bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of E1A-mediated growth regulation functions: binding of the 300-kilodalton cellular product correlates with E1A enhancer repression function and DNA synthesis-inducing activity
Authors: Stein R W , Corrigan M , Yaciuk P , Whelan J , Moran E
Published: January 2020

Adenovirus E1A transforming function requires two distinct regions of the protein. Transforming activity is closely linked with the presence of a region designated conserved domain 2 and the ability of this region to bind the product of the cellular retinoblastoma tumor suppressor gene. We have investigated the biological properties of the second transforming region of E1A, which is located near the N terminus. Transformation-defective mutants containing deletions in the N terminus (deletion of residues between amino acids 2 and 36) were deficient in the ability to induce DNA synthesis and repress insulin enhancer-stimulated activity. The function of the N-terminal region correlated closely with binding of the 300-kilodalton E1A-associated protein and not with binding of the retinoblastoma protein. These results indicate that transformation by E1A is mediated by two functionally independent regions of the protein which interact with different specific cellular proteins and suggest that the 300-kilodalton E1A-associated protein plays a major role in E1A-mediated cell growth control mechanisms.
Preferred translation of human hepatitis B virus polymerase from core protein- but not from precore protein-specific transcript
Authors: Ou J H , Bao H , Shih C , Tahara S M
Published: January 2020

In the human hepatitis B virus (HBV) genome, the 5' end of the polymerase coding sequence overlaps with the 3' end of the core protein coding sequence. Recent results obtained from genetic studies have suggested that translation of HBV polymerase initiates from the first ATG codon of the polymerase reading frame and is not a result of frameshift translation from the core protein reading frame, as in the case of retroviruses. By using in vitro-synthesized SP6 RNA transcripts, we now demonstrate that HBV core protein-specific mRNA can direct the synthesis of polymerase from the internal polymerase ATG codon in rabbit reticulocyte lysates and Xenopus oocytes. A related message with an additional 60 nucleotides at the 5' end (pre-core protein mRNA) was not as efficient as the core protein mRNA for translation of polymerase. Furthermore, translation of polymerase from the core protein mRNA was not inhibited by the cap analog m7GpppG. This result, together with the results described above, indicates that translation of HBV polymerase occurs in a novel, cap-independent manner.
Effective clearance of mouse hepatitis virus from the central nervous system requires both CD4+ and CD8+ T cells
Authors: Williamson J S , Stohlman S A
Published: January 2020

Both CD4+ and CD8+ T cells are required for the clearance of virus from the central nervous system following infection with the JHM strain of mouse hepatitis virus. Development of antiviral antibodies requires the presence of CD4+ T cells but appears to play a minimal role in the reduction of virus. The data presented are consistent with the hypothesis that clearance of JHM virus is mediated by virus-specific CD8+ T cells, which appear to require the presence of CD4+ T cells.
Conversion of Friend mink cell focus-forming virus to Friend spleen focus-forming virus by modification of the 3' half of the env gene
Authors: Watanabe N , Nishi M , Ikawa Y , Amanuma H
Published: January 2020

The 3' half of the env gene of the dualtropic Friend mink cell focus-forming virus was modified by replacing the restriction enzyme fragment of the genome DNA with the corresponding fragment of the acutely leukemogenic, polycythemia-inducing strain of Friend spleen focus-forming virus (F-SFFVP) genome DNA. Replacement with the fragment of F-SFFVP env containing the 585-bp deletion, the 6-bp duplication, and the single-base insertion converted the resulting chimeric genome so that the mutant had a pathogenic activity like that of F-SFFVP. Replacement with the fragment containing only the 585-bp deletion did not result in a pathogenic virus. However, when this virus pseudotyped by Friend murine leukemia virus was passaged in newborn DBA/2 mice, we could recover weakly pathogenic viruses with a high frequency. Molecular analysis of the genome of the recovered virus revealed the presence of a single-base insertion in the same T5 stretch where the wild-type F-SFFV env has the single-base insertion. These results provided evidence that the unique genomic structures present in the 3' half of F-SFFV env are the sole determinants that distinguish the pathogenicity of F-SFFV from that of Friend mink cell focus-forming virus. The importance of the dualtropic env-specific sequence present in the 5' half of F-SFFV env for the pathogenic activity was evaluated by constructing a mutant F-SFFV genome in which this sequence was replaced by the ecotropic env sequence of Friend murine leukemia virus and by examining its pathogenicity. The results indicated that the dualtropic env-specific sequence was essential to pathogenic activity.
Autoanti-idiotypes exhibit mimicry of myocyte antigens in virus-induced myocarditis
Authors: Paque R E , Miller R
Published: January 2020

Mice infected with coxsackievirus B develop immunologically mediated inflammatory myocarditis in heart tissue that results in the development of autoantibodies with multiple idiotypes. The specificity and temporal development of autoantibodies produced during coxsackievirus B3 infection were assessed. Antiviral idiotypes and anti-idiotypic antibodies against coxsackievirus B3 idiotypes were detected and quantitated over 21- and 42-day periods, respectively. Both polyclonal and monoclonal anti-idiotypes exhibited greater but nonspecific binding to heart, liver, kidney, and spleen cells from virus-exposed animals and normal tissue. Binding of anti-idiotypes was also demonstrated to myosin and to solubilized heart-associated antigens but not to virus. Western immunoblot analysis revealed that monoclonal and polyclonal anti-idiotypes selectively bound to hypertonic, salt-extracted, solubilized proteins of myocyte extracts of virus-exposed animals.
The promoter, transcriptional unit, and coding sequence of herpes simplex virus 1 family 35 proteins are contained within and in frame with the UL26 open reading frame
Authors: Liu F Y , Roizman B
Published: January 2020

