bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequences required for coordinate induction of adeno-associated virus p19 and p40 promoters by Rep protein
Authors: McCarty D M , Christensen M , Muzyczka N
Published: January 2020

A series of contiguous 30-bp deletions were introduced into the regions upstream of the p19 and p40 promoters of adeno-associated virus (AAV), and the effects of these deletions on induction of AAV transcription by the rep gene products was evaluated. A novel complementation system was devised for supplying wild-type Rep protein when mutations disrupted the trans activation activity of the Rep protein. Transcription from the p40 promoter was eliminated upon deletion of the TATA sequence located between -4 and -33 from the cap site. Deletions which removed sequences from -34 to -123 bp from the p40 mRNA start site substantially reduced Rep induction of p40 transcription. p19 transcription was also undetectable when the p19 TATA sequence between -4 and -33 was deleted. In contrast to the p40 region, two types of cis-active sequences were found associated with the p19 promoter. Sequences between -4 and -63 bp relative to the p19 cap site were essential for Rep induction only from the p19 promoter. Deletions between -94 and -153 bp relative to the p19 cap site reduced Rep induction of both the p19 and p40 promoters coordinately. These two noncontiguous regions were separated by a 30-bp sequence that was not essential for transcription control. Further deletion analysis delineated a second cis-active element, associated with the p5 promoter (AAV nucleotides 191 to 320), which was also necessary for coordinate Rep activation of both the p19 and p40 promoters. Finally, the dependence of p40 transcription on the Rep-responsive elements within the p5 and p19 regions could be overcome by the presence of the AAV terminal repeats, suggesting that the terminal repeats contained redundant Rep-responsive elements. These results implied an interdependence in cis between the three AAV promoters and suggested a novel mechanism for coordinate regulation of gene expression in response to the trans-activating Rep protein. Coordinate induction appeared to be the result of a simultaneous interaction between the Rep protein and sequence elements associated with two or all three of the AAV promoters.
Expression of rotavirus VP2 produces empty corelike particles
Authors: Labbé M , Charpilienne A , Crawford S E , Estes M K , Cohen J
Published: January 2020

The complete VP2 gene of bovine rotavirus strain RF has been inserted into the baculovirus transfer vector pVL941 under the control of the polyhedrin promoter. Cotransfection of Spodoptera frugiperda 9 cells with wild-type baculovirus DNA and transfer vector DNA led to the formation of recombinant baculoviruses which contain bovine rotavirus gene 2. Infection of S. frugiperda cells with this recombinant virus resulted in the production of a protein similar in size and antigenic properties to the authentic rotavirus VP2. The protein binds double-stranded RNA and DNA in an overlay protein blot assay. Expressed VP2 assembles in the cytoplasm of infected cells in corelike particles 45 nm in diameter. These corelike particles were purified by sucrose gradient centrifugation and found to be devoid of nucleic acid. Coexpression of VP2 and VP6 from heterologous rotavirus strains (bovine and simian) resulted in the formation of single-shelled particles. These results definitively show the existence of an innermost protein shell in rotavirus which is formed independently of other rotavirus proteins. These results have implications for schemes of rotavirus morphogenesis.
Expression of processed core protein of hepatitis C virus in mammalian cells
Authors: Harada S , Watanabe Y , Takeuchi K , Suzuki T , Katayama T , Takebe Y , Saito I , Miyamura T
Published: January 2020

A structural protein of hepatitis C virus (HCV) was expressed in monkey COS cells under the control of an exogenous promoter, and a protein of 22 kDa was identified by immunoblot analysis. This protein (p22), which was produced by processing in COS cells, reacted specifically to sera of chronic hepatitis C patients, and its coding region was mapped at the most amino-terminal part of the HCV polyprotein. These results suggested that the p22 protein is the nucleocapsid (core) protein of HCV. Moreover, the assay detecting antibody to p22 was found to be useful for early diagnosis of HCV infection.
The E1B 19,000-molecular-weight protein of group C adenoviruses prevents tumor necrosis factor cytolysis of human cells but not of mouse cells
Authors: Gooding L R , Aquino L , Duerksen-Hughes P J , Day D , Horton T M , Yei S P , Wold W S
Published: January 2020

