bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations responsible for adaptation of hepatitis A virus to efficient growth in cell culture
Authors: Emerson S U , McRill C , Rosenblum B , Feinstone S , Purcell R H
Published: January 2020

Chimeric genomes of hepatitis A virus strain HM-175 were constructed from cDNA clones of the wild-type virus and its cell culture-adapted variant. RNA transcribed in vitro from each construct was assayed for infectivity by transfection of cultured cells. RNA transcribed from the wild-type cDNA clone was minimally infectious and produced virus that grew inefficiently in vitro, whereas that transcribed from certain chimeric genomes consistently produced virus that grew efficiently in cultured cells. Mutations in the P2 region were found to be necessary for efficient virus growth in vitro, while mutations in the 5' noncoding region imparted a conditional enhancement of growth in vitro.
Transcriptional elongation by purified RNA polymerase II is blocked at the trans-activation-responsive region of human immunodeficiency virus type 1 in vitro
Authors: Bengal E , Aloni Y
Published: January 2020

It has previously been shown that the human immunodeficiency virus type 1 (HIV-1) trans-activation-responsive region (TAR) is contained in a stem-loop RNA structure. Moreover, the interaction of the RNA secondary structure with Tat, the trans-activator protein, seems to play a role in activation of transcription initiation and in preventing transcription attenuation. In this work, we have studied the ability of the HIV-1 TAR stem-loop to act as a specific attenuation signal for highly purified RNA polymerase II. We developed an in vitro system using dC-tailed DNA fragments of HIV-1 to study transcriptional control in the HIV-1 LTR. We have found that transcription in this system yields an attenuator RNA whose 3' end maps to the end of the TAR stem-loop, approximately 60 to 65 nucleotides downstream of the in vivo initiation site. Furthermore, transcription attenuation occurs only under conditions which cause displacement of the nascent transcript from the template DNA strand, thus allowing the RNA to fold into secondary structure. Evidence is provided that the purified polymerase II indeed recognizes stable RNA secondary structure as an intrinsic attenuation signal. The existence of this signal in the TAR stem-loop suggests that in vivo an antiattenuation factor, probably Tat, alone or in combination with other factors, acts to relieve the elongation block at the HIV-1 attenuation site.
Activation of the c-ski oncogene by overexpression
Authors: Colmenares C , Sutrave P , Hughes S H , Stavnezer E
Published: January 2020

The v-ski oncogene is a truncated version of the cellular proto-oncogene, c-ski, and lacks sequences coding for both the N- and C-terminal ends of the c-ski protein. In the region of overlap, v-ski and c-ski differ by only one amino acid. To determine whether these differences underlie v-ski's oncogenic activation, we have cloned cDNAs for several alternatively spliced c-ski mRNAs and introduced these cDNAs into replication-competent retroviral vectors. The biological activities of these c-ski constructs have been compared with those of v-ski. We found that all c-ski gene products, when expressed at high levels from the promoter in the retroviral long terminal repeat, can induce morphological transformation, anchorage independence, and muscle differentiation in avian cells. Cells that are susceptible to ski-induced transformation and myogenesis normally express endogenous c-ski at low levels. Thus, it appears that overexpression of ski is sufficient for oncogenic and myogenic activation.
Internal initiation of translation in retroviral vectors carrying picornavirus 5' nontranslated regions
Authors: Adam M A , Ramesh N , Miller A D , Osborne W R
Published: January 2020

Previous work has shown that picornavirus 5' nontranslated regions (NTRs) can initiate internal translation of downstream coding regions both in vitro and in transient in vivo assays. We have used 5' NTR sequences from encephalomyocarditis virus and poliovirus to construct retroviral vectors that are designed to express two proteins from a single mRNA. Inclusion of 5' NTR sequences did not adversely affect vector titer. Protein expression was studied with stable cell lines generated by vector infection of mouse NIH 3T3 cells and human and canine skin fibroblasts. Expression of a coding region in the downstream position was at levels from 25 to 100% of the same coding region in the upstream position. Expression of downstream coding regions in control vectors that did not contain the 5' NTR sequences was very low, in agreement with the predictions of the scanning model for eukaryotic translation. These experiments demonstrate coordinate expression of two coding regions from a single mRNA in stable cell lines and provide further support for the model of internal translation initiation by sequences in the 5' NTRs of picornaviruses.
Prevalence of the archetypal regulatory region and sequence polymorphisms in nonpassaged BK virus variants
Authors: Negrini M , Sabbioni S , Arthur R R , Castagnoli A , Barbanti-Brodano G
Published: January 2020

Since the first isolation and characterization of BK virus (BKV), a number of BKV variants which differ in genomic structure or antigenic determinants have been described. The regulatory region, in particular, the enhancer elements, show the most divergent sequences among different isolates. The structural organization of a putative ancestral prototype or archetype, from which all of the variants are probably derived, has been proposed. By sequencing the regulatory regions of 13 different isolates from the urine of bone marrow transplant recipients, we determined the structures and sequences of BKV variants diffused in the human population. The enhancer region was amplified by polymerase chain reaction to avoid passage in culture, and the product was directly sequenced. The structure most frequently observed is in agreement with the postulated archetype, containing a single enhancer element with no repeats. By sequence analysis we identified four hot spots of nucleotide variation. These variations are consistent with the existence of two consensus sequences. One sequence motif, observed in about 85% of the isolates, is referred to as the archetypal BKV, while a second motif, observed in the remaining 15% of the variants, is highly reminiscent of the AS strain.
The Lang strain of reovirus serotype 1 and the Dearing strain of reovirus serotype 3 differ in their sensitivities to beta interferon
Authors: Jacobs B L , Ferguson R E
Published: January 2020