The herpes simplex virus 1 (HSV-1) genome specifies an abundant capsid protein which in denaturing gels forms multiple bands designated family 35 proteins (D.K. Braun, B. Roizman, and L. Pereira, J. Virol. 49:142-153, 1984). Nucleotide-sequencing studies have assigned the coding sequences of these proteins to the open reading frame UL26 (D.J. McGeoch, M.A. Dalrymple, A.J. Davidson, A. Dolan, M.C. Frame, D. McNab, L.J. Perry, J.E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988). IN studies reported here, a series of plasmid constructs containing deletions or insertions of an alpha 4 promoter or of a sequence encoding a cytomegalovirus epitope reacting with a mouse monoclonal antibody revealed the following: the open reading frame previously designated UL26 encodes two proteins which share amino acid sequences, and each coding domain is contained in its own transcriptional unit that terminates at a common, unique poly(A) site. On the basis of the transcription initiation site (+1), it was predicted that the UL26 open reading frame encodes a protein of 635 amino acids, and a protein with an apparent molecular weight of approximately 75,000 has been identified. The second transcriptional unit, designated UL26.5, predicted to specify a protein of 329 amino acids, encodes the family 35 proteins; it is transcribed by an mRNA which initiates at approximately nucleotide +1000 of the UL26 transcription initiation site and is translated from the methionine initiation codon located at position +1099 of the UL26 transcriptional unit. The DNA fragment comprising the sequences downstream of the HpaI cleavage site (+832 of UL26) contains both the promoter and the coding sequence of family 35 proteins and is both competent and efficient in expressing the proteins in transfected cells superinfected with HSV-1 or HSV-2.
Sequences from myeloblastosis-associated virus MAV-2(O) and UR2AV involved in the formation of plaques and the induction of osteopetrosis, anemia, and ataxia
Authors: Aurigemma R E , Torgersen J L , Smith R E
Published: January 2020

Recombinant viruses were made between myeloblastosis-associated virus MAV-2(O) and UR2AV to examine the relationship between regions of the MAV-2(O) genome and disease induction. The env-long terminal repeat (LTR) portion of MAV-2(O), when substituted into UR2AV, was sufficient to induce osteopetrosis identical to that caused by the parent MAV-2(O). When this region was reduced to the gp37 and LTR of MAV-2(O), osteopetrosis more severe than that caused by the parent virus was induced. Recombinant viruses that contained all or part of the MAV-2(O) env gene in the absence of the MAV-2(O) LTR induced a severe, chronic anemia and late-onset osteopetrosis, leading to the conclusion that the MAV-2(O) LTR, in addition to env, was required for rapid induction of osteopetrosis. A viral recombinant, pEU, which contained the gp85 segment of UR2AV substituted into MAV-2(O), induced an ataxia/cerebellar dysfunction not seen during infection with the other chimeric or parent viruses. In vitro studies of the parent and recombinant viruses demonstrated that the ability to form plaques on chicken embryo fibroblasts correlated with the presence of the MAV-2(O) gp37 and LTR except for construct pEU. When the viruses were inoculated into 10-day-old chickens, chimeras containing the env-LTR of gp37-LTR region of MAV-2(O) induced severe regenerative anemia similar to that induced by MAV-2(O). pEU was the exception, suggesting that the unique configuration of this chimera is responsible for its unusual pathogenic properties.
Polyomavirus tumor induction in mice: effects of polymorphisms of VP1 and large T antigen
Authors: Freund R , Calderone A , Dawe C J , Benjamin T L
Published: January 2020

By testing recombinants between "high tumor" (inducing a high incidence of tumors) and "low tumor" (inducing a low incidence of tumors) strains of polyomavirus, we have previously shown that the key determinant(s) for induction of a high tumor profile resides in coding regions of the high tumor strain (R. Freund, G. Mandel, G. G. Carmichael, J. P. Barncastle, C. J. Dawe, and T. L. Benjamin, J. Virol. 61:2232-2239, 1987). Three single-amino-acid differences between the PTA (high tumor) and RA (low tumor) virus strains have now been identified by DNA sequencing, one each in the large T antigen, in the region common to the middle and small T antigens, and in the major capsid protein VP1. Further tests of appropriate recombinants and oligonucleotide-induced mutants show that VP1 of PTA is the major determinant for induction of a high tumor profile, including all tumors of epithelial origin. The differential effect of the VP1s of PTA and RA on the tumor profile is discussed in terms of a likely contribution of the polymorphic region of VP1 to binding of receptors and infection of different cell types in the animal. The polymorphism in the large T antigen has a more restricted action, which is seen only when tested in virus carrying the VP1 type of PTA; the PTA large T antigen then promotes more rapid growth of tumors of salivary gland and thymus than the RA large T antigen.
The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells
Authors: Halbert C L , Demers G W , Galloway D A
Published: January 2020