Tumor necrosis factor (TNF) is a multifunctional immunoregulatory protein that is secreted by activated macrophages and is believed to have antiviral activities. We reported earlier that when mouse C3HA fibroblasts are infected with human adenoviruses, the 289R and 243R proteins encoded by region E1A render the cells susceptible to lysis by TNF, and a 14,700-molecular-weight protein (14.7K protein) encoded by region E3 protects the cells against lysis by TNF. We now report that the 19,000-molecular-weight (19K) (176R) protein encoded by the E1B transcription unit can protect human HEL-299 fibroblasts and human ME-180 cervical carcinoma cells against lysis by TNF. This was determined by infecting cells with adenovirus double mutants that lack region E3 and do or do not express the E1B-19K protein and by measuring cytolysis by using a short-term (18-h) 51Cr-release assay. Under these assay conditions, the 51Cr release was specific to TNF and was not a consequence of the cyt phenotype associated with E1B-19K protein-negative mutants. Also, by using virus double mutants that lack E3 in combination with other early regions, we found that E1A, the E1B-55K protein-encoding gene, E3, and E4 are not required to protect HEL-299 cells against TNF cytolysis. Three additional human cancer cell lines (HeLa, HCT8, and RC29) and a simian virus 40-transformed WI38 cell line (VA-13) also required E1B for protection against TNF cytolysis, indicating that the E1B-19K protein is required to protect many if not all human cell types against lysis by TNF when infected by adenovirus. The E1B-19K protein was not able to protect six different adenovirus-infected mouse cell lines against TNF lysis, even though the protein was shown to be efficiently expressed in one of the cell lines. HEL-299 or ME-180 cells infected by a mutant that lacks the E1B-19K protein but retains region E3 were not lysed by TNF, indicating that one or more of the E3 proteins can protect these cells against TNF lysis in the absence of the E1B-19K protein. Thus, the E3-14.7K but not the E1B-19K protein can protect adenovirus-infected mouse cells against TNF cytolysis, whereas the E1B-19K protein as well as one or more of the E3 proteins can protect adenovirus-infected human cells against TNF cytolysis.
Herpes simplex virus type 1 origin-specific binding protein: oriS-binding properties and effects of cellular proteins
Authors: Dabrowski C E , Schaffer P A
Published: January 2020

The herpes simplex virus type 1 (HSV-1) origin of replication, oriS, contains three highly homologous sequences, sites I, II, and III. The HSV-1 origin-binding protein (OBP), the product of the UL9 gene, has been shown to bind specifically to sites I and II. In this study, gel shift analysis was used to characterize interactions between site I DNA and proteins in infected and uninfected cell extracts. The formation of two protein-DNA complexes, bands A and B, was demonstrated with infected cell extracts, and one predominant protein-DNA complex, band M, was identified with mock-infected extracts. Protein interactions with the highly homologous site II and III DNAs were also characterized. Incubation of infected cell extracts with the lower-affinity site II DNA as a probe resulted in the appearance of two protein-DNA complexes with mobilities identical to those of the A and B complexes, while incubation with site III DNA resulted in the formation of a single complex with the mobility of band B; no A-like band was observed. Incubation of high concentrations of partially purified OBP with site I DNA resulted in the formation of two novel complexes, bands 9-1 and 9-2. Addition of uninfected or HSV-1-infected cell extracts to the purified OBP-site I DNA mix significantly enhanced the formation of complex 9-1. The enhanced formation of complex 9-1 by uninfected cell extracts implicates a cellular factor or factors in the formation or stabilization of the OBP-site I DNA complex.
Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein
Authors: Luban J , Goff S P
Published: January 2020

We have expressed the human immunodeficiency virus type 1 (HIV-1) gag polyprotein (Pr55gag) in bacteria under the control of the T7 phage gene 10 promoter. When the gene encoding the viral protease is included in cis, in the -1 reading frame, the expected proteolytic cleavage products MA and CA are produced. Disruption of the protease-coding sequence prevents proteolytic processing, and full-length polyprotein is produced. Pr55gag, separated from bacterial proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immobilized on nitrocellulose membranes, binds RNA containing sequences from the 5' end of the HIV-1 genome. This binding is tolerant of a wide range of pH and temperature but has distinct salt preferences. Conditions were identified which prevented nonspecific binding of RNA to bacterial proteins but still allowed binding to Pr55gag. Under these conditions, irrelevant RNA probes lacking HIV-1 sequences bound Pr55gag less efficiently. Quantitation of binding to Pr55gag by HIV-1 RNA probes with deletions mutations demonstrated that there are two regions lying within the HIV-1 gag gene which independently promote binding of RNA to Pr55gag.
Human immunodeficiency virus infection and syncytium formation in HeLa cells expressing glycophospholipid-anchored CD4
Authors: Kost T A , Kessler J A , Patel I R , Gray J G , Overton L K , Carter S G
Published: January 2020