Replication of the Dearing strain of reovirus serotype 3 in mouse L cells was decreased 17- to 100-fold when a saturating dose of beta interferon (1,000 IU/ml) was used. Replication of the Lang strain of reovirus serotype 1 was inhibited only two- to threefold under similar conditions. It therefore appears that closely related strains of reovirus differ in their sensitivities to beta interferon treatment of mouse L cells.
Overexpression of the gag-pol precursor from human immunodeficiency virus type 1 proviral genomes results in efficient proteolytic processing in the absence of virion production
Authors: Park J , Morrow C D
Published: January 2020

The expression of the gag-pol polyprotein of human immunodeficiency virus type 1 (HIV-1) occurs via ribosomal frameshifting between the gag and pol genes. Because low levels of the gag-pol precursor are naturally produced in HIV-1-infected cells, a limited amount of information is available on the biology of this molecule. To further study this polyprotein, two mutant HIV-1 proviral genomes were created to position the gag and pol genes in the same translational reading frame. The mutations inserted a single thymidine nucleotide at the site of ribosomal frameshifting (nucleotide 1635), which results in the addition of a phenylalanine residue (frameshift 1 [FS1]), or a single adenine nucleotide, which results in the addition of a leucine residue (frameshift 2 [FS2]). Transfection of the mutant proviral genomes into COS-1 cells resulted in the expression of the p160gag-pol polyprotein precursor as well as the proteolytically processed gag and pol gene products. Metabolic labeling of the transfected cells with [3H]myristic acid revealed that the p160gag-pol and p17gag proteins expressed from the mutant genomes were myristylated. While the supernatants from COS-1 cells transfected with wild-type or mutant proviral genomes contained similar amounts of p24 antigen, the levels of reverse transcriptase were, on the average, 10 times greater in the supernatants from cells transfected with the FS1 and FS2 proviral genomes. The cells transfected with the wild-type proviral genome released infectious viral particles, while the mutant proviral genomes released p24 and reverse transcriptase in the absence of detectable particle formation. The mutant proviral genomes were completely noninfectious as determined by coculture of the transfected COS-1 cells with SupT1 cells. These results demonstrate that the gag-pol polyprotein of HIV-1 contains the appropriate signals for proteolytic processing and association with intracytoplasmic membranes in the absence of virion formation.
Kinetics of soluble CD4 binding to cells expressing human immunodeficiency virus type 1 envelope glycoprotein
Authors: Dimitrov D S , Hillman K , Manischewitz J , Blumenthal R , Golding H
Published: January 2020

The high-affinity interaction between the envelope glycoprotein (gp120-gp41) of the human immunodeficiency virus type 1 and its receptor, CD4, is important for viral entry into cells and therapeutical approaches based on the soluble form of CD4 (sCD4). Using flow cytometry, we studied the kinetics of binding of sCD4 to gp120-gp41 expressed on the cell surface. sCD4 binding was dependent on sCD4 concentration and temperature and exhibited bimolecular reaction kinetics. Binding was very slow at low sCD4 concentrations (below 0.2 micrograms/ml) and low temperatures (below 13 degrees C) but increased sharply with increasing temperature. The rate constant for association at 37 degrees C (1.5 x 10(5) M-1 s-1) was 14-fold higher than at 4 degrees C, but the affinity of sCD4 to membrane-bound gp120-gp41 was not significantly affected. The activation energy at higher temperatures (28 to 37 degrees C) was less than at lower temperatures (4 to 13 degrees C). After long periods of incubation, we observed a decrease of surface-bound sCD4 and gp120, even at low temperatures, which was attributed to sCD4-induced shedding of gp120. The rate of gp120 shedding was much lower than the rate of sCD4 binding and was dependent on sCD4 concentration and temperature. The finding that sCD4 binding is slow, especially at low sCD4 concentrations, can be of critical importance for efficient blocking of viral infection by sCD4 and should be considered when designing new protocols in the therapy of AIDS patients.
Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein
Authors: Schwartz S , Felber B K , Pavlakis G N
Published: January 2020