The contribution of the E6 and E7 open reading frames of human papillomavirus type 6b (HPV6b) and HPV16 to immortalization of human keratinocytes was evaluated by using amphotropic recombinant retroviruses. The HPV16 E7 gene could immortalize primary human keratinocytes without the cooperation of the viral E6 gene; however, E6 was able to contribute significantly to the efficiency of the E7 immortalizing function. Infection of HFE cells with retroviruses carrying the 16E6, 6bE6, or 6bE6E7 open reading frame did not result in immortalization.
The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces
Authors: Lai C F , Gong S C , Esteban M
Published: January 2020

The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined. A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:1569-1577, 1990). To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32. The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein. Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E. coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability. Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor. The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor.
Biological basis of rabies virus neurovirulence in mice: comparative pathogenesis study using the immunoperoxidase technique
Authors: Jackson A C
Published: January 2020

The CVS strain of fixed rabies virus causes acute, fatal encephalomyelitis in young adult ICR mice. Variant RV194-2, which was selected from CVS virus in cell culture with a neutralizing antiglycoprotein monoclonal antibody, has a single amino acid change in the glycoprotein. The infections caused by CVS virus and RV194-2 virus were compared in mice for 14 days postinoculation of 5 x 10(7) PFU into the right masseter muscle. All CVS virus-infected mice died (mean time to death, 7.9 days), compared with a mortality rate of 8.5% for RV194-2 virus-infected mice. RV194-2 virus spread to the ipsilateral trigeminal ganglion during the first 2 days postinoculation, and both viruses spread to the ipsilateral motor nucleus of the trigeminal nerve in the pons. Both viruses spread centrifugally and caused infection of bilateral trigeminal ganglia on day 3. The viruses spread throughout the central nervous system (CNS) at similar rates, but CVS virus infected many more neurons than did RV194-2 virus. Rabies virus antigen was observed in only occasional CNS neurons after day 6 of RV194-2 virus infection. By this time, CVS virus had caused severe widespread infection. In this model, virulence depends on improved efficiency of viral spread between CNS neurons rather than the rate of spread or topographical distribution of the infection.
Transdominant repressors for human T-cell leukemia virus type I rex and human immunodeficiency virus type 1 rev function
Authors: Böhnlein S , Pirker F P , Hofer L , Zimmermann K , Bachmayer H , Böhnlein E , Hauber J
Published: January 2020

Human T-cell leukemia virus type I (HTLV-I) encodes a 27-kDa trans-acting gene product (Rex) which is involved in the regulated expression of transcripts coding for the viral structural proteins. We used oligonucleotide-directed mutagenesis to generate a series of mutant HTLV-I rex genes. Transient expression experiments demonstrated that 3 of 28 mutant proteins are functionally inactive on the homologous HTLV-I rex response element, whereas an additional 2 mutant proteins are functionally inactive on the heterologous human immunodeficiency virus type 1 rev response element. One of these mutants is able to suppress the function of the wild-type HTLV-I Rex protein in trans on the homologous rex response element sequence. Furthermore, all of these mutants are able to inhibit Rex function on the heterologous rev response element sequence. Intriguingly, only three of these mutants are able to inhibit the human immunodeficiency virus type 1 Rev protein in a dominant-negative manner.
The herpes simplex virus 1 gene encoding a protease also contains within its coding domain the gene encoding the more abundant substrate
Authors: Liu F Y , Roizman B
Published: January 2020

The herpes simplex virus 1 open reading frames UL26 and UL26.5 are 3' coterminal. The larger, UL26 open reading frame encodes a protein approximately 80,000 in apparent molecular weight and contains the promoter and coding sequence of the UL26.5 gene, which specifies a capsid protein designated infected cell protein 35. The larger product contains in its entirety the amino acid sequence of the smaller protein. We report that the UL26 gene encodes a protease which catalyzes its own cleavage and that of the more abundant product of UL26.5. By inserting the coding sequence of an epitope to a cytomegalovirus monoclonal antibody and homologs of the immunoglobulin G binding domain of staphylococcal protein A into the 3' termini of the coding domains of the two open reading frames, we identified both products of the cleavage and determined that the cleavage site is approximately 20 amino acids from the carboxyl termini of both proteins.
Zidovudine-resistant human immunodeficiency virus selected by passage in cell culture
Authors: Larder B A , Coates K E , Kemp S D
Published: January 2020

Variants of human immunodeficiency virus (HIV) with reduced sensitivity to zidovudine (3'-azido-3'-deoxythymidine) have been selected by passage of virus in cell culture in the presence of drug. Wild-type, sensitive virus became partially resistant to zidovudine by passage 12 (50% inhibitory dose values measured in HeLa CD4+ cells increased from 0.014 to 0.2 microM), and genetic analysis using the polymerase chain reaction revealed that mutations in the reverse transcriptase coding region identical to those seen in clinical isolates from treated individuals had occurred. The order of appearance of these resistance mutations in passaged virus was also similar to that in clinical isolates. The partially resistant strain, HIVRTMC/F, became highly zidovudine resistant by passage 12 (50% inhibitory dose values increased from 0.4 to 2.5 microM during passages 7 to 11). Nucleotide sequence analysis of the reverse transcriptase from this variant revealed a novel amino acid substitution (Lys----Glu) at codon 219. A different substitution at this codon (Lys----Gln) had been seen previously in clinical isolates. When this mutation was created in HIVRTMC/F by site-directed mutagenesis, the resulting partially resistant virus became highly resistant, thus confirming the significance of this change. In view of the possibility that this mutation might occur in HIV isolates during treatment of patients, we adapted our selective polymerase chain reaction procedure to enable screening for this change in clinical samples. The virus passage procedure described here may be useful for gaining further insight into the mutational events occurring during the development of resistance to zidovudine and other HIV inhibitors.
Mutational analysis of the simian immunodeficiency virus SIVmac nef gene
Authors: Binninger D , Ennen J , Bonn D , Norley S G , Kurth R
Published: January 2020