The CD4 molecule, a glycoprotein expressed primarily on the cell surface of specific T lymphocytes, is thought to function in T-cell antigen recognition and activation. In addition, CD4 serves as a receptor for human immunodeficiency virus type 1 (HIV-1) by a direct interaction with the HIV-1 surface glycoprotein (gp120). To further characterize the HIV-1-cell interaction, a HeLa cell line was established that expressed a chimeric molecule of CD4 and decay-accelerating factor (DAF). In the chimeric CD4-DAF molecule the transmembrane and cytoplasmic domains of CD4 were deleted and replaced with the carboxy-terminal 37 amino acids of DAF. This resulted in the anchoring of the extracellular domain of CD4 to the cell membrane via a glycophospholipid linkage. The glycophospholipid-anchored CD4 had a molecular size of approximately 56 to 62 kDa and was released following treatment of the cells with phosphatidylinositol-specific phospholipase C. HeLa cells expressing the CD4-DAF hybrid could be infected with HIV-1, as evidenced by reverse transcriptase activity, p24 core antigen content, and infectious virus production. In addition, transfection of the HeLa CD4-DAF cells with a plasmid that directs the synthesis of HIV-1 envelope glycoproteins or cocultivation with HeLa cells expressing the virus glycoproteins resulted in syncytium formation. These results indicate that the transmembrane and cytoplasmic domains of the CD4 molecule are dispensable for both HIV infection and syncytium formation.
Efficient polyadenylation within the human immunodeficiency virus type 1 long terminal repeat requires flanking U3-specific sequences
Authors: Brown P H , Tiley L S , Cullen B R
Published: January 2020

During transcription of the human immunodeficiency virus type 1 provirus, polyadenylation signals present in the 5' long terminal repeat (LTR) are disregarded while the identical polyadenylation signals present in the 3' LTR are utilized efficiently. As both transcribed LTR sequences contain all signals known to be required for efficient polyadenylation, the basis for this differential utilization has been unclear. Here, we describe experiments that suggest that transcribed sequences present within the human immunodeficiency virus type 1 LTR U3 region act in cis to enhance polyadenylation within the 3' LTR.
Transgenic thymocytes are refractory to transformation by the human T-cell leukemia virus type I tax gene
Authors: Nerenberg M I , Minor T , Price J , Ernst D N , Shinohara T , Schwarz H
Published: January 2020

Previous transgenic work demonstrated transforming activity of the human T-cell leukemia virus type I Tax protein in fibroblasts. In the present study, a Thy-1-based vector was used to express Tax in thymocytes. These mice developed no functional or neoplastic abnormalities of T cells but developed fibroblastic tumors with a longer latency than in the previous model.
Raccoon poxvirus recombinants expressing the rabies virus nucleoprotein protect mice against lethal rabies virus infection
Authors: Lodmell D L , Sumner J W , Esposito J J , Bellini W J , Ewalt L C
Published: January 2020

Raccoon poxvirus (RCN) recombinants expressing the rabies virus internal structural nucleoprotein (RCN-N) protected A/WySnJ mice against a lethal challenge with street rabies virus (SRV). Maximum survival was achieved following vaccination by tail scratch and footpad (FP) SRV challenge. RCN-N-vaccinated mice inoculated in the FP with SRV were resistant to infection for at least 54 weeks postvaccination. Protection was also elicited by RCN recombinants expressing the rabies virus glycoprotein (RCN-G). Vaccination with RCN-G evoked rabies virus neutralizing antibody. Rabies virus neutralizing antibody was not detected in RCN-N-vaccinated mice prior to or following SRV infection. Radioimmunoprecipitation assays showed that sera from RCN-N-vaccinated mice which survived SRV infection did not contain antibody to SRV structural protein G, M, or NS. The mechanism(s) of N-induced resistance appears to correlate with the failure of peripherally inoculated SRV to enter the central nervous system (CNS). Support for this correlation with resistance was documented by the observations that SRV-inoculated RCN-N-vaccinated mice did not develop clinical signs of CNS rabies virus infection, infectious SRV was not detected in the spinal cord or brain following FP challenge, and all RCN-N-vaccinated mice died following direct intracranial infection of the CNS with SRV. These results suggest that factors other than anti-G neutralizing antibody are important in resistance to rabies virus and that the N protein should be considered for incorporation with the G protein in recombinant vaccines.
Tissue culture system for infection with human hepatitis delta virus
Authors: Sureau C , Jacob J R , Eichberg J W , Lanford R E
Published: January 2020