The expression of Gag, Pol, Vif, Vpr, Vpu, and Env proteins from unspliced and partially spliced human immunodeficiency virus type 1 (HIV-1) mRNAs depends on the viral protein Rev, while the production of Tat, Rev, and Nef from multiply spliced mRNAs does not require Rev. To investigate the difference between gag and tat mRNAs, we generated plasmids expressing tat-gag hybrid mRNAs. Insertion of the gag gene downstream of the tat open reading frame in the tat cDNA resulted in the inhibition of Tat production. This inhibition was caused, at least in part, by a decrease in the stability of the produced mRNA. Deletions in gag defined a 218-nucleotide inhibitory sequence named INS-1 and located at the 5' end of the gag gene. Further experiments indicated the presence of more than one inhibitory sequence in the gag-protease gene region of the viral genome. The inhibitory effect of INS-1 was counteracted by the positive effect mediated by the Rev-Rev-responsive element interaction, indicating that this sequence is important for Rev-regulated gag expression. The INS-1 sequence did not contain any known HIV-1 splice sites and acted independently of splicing. It was found to have an unusually high AU content (61.5% AU), a common feature among cellular mRNAs with short half-lives. These results suggest that HIV-1 and possibly other lentiviruses have evolved to express unstable mRNAs which require additional regulatory factors for their expression. This strategy may offer the virus several advantages, including the ability to enter a state of low or latent expression in the host.
Native but not denatured recombinant human immunodeficiency virus type 1 gp120 generates broad-spectrum neutralizing antibodies in baboons
Authors: Haigwood N L , Nara P L , Brooks E , Van Nest G A , Ott G , Higgins K W , Dunlop N , Scandella C J , Eichberg J W , Steimer K S
Published: January 2020

The protection of individuals from human immunodeficiency virus type 1 (HIV-1) infection with an envelope subunit derived from a single isolate will require the presentation of conserved epitopes in gp120. The objective of the studies presented here was to test whether a native recombinant gp120 (rgp120) immunogen would elicit responses to conserved neutralization epitopes that are not present in a denatured recombinant gp120 antigen from the same virus isolate. In a large study of 51 baboons, we have generated heterologous neutralizing activity with native, glycosylated rgp120SF2 but not with denatured, nonglycosylated env 2-3SF2. After repeated exposure to rgp120SF2 formulated with one of several adjuvants, virus isolates from the United States, the Caribbean, and Africa were neutralized. The timing of the immunization regimen and the choice of adjuvant affected the virus neutralization titers both quantitatively and qualitatively. These results suggest that vaccination with native, glycosylated rgp120 from a single virus isolate, HIV-SF2, may elicit a protective immune response effective against geographically and sequentially distinct HIV-1 isolates.
Molecular cloning and characterization of the RNA packaging-defective retrovirus SE21Q1b
Authors: Anderson D J , Lee P , Levine K L , Sang J S , Shah S A , Yang O O , Shank P R , Linial M L
Published: January 2020

The nonconditional RNA packaging mutant SE21Q1b contains cis- and trans-acting defects which cause cellular mRNA, rather than viral genomic RNA, to be nonspecifically packaged into SE21Q1b viral particles. Using genomic libraries of the c-SE21Q1b quail cell line, we have been able to construct a molecular clone of the SE21Q1b provirus. Upon transfection into primary quail embryo fibroblasts, the SE21Q1b molecular clone is able to recapitulate the nonspecific RNA packaging phenotype of the c-SE21Q1b cell line. The RNA packaging phenotypes displayed by several SE21Q1b/avian sarcoma-leukemia virus hybrid provirus constructs have further indicated that sequences responsible for the altered RNA packaging phenotype of SE21Q1b are localized in the left third of the SE21Q1b proviral genome. DNA sequence analysis of this region has revealed that the 5' SE21Q1b deletion has removed 179 bp from the SE21Q1b left long terminal repeat and leader regions. Several differences were detected at the carboxyl terminus of the deduced SE21Q1b nucleocapsid protein sequence in comparison with that of Rous sarcoma virus PR-C. Results of site-directed oligonucleotide mutagenesis experiments indicate, however, that the presence of these residues in the nucleocapsid protein alone is not responsible for the decreased RNA packaging specificity of SE21Q1b.
Structural organization, expression, and functional characterization of the murine cytomegalovirus immediate-early gene 3
Authors: Messerle M , Bühler B , Keil G M , Koszinowski U H
Published: January 2020

We have previously defined ie3 as a coding region located downstream of the ie1 gene which gives rise to a 2.75-kb immediate-early (IE) transcript. Here we describe the structural organization of the ie3 gene, the amino acid sequence of the gene product, and some of the functional properties of the protein. The 2.75-kb ie3 mRNA is generated by splicing and is composed of four exons. The first three exons, of 300, 111, and 191 nucleotides (nt), are shared with the ie1 mRNA and are spliced to exon 5, which is located downstream of the fourth exon used by the ie1 mRNA. Exon 5 starts 28 nt downstream of the 3' end of the ie1 mRNA and has a length of 1,701 nt. The IE3 protein contains 611 amino acids, the first 99 of which are shared with the ie1 product pp89. The IE3 protein expressed at IE times has a relative mobility of 88 kDa in gels, and a mobility shift to 90 kDa during the early phase is indicative of posttranslational modification. Sequence comparison reveals significant homology of the exon 5-encoded amino acid sequence with the respective sequence of UL 122, a component of the IE1-IE2 complex of human cytomegalovirus (HCMV). This homology is also apparent at the functional level. The IE3 protein is a strong transcriptional activator of the murine cytomegalovirus (MCMV) e1 promoter and shows an autoregulatory function by repression of the MCMV ie1/ie3 promoter. The high degree of conservation between the MCMV ie3 and HCMV IE2 genes and their products with regard to gene structure, amino acid sequence, and protein functions suggests that these genes play a comparable role in the transcriptional control of the two cytomegaloviruses.
Herpes simplex virus type 1 recombination: role of DNA replication and viral a sequences
Authors: Dutch R E , Bruckner R C , Mocarski E S , Lehman I R
Published: January 2020