We are using site-directed mutagenesis of single viral genes to identify and analyze the genetic determinants of human and simian immunodeficiency virus pathogenicity. In a first approach, we have constructed a series of simian immunodeficiency virus SIVmac nef mutants by partial deletion and insertions in the nef gene, as this gene is a candidate gene for the establishment and maintenance of latency. nef insertion mutants replicated faster than wild-type SIVmac, suggesting that the nef gene product acts as a negative factor for replication. Surface phenotyping revealed that cultures permanently infected with nef mutants exhibit an enhanced expression of viral proteins on the outer cell surface. We have analyzed the properties of the mutant viruses in cell culture and intend to use rapidly replicating mutants (putatively unable to undergo latency) as model vaccine viruses in the rhesus monkey.
Cloning of the latency gene and the early protein 0 gene of pseudorabies virus
Authors: Cheung A K
Published: January 2020

A collection of overlapping cDNA clones encoding the latency transcript of pseudorabies virus and the DNA nucleotide sequence of the latency gene has been obtained. The transcript is spliced with 4.6 kb of intervening sequences. This mRNA, designated the large latency transcript, is 8.5 kb. It is polyadenylated and contains a large open reading frame capable of coding for a 200-kDa polypeptide. The direction of transcription is antiparallel to that of the immediate-early gene IE180 and a newly identified early gene, EP0. The latency transcript overlaps the entire IE180 gene and most of the EP0 gene. The EP0 mRNA is 1.75 kb and polyadenylated. The deduced amino acid sequence revealed the presence of cysteine-rich zinc finger domain similar to that of the immediate-early gene ICP0 of herpes simplex virus type 1 and the gene 61 polypeptide of varicella-zoster virus. On the basis of the biological functions, conserved protein domains, and unique spatial arrangements of the homologous polypeptides (IE180 versus ICP4 and EP0 versus ICP0) between pseudorabies virus and herpes simplex virus type 1, it is predicted that a homologous protein domain is also encoded by the 8.5-kb large latency transcripts of these two viruses.
Novel regulatory properties of the IE1 and IE0 transactivators encoded by the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus
Authors: Kovacs G R , Guarino L A , Summers M D
Published: January 2020

The baculovirus Autographa californica multicapsid nuclear polyhedrosis virus expresses two immediate-early genes from the HindIII-G region (map units 90.4 to 96.8) of the genome. During the early phase of infection, nonspliced 1.9-kb and spliced 2.1-kb transcripts are expressed which encode the IE1 and IE0 (spliced IE1) gene products, respectively. These two gene products differ only in that IE0 contains an additional 54 amino acids at the amino terminus. RNA analysis of these two genes during infection revealed that they were differentially expressed. IE1 was expressed early and late, whereas IE0 was expressed only early in infection. The regulation of these two immediate-early genes was analyzed by transient expression assays. The IE1 gene product stimulated expression of IE1 promoter-directed expression but down-regulated expression from the IE0 promoter. The IE0 gene product also transactivated the IE1 promoter but did not affect expression from its own promoter. Unlike IE1, which transactivates the delayed early 39K gene in the presence and absence of the homologous region (hr) enhancers, IE0 transactivated the 39K promoter only in the presence of cis-linked hr5 enhancer. The results of this study in conjunction with previous results suggest that the IE1 gene encodes a multifunctional gene product that may be involved in (i) repression of immediate-early gene expression, (ii) continued expression of its own gene product during infection, and (iii) transactivation of the delayed early and late classes of genes.
Identification and characterization of intragenic sequences which repress human immunodeficiency virus structural gene expression
Authors: Cochrane A W , Jones K S , Beidas S , Dillon P J , Skalka A M , Rosen C A
Published: January 2020

Examination of the life cycle of the human immunodeficiency virus (HIV) has shown that multiple levels of regulation exist, including some which require the virus-encoded Rev protein. In the absence of Rev, mRNAs encoding the structural proteins remain untranslated, a phenomenon which appears, in part, to be caused by nuclear entrapment of these RNA species. To examine the basis for repression of structural gene mRNA expression, a heterologous assay system was utilized to determine whether regions present within gag and pol contain elements capable of suppressing gene expression when present in cis. Both genes were found to contain cis-acting repressor sequences (CRS) that block gene expression when present within the 3' untranslated portion of a heterologous gene transcript. The element within pol was found to have the strongest repressive effect. While Rev alone was unable to reverse the repression observed with the pol sequence, addition of the env Rev-responsive element (RRE) in cis and Rev in trans did cause reversal of inhibition. Deletion mutagenesis defined a 260-bp element within the 3' portion of pol that contains a potent CRS which functions when present in the sense orientation. The corresponding region in HIV-2 pol was found to contain a functionally similar CRS element. To examine the mechanism of repression, the effects of the CRS elements on both the abundance and subcellular distribution of the mRNAs were examined. Neither was dramatically altered when examined in the context of a heterologous reporter (chloramphenicol acetyltransferase) mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
A bovine papillomavirus E1-related protein binds specifically to bovine papillomavirus DNA
Authors: Wilson V G , Ludes-Meyers J
Published: January 2020