An in vitro culture system was developed for assaying the infectivity of the human hepatitis delta virus (HDV). Hepatocytes were isolated from chimpanzee liver and grown in a serum-free medium. Cells were shown to be infectible by HDV and to remain susceptible to infection for at least 3 weeks in culture, as evidenced by the appearance of RNA species characteristic of HDV replication as early as 6 days postinfection. When repeated experiments were carried out on cells derived from an animal free of hepatitis B virus (HBV), HDV infection occurred in a consistent fashion but there was no indication of infection with the HBV that was present in the inoculum. Despite numerous attempts with different sources of HBV inocula free of HDV, there was no evidence that indicated susceptibility of these cells to HBV infection. This observation may indicate that HBV and HDV use different modes of entry into hepatocytes. When cells derived from an HBV-infected animal were exposed to HDV, synthesis and release of progeny HDV particles were obtained in addition to HBV replication and production of Dane particles. Although not infectible with HBV, primary cultures of chimpanzee hepatocytes are capable of supporting part of the life cycle of HBV and the entire life cycle of HDV.
Mutational analysis of the equine infectious anemia virus Tat-responsive element
Authors: Carvalho M , Derse D
Published: January 2020

A hairpinlike structure is predicted to exist at the 5' end of equine infectious anemia virus (EIAV) RNA which is similar in many ways to the human immunodeficiency type 1 (HIV-1) Tat-responsive element (TAR). In EIAV, this structure has a shorter stem than in HIV-1 and lacks the uridine bulge. Primer extension analysis of EIAV RNA was used to identify the transcriptional start site in the viral long terminal repeat. Premature termination of primer elongation at the predicted double-stranded RNA region was frequently observed and suggests that the inferred hairpin structure exists under these conditions. We have functionally characterized EIAV TAR by site-directed mutagenesis and transient gene expression analysis. It is demonstrated here that the secondary structure of this element is essential for Tat action. Mutations that disrupted base pairing abolished TAR function, and compensatory mutations that restored the stem structure resulted in Tat activation. The TAR loop appears to be closed by two U.G base pairs that are likely to provide a unique structural motif recognized by the Tat protein. With one exception, substitutions of nucleotides within the EIAV loop sequence decreased TAR function. All nucleotide substitutions of the cytidine at position +14 increased EIAV Tat responsiveness; however, its deletion abolished trans activation. Our results lead us to propose that the EIAV and HIV-1 Tat systems employ closely related cis- and trans-acting components that probably act by the same mechanism.
Generation of a chimeric human and simian immunodeficiency virus infectious to monkey peripheral blood mononuclear cells
Authors: Shibata R , Kawamura M , Sakai H , Hayami M , Ishimoto A , Adachi A
Published: January 2020

We constructed five chimeric clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIVMAC) and four SIVMAC mutants by recombinant DNA techniques. Three chimeric clones and all mutants with an alteration in either the vif, vpx, vpr, or nef gene were infectious to human CD4-positive cell lines. The susceptibility of macaque monkey peripheral blood mononuclear cells (PBMC) to infection by these mutants and chimeras was examined in vitro. Macaque PBMC supported the replication of wild-type and vpx, vpr, and nef mutant SIVMAC strains. A chimera carrying the long terminal repeats (LTRs), gag, pol, vif, and vpx of SIVMAC and tat, rev, vpu, and env of HIV-1 was also replication competent in PBMC. In contrast, HIV-1, the vif mutant of SIVMAC, a chimera containing rev and env of SIVMAC, and a chimera containing vpx, vpr, tat, rev, and env of SIVMAC did not grow in PBMC. Western immunoblotting analysis of the replicating chimera in PBMC confirmed the hybrid nature of the virus. These data strongly suggested that the sequence important for macaque cell tropism lies within the LTR, gag, pol, and/or vif sequences of the SIVMAC genome.
Inhibition of Epstein-Barr virus infection in vitro and in vivo by soluble CR2 (CD21) containing two short consensus repeats
Authors: Moore M D , Cannon M J , Sewall A , Finlayson M , Okimoto M , Nemerow G R
Published: January 2020