During the course of infection, elements of the herpes simplex virus type 1 (HSV-1) genome undergo inversion, a process that is believed to occur through the viral a sequences. To investigate the mechanism of this recombinational event, we have developed an assay that detects the deletion of DNA segments flanked by directly repeated a sequences in plasmids transiently maintained in Vero cells. With this assay, we have observed a high frequency of recombination (approximately 8%) in plasmids that undergo replication in HSV-1-infected cells. We also found a low level of recombination between a sequences in plasmids introduced into uninfected cells and in unreplicated plasmids in HSV-1-infected cells. In replicating plasmids, recombination between a sequences occurs at twice the frequency seen with directly repeated copies of a different sequence of similar size. Recombination between a sequences appears to occur at approximately the same time as replication, suggesting that the processes of replication and recombination are closely linked.
Similar replication capacities of primary human immunodeficiency virus type 1 isolates derived from a wide range of clinical sources
Authors: Lu W , Andrieu J M
Published: January 2020

Numerous studies have suggested that there are significant differences in replication capacities and cytopathicities among human immunodeficiency virus type 1 (HIV-1) isolates and that these differences correlate with the clinical status and geographical origin of infected individuals. However, it has been difficult to assess whether reported distinctions could be attributed to the methods used or whether they imply a true disparity between viral isolates. We thus attempted to characterize the replication properties of HIV-1 isolates directly recovered from infected patients (primary isolates) by using a standardized infection assay. Viruses were isolated from patients' peripheral blood mononuclear cells (PBMC) by a single coculture with normal donor PBMC stimulated with phytohemagglutinin. Replication curves and cytopathic effect of a standard inoculum (1 ng of p24) of 66 primary HIV-1 isolates were similar regardless of clinical stage of the patient (asymptomatic, AIDS-related complex, or AIDS) and evolutive feature (rate of progression to AIDS). There was no difference between viruses derived from patients sensitive to zidovudine and those derived from patients resistant to zidovudine. Moreover, no difference was found among viral isolates of different geographical origins (Central Africa, Zaire, Brazil, or France). Similarly, the replication patterns and cytopathicities of isolates from bronchoalveolar lymphocytes did not differ from those of isolates derived from PBMC. In contrast, the same amount of viral inoculum of five laboratory HIV-1 strains (HIV-1, EL1, SF, MN, and RF) produced different replication curves and were much less cytopathic. In contrast to laboratory viral strains, it appears that the primary HIV-1 isolates tested, whatever their clinical status and source, exhibited similar replication capacities and cytopathicities in allogeneic donor PBMC.
Subunit interactions of vesicular stomatitis virus envelope glycoprotein stabilized by binding to viral matrix protein
Authors: Lyles D S , McKenzie M , Parce J W
Published: January 2020

The mechanism by which viral glycoproteins are incorporated into virus envelopes during budding from host membranes is a major question of virus assembly. Evidence is presented here that the envelope glycoprotein (G protein) of vesicular stomatitis virus binds to the viral matrix protein (M protein) in vitro with the specificity, reversibility, and affinity necessary to account for virus assembly in vivo. The assay for the interaction is based on the ability of M protein to stabilize the interaction of G protein subunits, which exist as trimers of identical subunits in the virus envelope. The interaction with M protein was shown by using G proteins labeled with fluorescent probes capable of detecting subunit dissociation and reassociation in vitro. The results show that the M protein isolated from virions either as purified soluble protein or as nucleocapsid-M protein complexes interacts with the G protein in vitro and that the reaction is reversible. The interaction between the G and M proteins was not serotype specific, but no interaction between the vesicular stomatitis virus M protein and the influenza virus hemagglutinin could be detected. These results support the conclusion that the interactions described here are the ones that govern assembly of G protein into virus envelopes in vivo.
Hepatitis B virus p25 precore protein accumulates in Xenopus oocytes as an untranslocated phosphoprotein with an uncleaved signal peptide
Authors: Yang S Q , Walter M , Standring D N
Published: January 2020

We have analyzed the translocation of hepatitis B virus (HBV) precore (PC) proteins by using Xenopus oocytes injected with a synthetic PC mRNA. The PC region is a 29-amino-acid sequence that precedes the 21.5-kDa HBV capsid or core (C) protein (p21.5) and directs the secretion of core-related proteins. The first 19 PC amino acids provide a signal peptide that is cleaved with the resultant translocation of a 22.5-kDa species (p22.5), in which the last 10 PC residues precede the complete p21.5 C polypeptide. Most p22.5 is matured to 16-20 kDa species by carboxyl-terminal proteolytic cleavage prior to secretion. Here we show that some four unexpected PC proteins of 24 to 25 kDa are produced in addition to the secretion products described above. Protease protection and membrane cosedimentation experiments reveal that all PC proteins behave as expected for proteins that are translocated into the lumen of the endoplasmic reticulum except for the single largest PC protein (p25), which is not translocated. Like p21.5, p25 is a phosphoprotein that localizes to the oocyte cytosol and nucleus, and protease digestion studies suggest that the two molecules have similar two-domain structures. Radiosequencing of immobilized p25 demonstrates that it contains the intact PC signal peptide and represents the unprocessed translation product of the entire PC/C locus. Thus, while many HBV PC protein molecules are correctly targeted to intracellular membranes and translocated, a significant fraction of these molecules can evade translocation and processing.
Enhancement of transforming potential of human insulinlike growth factor 1 receptor by N-terminal truncation and fusion to avian sarcoma virus UR2 gag sequence
Authors: Liu D , Rutter W J , Wang L H
Published: January 2020