The E1 open reading frame of bovine papillomavirus (BPV) was expressed as a RecA-E1 fusion protein in Escherichia coli. The bacterially expressed RecA-E1 protein exhibited sequence-specific DNA binding activity; strong binding to the region from nucleotides 7819 to 93 on the BPV genome (designated region A) and weak binding to the adjacent region from nucleotides 7457 to 7818 (region B) were observed. The interaction between the BPV-derived RecA-E1 protein and region A appeared to be highly specific for BPV DNA, as no comparable binding was detected with heterologous papillomavirus DNAs. Binding to region A was eliminated by digestion of region A at the unique HpaI site, which suggests that the RecA-E1 binding site(s) was at or near the HpaI recognition sequence. Binding to region B but not region A was observed when nuclear extracts from ID13 cells were used as a source of E1 proteins. The absence of region A binding by ID13 extracts may reflect a negative regulation of E1 DNA binding activity.
Immune response and resistance to Rous sarcoma virus challenge of chickens immunized with cell-associated glycoproteins provided with a recombinant avian leukosis virus
Authors: Chebloune Y , Rulka J , Cosset F L , Valsesia S , Ronfort C , Legras C , Drynda A , Kuzmak J , Nigon V M , Verdier G
Published: January 2020

The Rous-associated virus 1 env gene, which encodes the envelope gp85 and gp37 glycoproteins, was isolated and inserted in place of the v-erbB oncogene into an avian erythroblastosis virus-based vector, carrying the neo resistance gene substituted for the v-erbA oncogene, to generate the pNEA recombinant vector. A helper-free virus stock of the pNEA vector was produced on an avian transcomplementing cell line and used to infect primary chicken embryo fibroblasts (CEFs) or quail QT6 cells. These infected cells, selected with G418 (CEF/NEA and QT6/NEA, respectively) were found to be resistant to superinfections with subgroup A retroviruses. The CEF/NEA preparations were used as a cell-associated antigen to inoculate adult chickens by the intravenous route compared with direct inoculations of NEA recombinant helper-free virus used as a cell-free antigen. Chickens injected with the cell-associated antigen (CEF/NEA) exhibited an immune response demonstrated by induction of high titers of neutralizing antibodies and were found to be protected against tumor production after Rous sarcoma virus A challenge. Conversely, no immune response and no protection against Rous sarcoma virus A challenge were observed in chickens directly inoculated with cell-free NEA recombinant virus or in sham-inoculated chickens.
Neuroattenuation of an avirulent bunyavirus variant maps to the L RNA segment
Authors: Endres M J , Griot C , Gonzalez-Scarano F , Nathanson N
Published: January 2020

The derivation and characterization of a neuroattenuated reassortant clone (RFC 25/B.5) of California serogroup bunyavirus was described previously (M. J. Endres, A. Valsamakis, F. Gonzalez-Scarano, and N. Nathanson, J. Virol. 64:1927-1933, 1990). To map the RNA segment responsible for this attenuation, a panel of reassortants was constructed between the attenuated clone B.5 (genotype TLL) and a virulent clone (B1-1a) of reciprocal genotype (LTT). Parent viruses and clones representing all of the six possible reassortants were examined for neurovirulence by intracerebral injection in adult mice. Reassortants bearing the large RNA segment from the virulent parent were almost as virulent as the virulent parent virus, while reassortants bearing the large RNA segment from the avirulent parent virus exhibited low or intermediate virulence. These results indicate that the large RNA segment is the major determinant of neuroattenuation of clone B.5. In addition to its neuroattenuation, clone B.5 was temperature sensitive and exhibited an altered plaque morphology. These phenotypes also segregated with the large RNA segment. The importance of the large RNA segment (which encodes the viral polymerase) in neurovirulence contrasts with prior studies which indicate that the ability to cause lethal encephalitis after peripheral injection of suckling mice (neuroinvasiveness) is primarily determined by the middle-sized RNA segment, which encodes the viral glycoproteins.
Inhibition of human immunodeficiency virus type 1 multiplication by antisense and sense RNA expression
Authors: Joshi S , Van Brunschot A , Asad S , van der Elst I , Read S E , Bernstein A
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4+ lymphocytes and macrophages and causes AIDS in humans. Retroviral vectors allowing neomycin phosphotransferase (npt) gene expression were engineered to express 5' sequences of HIV-1 RNA in the antisense or sense orientation and used to transform the human CD4+ lymphocyte-derived MT4 cell line. Cells expressing antisense or sense RNA to the HIV-1 tat mRNA leader sequence, as part of the 3' untranslated region of the npt mRNA, remained sensitive to HIV-1 infection. In contrast, resistance to HIV-1 infection was observed in cells expressing antisense RNA to the HIV-1 primer-binding site or to the region 5' to the primer-binding site as part of the 3' region of the npt mRNA. Cells expressing the tat mRNA leader sequence in the sense orientation as a precise replacement of the 5' untranslated region of npt mRNA were also resistant to HIV-1. These results indicate that sense and antisense approaches can be used to interfere with HIV-1 multiplication.
The flavivirus envelope protein E: isolation of a soluble form from tick-borne encephalitis virus and its crystallization
Authors: Heinz F X , Mandl C W , Holzmann H , Kunz C , Harris B A , Rey F , Harrison S C
Published: January 2020