The extracellular domain of CR2, the Epstein-Barr virus (EBV)/C3d receptor of B lymphocytes, contains 15 or 16 tandemly arranged short consensus repeat elements (SCR). Recombinant CR2 proteins containing SCR 1 and 2 fused to Staphylococcus aureus protein A (PA-CR2) and to murine complement factor H SCR 20 (CR2FH) were expressed in Escherichia coli and in insect cells, respectively. These recombinant CR2 molecules retained functional activity as indicated by their ability to bind to C3dg in an enzyme-linked immunosorbent assay and to inhibit EBV gp350/220 binding to B cells. PA-CR2 and CR2FH were as efficient in blocking EBV gp350/220 binding as the full-length CR2 extracellular domain, indicating that the first two SCR of CR2 contain the majority of the ligand binding activity of the receptor. PA-CR2 and CR2FH inhibited EBV-induced B-cell proliferation in vitro and blocked the development of EBV-induced lymphoproliferative disease in severe combined immunodeficient mice reconstituted with human lymphocytes. These studies indicate that soluble forms of truncated CR2 proteins may have potential therapeutic value in the treatment of EBV-induced lymphoproliferative disorders in humans that involve viral replication.
Molecular characterization of the vaccinia virus hemagglutinin gene
Authors: Brown C K , Turner P C , Moyer R W
Published: January 2020

The vaccinia virus hemagglutinin (HA) is a glycoprotein found on the plasma membrane of infected cells and the envelope of extracellular virus. Two forms of HA (85 and 68 kDa) are detected by immunoblot analysis. Although hemagglutination activity is only readily detectable late in infection, the 85-kDa HA appears early and accumulates throughout infection, whereas the 68-kDa form appears only late in the cycle. Production of the 68-kDa HA but not the 85-kDa HA was inhibited by either cytosine arabinoside or rifampin. Analysis of HA gene expression reveals a complex pattern of expression. The HA gene is transcribed early to yield a 1.65-kb dicistronic early transcript, consisting of the 945-bp HA open reading frame (ORF) fused to a 453-bp downstream ORF. Transcription from this site initiates 7 bases upstream of the AUG initiating codon of the HA ORF. Due to the discrepancy between the calculated size of the HA protein (33 kDa) and that reported for the unglycosylated HA protein derived from in vitro translation (58 kDa), we placed an early transcription termination signal (TTTTTAT) directly downstream of the 945-bp HA ORF. This led to a reduction in size of the early HA mRNA to 1.2 kb, as expected, but had no effect on the formation of either the 85- or 68-kDa protein. Transcripts originating from the early promoter are found throughout the infection cycle. However, after DNA replication, transcription from a second, late promoter ensues. The transcriptional start site of the late promoter is within a consensus TAAATG sequence located 135 bases upstream of the transcriptional start site of the first promoter. The late transcriptional start site is also found within an upstream ORF.
In vitro binding of human T-cell leukemia virus rex proteins to the rex-response element of viral transcripts
Authors: Grassmann R , Berchtold S , Aepinus C , Ballaun C , Boehnlein E , Fleckenstein B
Published: January 2020

Human T-cell leukemia virus (HTLV-I, HTLV-II) rex protein function is required for the cytoplasmic expression of incompletely spliced viral transcripts encoding structural proteins. The effect is mediated by a cis-acting rex-response element (RRX) which is located near the 3' end of all viral mRNAs. We show that rex polypeptides of HTLV-I and HTLV-II expressed in Escherichia coli are capable of specifically binding RRX-containing transcripts of both viruses in cell-free assays. Binding analyses with deletion variants of rex proteins revealed a domain with RNA-binding activity in the first 77 N-terminal amino acids. Removal of a basic peptide of 19 amino acids from the N terminus abrogated RNA binding, whereas a beta-galactosidase fusion protein containing this peptide bound to the RRX. These results suggest that direct binding of rex protein to the RRX is important for rex-mediated regulation of viral gene expression and that a short stretch of positively charged amino acids contributes to the specific binding of rex to its target RNA.
gp160, the envelope glycoprotein of human immunodeficiency virus type 1, is a dimer of 125-kilodalton subunits stabilized through interactions between their gp41 domains
Authors: Thomas D J , Wall J S , Hainfeld J F , Kaczorek M , Booy F P , Trus B L , Eiserling F A , Steven A C
Published: January 2020