The human insulinlike growth factor 1 (hIGF-1) receptor (hIGFR) is a transmembrane protein tyrosine kinase (PTK) molecule which shares high sequence homology in the PTK domain with the insulin receptor and, to a lesser degree, the ros transforming protein of avian sarcoma virus UR2. To assess the transforming potential of hIGFR, we introduced the intact and altered hIGFR into chicken embryo fibroblasts (CEF). The full-length hIGFR cDNA (fIGFR) was cloned into a UR2 retroviral vector, replacing the original oncogene v-ros. fIGFR was able to promote the growth of CEF in soft agar and cause morphological alteration in the absence of added hIGF-1 to medium containing 11% calf and 1% chicken serum. The transforming ability of hIGFR was not further increased in the presence of 10 nM exogenous hIGF-1. The 180-kDa protein precursor of hIGFR was synthesized and processed into alpha and beta subunits. The overexpressed hIGFR in CEF bound hIGF-1 with high affinity (Kd = 5.4 x 10(-9) M) and responded to ligand stimulation with increased tyrosine autophosphorylation. The cDNA sequence coding for part of the beta subunit of hIGFR, including 36 amino acids of the extracellular domain and the entire transmembrane and cytoplasmic domains, was fused to the 5' portion of the gag gene in the UR2 vector to form an avian retrovirus. The resulting virus, named UIGFR, was able to induce morphological transformation and promote colony formation of CEF with a stronger potency than did fIGFR. The UIGFR genome encodes a membrane-associated, glycosylated gag-IGFR fusion protein. The specific tyrosine phosphorylation of the mature form of the fusion protein, P75, is sixfold higher in vitro and threefold higher in vivo than that of the native IGFR beta subunit, P95. In conclusion, overexpression of the native or an altered hIGFR can induce transformation of CEF with the gag-IGFR fusion protein possessing enhanced transforming potential, which is consistent with its increased in vitro and in vivo tyrosine phosphorylation.
EBNA1 can link the enhancer element to the initiator element of the Epstein-Barr virus plasmid origin of DNA replication
Authors: Middleton T , Sugden B
Published: January 2020

The plasmid origin of DNA replication of Epstein-Barr virus, oriP, is replicated once per cell division, employing cellular replication machinery and only one viral protein. To understand how replication from this origin is initiated and regulated, we purified this viral protein, EBNA1. EBNA1 was expressed in CV-1p cells by using an infectious simian virus 40 vector containing the EBNA1 gene. It was purified in two chromatographic steps to apparent homogeneity. The purified protein is capable of supporting transcription of the luciferase gene from a reporter plasmid carrying the FR enhancer element to which EBNA1 binds. EBNA1 does not have oriP-dependent ATPase activity, indicating that it does not carry out an energy-dependent step in the initiation of DNA replication. However, EBNA1 does mediate an association between the two elements of oriP. We measured this association by binding one of the elements, the enhancer element, to a solid matrix and measuring retention by this element of the other one, the initiator element, in the presence of EBNA1. This retention is specific for DNA fragments containing EBNA1-binding sites. EBNA1 thus can link the two elements of the origin, providing a locally high concentration of EBNA1 at the site of initiation of DNA replication. We propose that this association is important either (i) to affect DNA structure to allow a cellular helicase to initiate DNA strand separation or (ii) to bind replication proteins to bring them to the origin of replication.
The central hydrophobic domain of the bovine papillomavirus E5 transforming protein can be functionally replaced by many hydrophobic amino acid sequences containing a glutamine
Authors: Kulke R , Horwitz B H , Zibello T , DiMaio D
Published: January 2020

The 44-amino-acid E5 transforming protein of bovine papillomavirus can induce growth transformation of cultured rodent fibroblast cell lines. Previous studies revealed that efficient transformation of mouse C127 cells by the E5 protein required a central core of hydrophobic amino acids and several specific carboxyl-terminal amino acids. Although a randomly derived sequence of hydrophobic amino acids could functionally replace the wild-type hydrophobic core, most such sequences could not. We show here that the conserved glutamine at position 17 in the hydrophobic domain is also important for transformation and that insertion of the glutamine can rescue the transforming activity of many but not all otherwise defective mutants containing random hydrophobic sequences. However, a class of mutants was identified that transform efficiently even in the absence of glutamine, demonstrating that the presence of this amino acid is not absolutely required for efficient transformation. E5 proteins containing the glutamine appear to display increased homodimer formation compared with mutant proteins lacking the glutamine, but this amino acid has no apparent effect on protein stability.
Identification of VP7 epitopes associated with protection against human rotavirus illness or shedding in volunteers
Authors: Green K Y , Kapikian A Z
Published: January 2020