By the use of limited trypsin digestion of purified virions, we generated a membrane anchor-free and crystallizable form of the tick-borne encephalitis virus envelope glycoprotein E. It retained its reactivity with a panel of monoclonal antibodies, and only subtle structural differences from the native protein E were recognized. Treatment with the bifunctional cross-linker dimethylsuberimidate resulted in the formation of a dimer. Crystallization experiments yielded hexagonal rod-shaped crystals suitable for X-ray diffraction analysis.
The T/t common region of simian virus 40 large T antigen contains a distinct transformation-governing sequence
Authors: Marsilio E , Cheng S H , Schaffhausen B , Paucha E , Livingston D M
Published: January 2020

Simian virus 40 large T antigen (T) can transform cultured cells, but the mechanisms by which it functions are not entirely understood. Several lines of evidence have suggested that the amino-terminal approximately 130 residues of T may be sufficient to confer the transforming capability. Oligonucleotide-directed mutagenesis was used to generate a series of deletion and substitution mutants within the amino-terminal 82 residues of T, the segment which is shared with simian virus 40 small t antigen (t). Results of stability and transformation assays of these mutants strongly suggest that the 1-to-82 region of T contains sequences which govern T transforming activity and affect in vivo stability. Instability and a defect in transforming activity could be separated from one another genetically. Thus, the 1-to-82 region appears to contain a specific region that contributes to the transforming function of the protein. This segment operates by means other than the simple binding of pRb and/or p107.
Identification of an immunodominant epitope within the phosphoprotein of rabies virus that is recognized by both class I- and class II-restricted T cells
Authors: Larson J K , Wunner W H , Otvos L , Ertl H C
Published: January 2020

Immunization of H-2k mice with live rabies virus induces cytolytic T lymphocytes to the phosphoprotein of rabies virus. The antigenic determinant responsible for stimulating this class I-restricted cytolytic response was mapped to 50 amino acids (residues 180 to 229) of the phosphoprotein by using vaccinia virus recombinants expressing either the full-length phosphoprotein or C-terminal truncations of the phosphoprotein. The epitope was more finely mapped to residues 191 to 206 by using synthetic peptides. Several CD4+, class II-restricted T-cell lines were isolated from splenocytes of H-2k mice immunized with the vaccinia virus-rabies virus phosphoprotein recombinant virus. These lines were specifically stimulated by the phosphoprotein, and in addition, each line proliferated and released lymphokines in response to the same synthetic peptide shown to stimulate phosphoprotein-specific, class I-restricted cytolytic T cells.
Hepatitis E virus: identification of type-common epitopes
Authors: Yarbough P O , Tam A W , Fry K E , Krawczynski K , McCaustland K A , Bradley D W , Reyes G R
Published: January 2020

Large epidemic outbreaks of enterically transmitted non-A, non-B viral hepatitis (ET-NANBH) have been documented in developing countries. A molecular clone derived from the causative agent, the hepatitis E virus (HEV), has recently been described (G.R. Reyes, M.A. Purdy, J.P. Kim, K.-C. Luk, L.M. Young, K.E. Fry, and D. Bradley, Science 247:1335-1339, 1990). We now report the isolation, by serologic screening, of two cDNA clones derived from a fecal sample collected during a 1986 outbreak of ET-NANBH in Telixtac, Mexico. The cDNA clones encode epitopes that specifically reacted with acute- and convalescent-phase sera collected during five different ET-NANBH epidemics and represent the initial cloning of the Mexico strain of HEV. Recombinant fusion proteins expressed from these clones were also recognized by antibodies from cynomolgus macaques experimentally infected with HEV. The cDNA clones were shown to be derived from HEV by their specific hybridization to the previously recognized full-length genomic RNA transcript of approximately 7.5 kb. In addition, however, subgenomic polyadenylated transcripts of approximately 2.0 and approximately 3.7 kb were also identified in HEV-infected cynomolgus monkey liver. Sequences homologous to the epitope clones were isolated from the Burma strain of the virus, and these demonstrated reactivity comparable to that seen with the Mexico strain epitopes. When compared with the available full-length sequence of the Burma strain of HEV, it was discovered that the cDNA clones were encoded in different open reading frames (ORFs). The comparison between Mexico and Burma HEV strains indicated amino acid homologies of 90.5 and 73.5% for these epitope-encoding clones derived from ORF2 and ORF3, respectively. The identification of these clones not only has provided insight into the expression strategy of HEV but has also resulted in a source of recombinant protein useful in the diagnosis of HEV-induced hepatitis.
Mechanisms of receptor-mediated rhinovirus neutralization defined by two soluble forms of ICAM-1
Authors: Greve J M , Forte C P , Marlor C W , Meyer A M , Hoover-Litty H , Wunderlich D , McClelland A
Published: January 2020