The molecular masses, carbohydrate contents, oligomeric status, and overall molecular structure of the env glycoproteins of human immunodeficiency virus type 1--gp120, gp160, and gp41--have been determined by quantitative electron microscopy. Using purified gp160s, a water-soluble form of env purified from a recombinant vaccinia virus expression system, we have measured the masses of several hundred individual molecules by dark-field scanning transmission electron microscopy. When combined with sequence-based information, these mass measurements establish that gp160s is a dimer of subunits with an average monomer mass of 123 kDa, of which approximately 32 kDa is carbohydrate and 91 kDa is protein. Similarly, gp120 was found to be a monomer of 89 kDa and to contain virtually all of env's glycosylation. gp41 is glycosylated only slightly, if at all, and is responsible for the interactions that stabilize the gp160s dimer. A molecular mass map of gp160s derived by image processing depicts an asymmetric dumbbell whose two domains have masses of approximately 173 and approximately 73 kDa, corresponding to a gp120 dimer and a gp41 dimer, respectively. We infer that the average monomer mass of native gp160 is 125 kDa and that in situ, env is either a dimer or a tetramer but is most unlikely to be a trimer.
Equine H7N7 influenza A viruses are highly pathogenic in mice without adaptation: potential use as an animal model
Authors: Kawaoka Y
Published: January 2020

Equine H7N7 influenza A viruses, representing a broad range of isolates, were lethal in mice without adaptation. After repeated passages, A/Equine/London/1416/73 acquired neurotropism upon intranasal infection. Thus, mice infected with equine influenza A viruses provide a model system for the study of highly virulent mammalian influenza viruses.
Dispensable role of the human immunodeficiency virus type 2 Vpx protein in viral replication
Authors: Marcon L , Michaels F , Hattori N , Fargnoli K , Gallo R C , Franchini G
Published: January 2020

Human immunodeficiency virus type 2 (HIV-2) is similar in genetic organization to HIV-1 but contains a unique gene (vpx) that encodes a 16-kDa protein. A replication-competent molecular clone of HIV-2 (HIV-2sbl/isy) that infects human primary cells in vitro and rhesus monkeys was used to generate three mutations in the vpx gene. In the first mutant, the vpx open reading frame was truncated at amino acid 20; the second mutant was tailored to eliminate the proline-rich carboxyl terminus of the protein; and the third mutant was obtained by addition of four amino acids (KDEL) to the carboxyl terminus of the protein to provide a retention signal in the endoplasmic reticulum. The viral infection kinetics of the three mutant viruses and isogeneic HIV-2sbl/isy in the SupT1 cell line were similar. Slight impairment in the early phases of viral replication was observed during infection of primary human peripheral blood mononuclear cells with the vpx mutant viruses. All of the vpx mutant viruses readily infected macrophages, indicating that vpx expression is dispensable for HIV-2 infection and replication in human macrophages.
Antigenic stability of foot-and-mouth disease virus variants on serial passage in cell culture
Authors: Gonzalez M J , Saiz J C , Laor O , Moore D M
Published: January 2020

Two neutralizing monoclonal antibody (MAb)-resistant variants selected from an isolate of foot-and-mouth disease virus (FMDV) type A5 were repeatedly passaged in cell culture and monitored for susceptibility to neutralization by the selecting MAb. A variant isolated with a MAb to a conformational epitope (1-OG2) lost resistance in 20 passages, while a variant isolated with a MAb to a linear epitope (1-HA6) persisted for 30 passages. In both cases, the virus population emerging after passage was antigenically and genetically indistinguishable from the original wild-type parental virus (FMDV A5 Spain-86). Coinfection assays with the wild type and each variant, and between the variants, showed rapid conversion to a homogeneous population. Wild-type virus prevailed over the variants and for coinfection between the variants, the linear epitope variant 1-HA6. While both variants arose from a single nucleotide substitution and reversion to wild type occurred for each, it appears that the variant based on the continuous epitope (1-HA6) was more stable. We discuss the implications of these results for the antigenic diversity of FMDV and its relationship to virus evolution.
Alpha interferon suppresses virion but not soluble human immunodeficiency virus antigen production in chronically infected T-lymphocytic cells
Authors: Fernie B F , Poli G , Fauci A S
Published: January 2020

