-1 envelope. The mutant viruses express novel glycosylated fusion proteins that are immunoprecipitated by polyvalent sera specific for gIII, as well as acquired immunodeficiency syndrome patient sera. The levels of expression are lower than expected due primarily to instability or altered processing of the hybrid mRNA. We could not detect cleavage of chimeric proteins carrying the gp120-gp41 protease processing site. The use of localization signals contained within herpesvirus membrane proteins to direct chimeric proteins to desired cellular locations is discussed.
Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells
Authors: Fitzpatrick D R , Zamb T , Parker M D , van Drunen Littel-van den Hurk S , Babiuk L A , Lawman M J
Published: January 2020

Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK- cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1-infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1-infected bovine cells. For gI, a deficiency in N-linked glycosylation of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected or BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes. Immunization of mice with the transfected cells elicited BHV-1-specific virus-neutralizing antibody, thus verifying the antigenic authenticity of the recombinant glycoproteins and the important role of gI and gIII as targets of the immune response to BHV-1 in this murine model system.
Interactions between coronavirus nucleocapsid protein and viral RNAs: implications for viral transcription
Authors: Baric R S , Nelson G W , Fleming J O , Deans R J , Keck J G , Casteel N , Stohlman S A
Published: January 2020

The interaction of the mouse hepatitis virus (MHV) nucleocapsid protein (N) and viral RNA was examined. Monoclonal antibody specific for N protein coimmunoprecipitated MHV genomic RNA as well as all six MHV subgenomic mRNAs found in MHV-infected cells. In contrast, monoclonal antibodies to the MHV E2 or E1 envelope glycoproteins, an anti-I-A monoclonal antibody, and serum samples from lupus patients did not immunoprecipitate the MHV mRNAs. Moreover, the anti-N monoclonal antibody did not coimmunoprecipitate vesicular stomatitis virus RNA or host cell RNA under conditions which immunoprecipitated all MHV RNAs. These data suggest a specific interaction between the N protein and the virus-specific mRNAs. Both the membrane-bound and cytosolic small MHV leader-specific RNAs of greater than 65 nucleotides long were immunoprecipitated only by anti-N monoclonal antibody. These data suggest that an N binding site is present within the leader RNA sequences at a site at least 65 nucleotides from the 5' end of genomic RNA and all six subgenomic mRNAs. The larger leader-containing RNAs originating from mRNA 1 and mRNA 6, as well as the MHV negative-stranded RNA, were also immunoprecipitated by the anti-N monoclonal antibody. These data indicate that the MHV N protein is associated with MHV-specific RNAs and RNA intermediates and may play an important functional role during MHV transcription and replication.
The carboxy-terminal 41 amino acids of herpes simplex virus type 1 glycoprotein B are not essential for production of infectious virus particles
Authors: Huff V , Cai W , Glorioso J C , Levine M
Published: January 2020

Glycoprotein B (gB) is a virally encoded protein that is found in the envelope of herpes simplex virus type 1 and membranes of cells infected with herpes simplex virus type 1. It is essential for the production of infectious virus particles. An amber mutation was introduced into the gB gene by oligonucleotide-directed mutagenesis at the codon for amino acid 863 of the protein. Virus carrying this mutation should synthesize gB molecules lacking the last 41 amino acids of the cytoplasmic domain. Immunoprecipitation of infected cell extracts demonstrated the synthesis of appropriately truncated gB molecules. Characterization of the mutant virus indicated that the loss of the carboxy-terminal 41 amino acids has little effect on gB function.
Identification of amino acids relevant to three antigenic determinants on the fusion protein of Newcastle disease virus that are involved in fusion inhibition and neutralization
Authors: Toyoda T , Gotoh B , Sakaguchi T , Kida H , Nagai Y
Published: January 2020

Nucleotide sequence analysis of F protein antigenic variants of Newcastle disease virus mapped three distinct antigenic determinants to positions 343, 72, and 161 on the protein. The high fusion-inhibiting and neutralizing capacities of all of the monoclonal antibodies used for selection suggested close functional and structural relationships of the three positions with the fusion-inducing N-terminal region of the F1 subunit. The former two positions were located at the cysteine cluster domain near the C terminus of the F1 subunit and at the major hydrophilic domain in the F2 subunit, respectively, and both domains appeared to represent the major antigenic determinants of paramyxovirus F protein.
Methylation of the promoter for an immediate-early frog virus 3 gene does not inhibit transcription
Authors: Thompson J P , Granoff A , Willis D B
Published: January 2020