The majority of human rhinoviruses use intercellular adhesion molecule 1 (ICAM-1) as a cell surface receptor. Two soluble forms of ICAM-1, one corresponding to the entire extracellular portion [tICAM(453)] and one corresponding to the two N-terminal immunoglobulin-like domains [tICAM(185)], have been produced, and their effects on virus-receptor binding, virus infectivity, and virus integrity have been examined. Results from competitive binding experiments indicate that the virus binding site is largely contained within the two N-terminal domains of ICAM-1. Virus infectivity studies indicate that tICAM(185) prevents infection by direct competition for receptor binding sites on virus, while tICAM(453) prevents infection at concentrations 10-fold lower than that needed to inhibit binding and apparently acts at the entry or uncoating steps. Neutralization by both forms of soluble ICAM-1 requires continual presence of ICAM-1 during the infection and is largely reversible. Both forms of soluble ICAM-1 can alter rhinovirus to yield subviral noninfectious particles lacking the viral subunit VP4 and the RNA genome, thus mimicking virus uncoating in vivo, although this irreversible modification of rhinovirus is not the major mechanism of virus neutralization.
Identification of cell membrane proteins that bind visna virus
Authors: Crane S E , Buzy J , Clements J E
Published: January 2020

Visna virus infects cells of ovine origin by attaching to a cell surface receptor via its envelope glycoprotein. The identity of the visna virus receptor is not known. To identify the molecule responsible for binding the virus to target cells, virus overlay protein blot assays were used to examine the molecular weights of cell surface molecules which bind purified virus. Molecules on the surface of goat synovial membrane (GSM) cells and sheep choroid plexus (SCP) cells of approximately 15, 30, and 50 kDa bound to visna virus. The binding of visna virus to these proteins was reduced by preincubating virus with neutralizing antibodies. 125I-labeled cell membrane preparations of GSM and SCP cells were used to affinity purify these virus-binding proteins. These proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had molecular masses of 15, 30, and 50 kDa. Antibodies to the 50-kDa protein bound to the surface of both live SCP and GSM cells in immunofluorescence assays. In addition, antibodies to the 50-kDa protein blocked the binding of [35S]methionine-labeled visna virus to SCP cells in culture. Antibodies raised against the 15- and 30-kDa proteins did not block virus binding to cells. The blocking activity of antibody of the 50-kDa protein provided data that this protein is the molecule which visna virus recognizes and binds to on the surface of target cells.
Characterization of a discontinuous human immunodeficiency virus type 1 gp120 epitope recognized by a broadly reactive neutralizing human monoclonal antibody
Authors: Thali M , Olshevsky U , Furman C , Gabuzda D , Posner M , Sodroski J
Published: January 2020

While one hypervariable, linear neutralizing determinant on the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein has been well characterized, little is known about the conserved, discontinuous gp120 epitopes recognized by neutralizing antibodies in infected individuals. Here, the epitope recognized by a broadly reactive neutralizing monoclonal antibody (F105) derived from an HIV-1-infected patient was characterized by examining the effects of changes in conserved gp120 amino acids on antibody reactivity. The F105 epitope was disrupted by changes in gp120 amino acids 256 and 257, 368 to 370, 421, and 470 to 484, which is consistent with the discontinuous nature of the epitope. Three of these regions are proximal to those previously shown to be important for CD4 binding, which is consistent with the ability of the F105 antibody to block gp120-CD4 interaction. Since F105 recognition was more sensitive to amino acid changes in each of the four identified gp120 regions than was envelope glycoprotein function, replication-competent mutant viruses that escaped neutralization by the F105 antibody were identified. These studies identify a conserved, functional HIV-1 gp120 epitope that is immunogenic in man and may serve as a target for therapeutic or prophylactic intervention.
Long-term in vivo expression of genes introduced by retrovirus-mediated transfer into mammary epithelial cells
Authors: Smith G H , Gallahan D , Zwiebel J A , Freeman S M , Bassin R H , Callahan R
Published: January 2020

Nonimmortalized mouse mammary epithelial cells expressing Escherichia coli beta-galactosidase from a murine amphotropic packaged retroviral vector were injected into the epithelium-divested mammary fat pads of syngeneic mice. Mammary glands formed from the injected mammary epithelial cells contained ductal and lobular cells, both of which expressed beta-galactosidase when examined in situ more than 12 months later. These results indicate that stable recombinant gene expression can be achieved in vivo in the mammary gland without altering the growth properties of normal mammary epithelium.
Analysis of Moloney murine leukemia virus revertants mutated at the gag-pol junction
Authors: Odawara T , Yoshikura H , Ohshima M , Tanaka T , Jones D S , Nemoto F , Kuchino Y , Iwamoto A
Published: January 2020

Among Moloney murine leukemia viruses (Mo-MuLVs) having stop codons other than UAG at the gag-pol junction, Mo-MuLV with UAA, but not with UGA, had a replication disadvantage. Mo-MuLV with a glutamine codon (CAG) at the junction did not replicate. A revertant of this virus consisted of the original virus and a virus with a deletion of the pol region. Protease and Pr65gag encoded by their respective genomes complemented each other.
Transactivation of human immunodeficiency virus type 1 long terminal repeats by cell surface tumor necrosis factor alpha
Authors: Tadmori W , Mondal D , Tadmori I , Prakash O
Published: January 2020