Sera from 17 of 18 adult volunteers challenged with a virulent serotype 1 rotavirus strain (D) were examined for prechallenge antibody levels against several well-defined rotavirus VP7 and VP4 neutralization epitopes by a competitive epitope-blocking immunoassay (EBA) in order to determine whether correlates of resistance to diarrheal illness could be identified. The presence of prechallenge serum antibody at a titer of greater than or equal to 1:20 that blocked the binding of a serotype 1 VP7-specific monoclonal antibody (designated 2C9) that maps to amino acid residue 94 in antigenic site A on the serotype 1 VP7 was significantly associated with resistance to illness or shedding (P less than 0.001) or illness and shedding (P less than 0.01) following challenge with the serotype 1 virus. In addition, an EBA antibody titer of greater than or equal to 1:20 in prechallenge serum against a serotype 3 VP7-specific epitope (defined by monoclonal antibody 954/159) that maps to amino acid 94 on the serotype 3 VP7 was also significantly associated with resistance to illness or shedding (P = 0.02), with a trend for protection against illness and shedding. A trend was also noted between the presence of EBA antibody against a cross-reactive VP4 epitope common to many human rotavirus strains, including the challenge virus, or a rhesus monkey rotavirus strain-specific VP4 antigenic site, and resistance to illness or shedding. These data confirm that the presence of serum antibody correlates with resistance to rotavirus illness or shedding but, in addition, demonstrate the association of antibody to a specific epitope with resistance to illness or shedding. These data also suggest that antigenic site A on the rotavirus VP7, composed of amino acids 87 to 96, may be involved in the formation of a major protective epitope. Further study of the role of this epitope in the development of homotypic and heterotypic immunity to rotaviruses following natural or vaccine-induced infection may be important in the development of strategies for control of rotavirus diarrheal disease.
Infection of cord blood monocyte-derived macrophages with human immunodeficiency virus type 1
Authors: Ho W Z , Lioy J , Song L , Cutilli J R , Polin R A , Douglas S D
Published: January 2020

We have investigated the susceptibility of cord blood monocyte-derived macrophages to human immunodeficiency virus type 1 (HIV-1) infection in vitro. Cord blood monocytes were maintained in vitro for 10 to 15 days and then infected with HIV-1. Syncytia were observed 14 days after infection by light microscopy. Viral proteins were detected by immunofluorescence assay. Electron microscopic examination demonstrated typical lentivirus particles within cytoplasmic vacuoles. The supernatants from the HIV-1-infected cultures also contained significant reverse transcriptase activity and p24 antigen. Like adult monocyte/macrophages, cord-derived monocyte/macrophages expressed the CD4 receptor molecule. Pretreatment with blocking antibody prior to infection with HIV-1 Bal significantly reduced or blocked infection of cord monocyte/macrophages. When cord and adult monocyte/macrophages were infected with HIV-1 Bal or Ada-M and directly compared, higher reverse transcriptase activities and p24 antigen expression were obtained with cord monocyte/macrophages. However, no significant difference was found between adult and cord monocyte/macrophages infected with HIV-1 IIIB. These observations suggest that cord monocyte-derived macrophages may be important in the pathogenesis of pediatric AIDS and that the increased susceptibility of cord monocyte/macrophages to HIV-1 infection in vitro may be relevant to the enhanced susceptibility of neonates to HIV-1 diseases in vivo.
p53 shares an antigenic determinant with proteins of 92 and 150 kilodaltons that may be involved in senescence of human cells
Authors: van den Heuvel S J , The S I , Klein B , Jochemsen A G , Zantema A , van der Eb A J
Published: January 2020

A panel of primary human cells and virus-transformed derivatives were tested for events that coincide with immortalization. In all primary and precrisis cells, two proteins of 92 and 150 kDa that shared an epitope with p53 were found; in most of their immortalized derivatives, however, they were absent. Expression of these proteins may be involved in senescence.
Human cytomegalovirus US3 and UL36-38 immediate-early proteins regulate gene expression
Authors: Colberg-Poley A M , Santomenna L D , Harlow P P , Benfield P A , Tenney D J
Published: January 2020

We have established the ability of the human cytomegalovirus (HCMV) UL36-38 and US3 immediate-early (IE) gene products to alter gene expression in human cells by using transient transfection assays. The cellular heat shock protein 70 (hsp70) promoter was transactivated following cotransfection with the HCMV IE regions in nonpermissive HeLa cells by UL36-38, US3, or IE1 and in permissive human diploid fibroblasts (HFF) by IE1 or IE2. Moreover, hsp70 expression was synergistically increased in HeLa cells cotransfected with US3 and UL36, with US3 and UL37, or with US3 and UL37x1. The synergistic transactivation of hsp70 expression by US3 and UL36-38 was not observed in HFF cells. Synergy was also not observed in HeLa cells between US3 and UL38, an early gene product encoded by the UL36-38 IE locus. Synergistic transactivation of hsp70 expression in HeLa cells required the syntheses of UL36-38 and US3 IE proteins, since nonsense mutants were not functional. hsp70 expression increased with increasing amounts of transfected US3 and UL37 DNA and occurred at the level of stable hsp70-promoted RNA. In contrast to the broad hsp70 response, promoters from the HCMV UL112 early gene and another cellular gene, brain creatine kinase, both responded strongly only to singly transfected IE2 in HeLa cells. Nevertheless, IE2 transactivation of the UL112 promoter was further stimulated by cotransfection of IE1 or of UL36-38 in both HeLa and HFF cells. Thus, different patterns of promoter transactivation and interactions between HCMV IE gene products in transactivation were found in HFF cells and in HeLa cells. These results establish the ability of the HCMV US3 and UL36-38 proteins to alter cellular and viral gene expression and are consistent with involvement of cellular transcription factors in HCMV IE regulation of gene expression.
Terminal regions of the NS-1 protein of the parvovirus minute virus of mice are involved in cytotoxicity and promoter trans inhibition
Authors: Legendre D , Rommelaere J
Published: January 2020