Methylation of critical sites within the promoter region of eucaryotic genes has been shown to inhibit transcription by RNA polymerase II. However, although the large DNA virus frog virus 3 (FV3) has a highly methylated genome, it uses host RNA polymerase II for at least the immediate-early stage of transcription. We have previously shown that an FV3-induced trans-acting protein allows transcription from adenovirus promoters inactivated by methylation. Since FV3 immediate-early genes are transcribed in the absence of de novo protein synthesis, it appears that the virus-induced trans-acting protein that allows transcription from methylated templates is not required for transcription of the immediate-early FV3 genes, possibly because they are not methylated in critical regulatory sequences. In this study, we used site-directed mutagenesis to alter the three CpG dinucleotide sequences in the promoter region of an immediate-early FV3 gene and thereby created sites recognized by bacterial methylases. Transient-expression assays demonstrated that neither the mutations nor methylation of the mutated sites inhibited transcription from the FV3 promoter in FV3-infected cells. These findings support the hypothesis that the immediate-early genes of FV3 do not contain methylatable sites in regions critical for transcription. The function of the virus-induced trans-acting protein that can override the inhibitory effect of methylation may therefore be to facilitate the transcription of methylated delayed-early or late FV3 genes.
Fine mapping of the immediate-early gene of the Indiana-Funkhauser strain of pseudorabies virus
Authors: Cheung A K
Published: January 2020

The immediate-early gene transcript of pseudorabies virus was found to be 5.6 kilobases and colinear with the DNA genome. Transcription initiation started in the BamHI E fragment, 45 nucleotides from the BamHI-E/BamHI-I junction. The first AUG codon was located in BamHI-I, 260 nucleotides from the mRNA initiation site, and a potential TGA termination codon was located in BamHI-J, 161 nucleotides from the BamHI-I/BamHI-J junction. The AATAAA polyadenylation signal was 30 bases from the stop codon, while the actual polyadenylation site was 18 bases further downstream.
Hepatitis B virus particles contain a polypeptide encoded by the largest open reading frame: a putative reverse transcriptase
Authors: Mack D H , Bloch W , Nath N , Sninsky J J
Published: January 2020

A segment of the largest open reading frame of hepatitis B virus (HBV) was inserted into an open reading frame vector directing the expression in Escherichia coli of a fusion molecule containing 143 HBV-encoded amino acids. The fusion protein was used to generate antiserum which served in immunoblots to identify a polypeptide with a molecular mass of 65 kilodaltons in HBV particles. Because of the small number of molecules in virus particles, unambiguous detection required the development of a highly sensitive immunoblot procedure.
Mapping of functional and antigenic domains of the alpha 4 protein of herpes simplex virus 1
Authors: Hubenthal-Voss J , Houghten R A , Pereira L , Roizman B
Published: January 2020

Monoclonal antibodies to alpha 4, the major regulatory protein of herpes simplex virus 1, have been shown to differ in their effects on the binding of the protein to its DNA-binding site in the promoter-regulatory domain of an alpha gene. To map the epitopes, we expressed truncated genes in transient expression systems. All 10 monoclonal antibodies tested reacted with the N-terminal 288-amino-acid polypeptide. To map the epitopes more precisely, 29 15-mer oligopeptides, overlapping by five amino acids at each end, were synthesized and reacted with the monoclonal antibodies. The nine reactive monoclonal antibodies were mapped to seven sites. Of the two monoclonal antibodies which blocked the binding of alpha 4 to DNA, one (H950) reacted with oligopeptide no. 3 near the N terminal of the protein, whereas the second (H942) reacted with oligopeptide no. 23 near the C terminus of the 288-amino-acid polypeptide. In further tests, oligopeptide no. 19 was found to compete with two host proteins, designated as alpha H1 and alpha H2-alpha H3, for binding to DNA as well as to retard DNA in a band shift assay, whereas oligopeptides no. 26, 27, and 28 enhanced the binding of alpha 4 to DNA. Moreover, oligopeptide no. 27 was also found to retard DNA in a band shift assay. Polypeptide no. 19 competed with alpha 4 for binding to DNA, whereas no. 27 neither enhanced nor competed with the binding of the host polypeptide alpha H1 to its binding site in the promoter-regulatory domain of an alpha gene, but did enhance the binding of the alpha H2-alpha H3 protein to its binding site. In contrast to these results, the truncated alpha 4 polypeptide, 825 amino acids long, bound to the viral DNA, whereas a shorter, 519-amino-acid-long, truncated polypeptide did not. The 825-amino-acid polypeptide was previously shown to induce in transient expression of a late (gamma 2) viral gene.