Tumor necrosis factor alpha (TNF-alpha) is expressed in secreted and cell surface (csTNF-alpha) forms by activated monocytic and T cells. In this report, we demonstrate that csTNF-alpha may predominantly regulate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) activation in the promonocytic cell line U937 and in the Epstein-Barr virus-transformed B-cell line BH1. Anti-TNF-alpha antibody suppressed both the constitutive expression of the HIV-1 LTR in BH1 cells and the expression induced by phorbol 12-myristate 13-acetate in U937 cells. This suppression was found to be mediated via csTNF-alpha. No correlation between the HIV-1 LTR activation and the secretion of TNF-alpha was evident in these cell lines. Suppression of TNF-alpha secretion by cyclosporin A or by a serine protease inhibitor did not suppress the HIV-1 LTR activation. These observations suggest a novel biological role for csTNF-alpha in the immunopathogenesis of AIDS.
Splice site skipping in polyomavirus late pre-mRNA processing
Authors: Luo Y , Carmichael G G
Published: January 2020

Polyomavirus late nuclear primary transcripts contain tandem repeats of the late strand of the viral genome, as a result of inefficient transcription termination and polyadenylation. Pre-mRNA processing involves the splicing of short noncoding late leader exons to each other (removing genome-length introns) and the splicing of the last leader to a coding body exon (such as for the major virion structural protein, VP1). As a result, cytoplasmic mRNAs contain 1 to 12 tandem leader exons at their 5' ends that are followed by a single coding exon. To understand more about how polyomavirus exons are spliced together, we studied a double-genome construct consisting of two tandem but nonidentical polyomavirus late transcription units. The alternating leader exons are distinguishable from one another but retain identical flanking RNA-processing signals, as for the alternating VP1 exons. We transfected this construct and derivatives of it into mouse cells and determined which leader exons are spliced to which others and which VP1 exons are utilized. Results showed that leader exons are almost never skipped during splicing and are spliced sequentially to one another. On the other hand, VP1 exons were often skipped, with the VP1 exon closest to the polyadenylation site splicing to the nearest upstream leader exon. Splice site replacement experiments showed that VP1 exon skipping is not due to a relative weakness of its 3' splice site or to any sequence upstream of the VP1 3' splice site. Exon skipping is also not the result of sequences within the VP1 exon. Rather, VP1 3' splice site skipping can be eliminated by replacing the inefficient late polyadenylation signal with an efficient one, or by inserting a 5' splice site between the VP1 3' splice site and the late polyadenylation site. Thus, sequences that compose the distal border of the VP1 exon can influence usage of the upstream 3' splice site.
Human T-cell leukemia virus type II Rex binding and activity require an intact splice donor site and a specific RNA secondary structure
Authors: Black A C , Ruland C T , Yip M T , Luo J , Tran B , Kalsi A , Quan E , Aboud M , Chen I S , Rosenblatt J D
Published: January 2020

The human T-cell leukemia virus type II (HTLV-II) regulatory protein Rex augments cytoplasmic levels of unspliced gag-pol mRNA by acting through a Rex-responsive element (RxRE) in the long terminal repeat. Purified Rex protein binds to long terminal repeat mRNA. Here, using an immunobinding assay to measure the binding of Rex protein to mutated RxRE RNAs, we show that efficient Rex binding requires a stem-bulge-loop RNA secondary structure (nucleotides [nt] 465 to 500) and specific sequences both within the stem-bulge (nt 470 to 476) and within a conserved upstream splice donor site (nt 449 to 455). Rex function in a transient transfection expression system correlates with Rex protein-RxRE RNA binding. The ability of HTLV-II Rex to interact directly with the HTLV-II splice donor site suggests that HTLV-II Rex may increase expression of unspliced gag-pol mRNA, in part, by inhibiting splicing.
Simian virus 40 late mRNA leader sequences involved in augmenting mRNA accumulation via multiple mechanisms, including increased polyadenylation efficiency
Authors: Chiou H C , Dabrowski C , Alwine J C
Published: January 2020

We have examined the contribution of 5' leader sequences to expression directed by the simian virus 40 (SV40) late promoter. These studies showed that addition of sequences which contain the late leader 3' splice site to the late promoter led to an increase in the accumulation of mRNA expressed by the promoter. No other sequences within the leader region, between SV40 positions 334 and 560, exhibited a substantial influence on mRNA accumulation. The increase was due, at least in part, to the creation of a spliceable mRNA transcript, since mutation of either the 5' or 3' splice site could attenuate the effect. However, sequences at or near the 3' splice site appeared to play a more important role than did the 5' splice site in bringing about this increase. In many instances, mutation of the 3' splice site also led to the accumulation of extended transcripts, whereas mutation of the 5' splice site did not produce this result in any instance. Analysis of these extended transcripts showed that they retained sequences normally lost upon cleavage and polyadenylation. This finding suggested that mutation of the 3' splice site sequence led to decreases in the efficiency of polyadenylation. We propose that the SV40 late leader sequences positively contribute to expression of the viral late genes by increasing mRNA accumulation via multiple mechanisms, including the enhancement of pre-mRNA polyadenylation efficiency.