The nonstructural (NS) transcription unit of minute virus of mice (MVMp) encodes proteins that are involved in viral DNA replication and in the regulation of homologous and heterologous promoters. Moreover, it has been shown that NS-protein accumulation is toxic for transformed cells. With the aim of identifying the NS-protein function(s) responsible for cytotoxicity, point mutations and deletions were introduced in the NS-protein-coding sequence of MVMp. This strategy indicated that in transformed human NBE cells, the NS-1 protein is indispensable for MVMp DNA replication, trans activation of the late parvoviral promoter P38, trans inhibition of the long terminal repeat promoter of the Rous sarcoma virus, and cytotoxicity. Moreover, some mutations led to the dissociation of the replicative and regulatory functions of the NS-1 protein and showed that cytotoxicity correlated with the latter, more particularly with the capacity to trans inhibit the heterologous promoter. The NS-1 sequences required for cytotoxicity were found to be restricted to the amino- and carboxy-terminal portions of the protein. Although the cytotoxicities of NS-1 extremities were weak when the extremities were tested separately, the cytotoxicities were comparable to that of the full protein when the extremities were fused. Interestingly, an overall negative charge can be predicted from the NS-1 sequence over about 100 amino acids at both ends. The conservation of this charge distribution among the NS proteins of different parvoviruses suggests that NS-1 may bear some similarities to acidic transcriptional activators.
Induction of AP-1 DNA-binding activity and c-fos mRNA by the adenovirus 243R E1A protein and cyclic AMP requires domains necessary for transformation
Authors: Gedrich R W , Bayley S T , Engel D A
Published: January 2020

The 243R E1A protein can act in synergy with cyclic AMP to induce AP-1 DNA-binding activity and c-fos mRNA in mouse S49 cells. A series of deletion mutants was used to identify two domains of the 243R protein that were required for these effects. Interestingly, these domains correlated precisely with regions known to be necessary for E1A-mediated transformation. One domain was located at the N terminus of E1A. The other domain spanned residues 36 to 81, corresponding to conserved region 1 of E1A. S49 cellular proteins that associate with E1A were coimmunoprecipitated with anti-E1A antibody. These included the previously identified proteins p300, p130, p107, p105Rb, and cyclin A. In addition, proteins of 90 kDa and a series of proteins in the 120- to 170-kDa range were identified. Binding of p300, p90, and the 120- to 170-kDa proteins was abolished in cells expressing mutants of E1A that were unable to induce AP-1 DNA-binding activity and c-fos mRNA. These data strongly suggest that specific cellular E1A-binding proteins are involved in the induction of AP-1 DNA-binding activity and c-fos mRNA by the synergistic action of the 243R E1A protein and cyclic AMP and that these transcriptional events are related to the transformation process.
Alterations in potential sites for glycosylation predominate during evolution of the simian immunodeficiency virus envelope gene in macaques
Authors: Overbaugh J , Rudensey L M
Published: January 2020

Genetic diversity is a hallmark of the human immunodeficiency virus (HIV) genome, but the role of distinct HIV variants in the development of AIDS is unclear. Envelope (env) is the most highly variable gene in HIV as well as in other retroviruses. We have previously demonstrated that variation in simian immunodeficiency virus (SIV) env is primarily localized in two regions (V1 and V4) during progression to simian AIDS. To determine whether there is a common genotype that evolves as AIDS develops, a total of 160 SIV env genes isolated directly from the tissue DNAs of four macaques infected with cloned virus were compared. Common amino acid sequence changes were identified within V1, V4, and, in the late stages of disease, near V3. At several positions, the same amino acid change was seen frequently in the variant genomes from all four animals. As AIDS developed, the majority of viruses evolved an extended sequence in V1 that was rich in serine and threonine residues and shared similarity with proteins modified by O-linked glycosylation. Several of the predominant common sequence changes in V1 and V4 created new sites for N-linked glycosylation. Thus, common features of the SIV variants that evolve during progression to AIDS are motifs that potentially allow for structural and functional changes in the env protein as a result of carbohydrate addition.
Fusion regulation proteins on the cell surface: isolation and characterization of monoclonal antibodies which enhance giant polykaryocyte formation in Newcastle disease virus-infected cell lines of human origin
Authors: Ito Y , Komada H , Kusagawa S , Tsurudome M , Matsumura H , Kawano M , Ohta H , Nishio M
Published: January 2020

Newcastle disease virus (NDV)-infected HeLa and FL cells showed small polykaryocytes at about 24 h postinfection, while the addition of anti-FL-cell rabbit, rat, or mouse serum to the NDV-infected cells gave rise to giant polykaryocytes at 15 h postinfection. We isolated three monoclonal antibodies (MAbs) (4-5-1, 6-1-13, and 7-2-1) capable of enhancing giant polykaryocyte formation in NDV-infected HeLa cells. These MAbs immunoprecipitated gp80 or gp135, which were detected mainly on the surface of HeLa cells. A functionally intact F protein was essential for antibody-enhanced cell fusion, and hemagglutinating (receptor-binding) activity of HN protein was involved in the fusion at an early stage; that is, the MAbs enhanced NDV-mediated syncytium formation. These molecules were considered to have the ability to regulate NDV-mediated cell fusion and thus were designated fusion regulation protein (FRP)-1 (gp80) and FRP-2 (gp135). Anti-FRP MAbs enhanced the susceptibility of cells to fusion activity of NDV. Anti-FRP-1 MAbs reacted with a molecule on the surface of every cell derived from humans and monkeys but showed no cross-reactivity with mouse or hamster cells. FRP-2 could be detected in limited cell lines of human origin.
cis-acting lesions targeted to the hydrophobic domain of a poliovirus membrane protein involved in RNA replication
Authors: Giachetti C , Hwang S S , Semler B L
Published: January 2020

The structural requirements of the hydrophobic domain contained in poliovirus polypeptide 3AB were studied by using a molecular genetic approach in combination with an in vitro biochemical analysis. We report here the generation and analysis of deletion, insertion, and amino acid replacement mutations aimed at decreasing the hydrophobic character of the domain. Our results indicated that the hydrophobicity of this region of 3AB is necessary to maintain normal viral RNA synthesis. However, in vitro membrane association assays of the mutated proteins did not establish a direct correlation between 3AB membrane association and viral RNA synthesis. Some of the lethal mutations we engineered produced polyproteins with abnormal P2- and P3-processing capabilities due to an alteration in the normal cleavage order of the polyprotein. A detailed analysis of these mutants suggests that P2 is not the major precursor for polypeptides 2A and 2BC and that P2 protein products are derived from P2-P3-containing precursors (most likely P2-P3 or P2-3AB). Such precursors are likely to result from primary polyprotein cleavage events that initiate a proteolytic cascade not previously documented. Our results also indicated that the function provided by the hydrophobic domain of 3AB cannot be provided in trans. We discuss the implications of these results on the formation of limited-diffusion replication complexes as a means of sequestering P2- and P3-region polypeptides required for RNA synthesis and protein processing.
Modulation of cellular and viral promoters by mutant human p53 proteins found in tumor cells
Authors: Deb S , Jackson C T , Subler M A , Martin D W
Published: January 2020

Wild-type p53 has recently been shown to repress transcription from several cellular and viral promoters. Since p53 mutations are the most frequently reported genetic defects in human cancers, it becomes important to study the effects of mutations of p53 on promoter functions. We, therefore, have studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen (PCNA) promoter and on several viral promoters, including the herpes simplex virus type 1 UL9 promoter, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and a plasmid containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. As expected, expression of the wild-type p53 inhibited promoter function. Expression of a p53 with a mutation at any one of the four amino acid positions 175, 248, 273, or 281, however, correlated with a significant increase of the PCNA promoter activity (2- to 11-fold). The viral promoters were also activated, although to a somewhat lesser extent. We also showed that activation by a mutant p53 requires a minimal promoter containing a lone TATA box. A more significant increase (25-fold) in activation occurs when the promoter contains a binding site for the activating transcription factor or cyclic AMP response element-binding protein. Using Saos-2 cells that do not express p53, we showed that activation by a mutant p53 was a direct enhancement. The mutant forms of p53 used in this study are found in various cancer cells. The activation of PCNA by mutant p53s may indicate a way to increase cell proliferation by the mutant p53s. Thus, our data indicate a possible functional role for the mutants of p53 found in cancer cells in activating several important loci, including PCNA.
Mouse hepatitis virus utilizes two carcinoembryonic antigens as alternative receptors
Authors: Yokomori K , Lai M M
Published: January 2020

The cellular receptor for the murine coronavirus mouse hepatitis virus (MHV) has been identified as a member of the murine carcinoembryonic antigen (CEA) family (R. K. Williams, G. S. Jiang, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 88:5533-5536, 1991). However, the receptor protein was not detected in some of the susceptible mouse tissues. We therefore examined whether other types of MHV receptor might exist. By polymerase chain reaction with the conserved sequences of murine CEA gene family members (mmCGM) as primers, we detected two CEA-encoding RNAs in the mouse liver. One of them (1.3 kb) encodes mmCGM1, which has previously been identified as the receptor for MHV, and the other one (0.8 kb) was shown to encode another member of mouse CGM, mmCGM2. The sequence analysis showed that mmCGM2 lacks 564 nucleotides in the middle of the gene compared with mmCGM1. These two CEA transcripts are probably derived from the same gene by an alternative splicing mechanism. Expression of either of these cDNA clones in COS-7 cells rendered these cells susceptible to MHV infection, suggesting that not only mmCGM1 but also mmCGM2 serves as a receptor for MHV. The mmCGM2 was the major CEA species in the mouse brain, which is a main target organ for the neurotropic strains of MHV. Very little mmCGM1 was detected in the mouse brain or in cells of the susceptible mouse astrocytoma cell line DBT. This result indicates that MHV may utilize different CEA molecules as the major receptor in the mouse brain and in the liver. This is a first identification of multiple receptors for a single virus. The presence of different receptors in different tissues may explain the target cell specificity of certain MHVs.