Alpha interferon (IFN-alpha) is effective in preventing the release of human immunodeficiency virus (HIV) from chronically infected T-lymphocytic (ACH-2) and promonocytic (U1) cell lines stimulated with the phorbol ester phorbol-12-myristate-13 acetate (PMA). In the present study, we observed that together with particle production, shedding of HIV antigen (p24gag) occurs in the T-cell line ACH-2 both constitutively and after stimulation with PMA. IFN-alpha, although effective in suppressing the release of HIV particles, did not inhibit shedding of p24gag into the culture supernatants of either unstimulated or PMA-stimulated cells. These observations may be of relevance in the evaluation of the in vivo efficacy of IFN-alpha treatment of HIV-infected individuals as determined by levels of p24 antigen in plasma.
Recombination between Sindbis virus RNAs
Authors: Weiss B G , Schlesinger S
Published: January 2020

The genome (49S RNA) of Sindbis virus is a positive-strand RNA of 11.7 kb that consists of two domains. The 5' two-thirds of the RNA codes for the proteins required for replication and transcription of the RNA. The 3' one-third codes for the structural proteins. The latter are translated from a 26S subgenomic RNA identical in sequence to the 3' one-third of the genome. The 26S RNA is transcribed by initiation from an internal promoter that spans the junction between the nonstructural and structural genes. We have used Sindbis virus RNAs transcribed from cloned cDNAs to demonstrate recombination between Sindbis virus RNAs in cultured cells. Several different combinations of deleted or mutationally altered RNAs gave rise to infectious recombinants. In 7 of 10 different crosses, the infectious recombinant RNAs were larger than wild-type 49S RNA. We sequenced the recombinant RNAs in the region spanning the junction between the nonstructural and structural protein genes from five different crosses. In three of the crosses, this is the only region within which recombination could have taken place to produce an infectious 49S RNA. Recombination also occurred in this region in the other two crosses. The recombinant RNAs were distinct from wild-type RNA and from each other. All contained sequence insertions derived from the parental RNAs. One contained a deletion and a rearrangement, and one also contained a stretch of 11 nucleotides not found in the Sindbis virus genome. When each of the parental RNAs contained a functional subgenomic RNA promoter, both promoters were present and functional in the recombinant RNA. Those recombinants with large sequence insertions showed evidence of evolution toward the wild-type single-junction RNA.
SL3-3 enhancer factor 1 transcriptional activators are required for tumor formation by SL3-3 murine leukemia virus
Authors: Hallberg B , Schmidt J , Luz A , Pedersen F S , Grundström T
Published: January 2020

The transcriptional enhancers of retroviruses that lack an oncogene are important determinants of their oncogenicity. However, no specific cellular transcriptional activator has yet been found to determine the oncogenicity for any of these viruses. The SL3-3 enhancer factor 1 (SEF1) cellular transcriptional activators are expressed preferentially in T lymphocytes. In the SL3-3 murine leukemia virus enhancer, two different sequences can bind SEF1 activators. We show that mutation of the SEF1 binding sites disrupts the disease potential of SL3-3 murine leukemia virus, implying that SEF1 transcriptional activators are required for tumor induction by SL3-3. The SEF1 site mutations did not appear to affect the pathogenicity of SL3-3 by impairment of virus multiplication, but rather by a specific defect in the ability of neoplastic transformation.
Intragenic suppression of a deletion mutation of the nonstructural gene of an influenza A virus
Authors: Treanor J J , Buja R , Murphy B R
Published: January 2020

The influenza A/Alaska/77 (H3N2) virus mutant 143-1 is temperature sensitive (ts) due to a spontaneous in-frame 36-nucleotide deletion in the nonstructural (NS) gene segment, which leads to a 12-amino-acid deletion in the NS1 protein. In addition, it has a small-plaque phenotype on MDCK cell monolayers. However, phenotypically revertant (i.e., ts+) viruses were isolated readily following replication of the 143-1 virus both in vitro and in vivo. In order to determine the genetic mechanism by which escape from the ts phenotype occurred, we performed segregational analysis and found that an intrasegmental suppressor mutation caused the loss of the ts phenotype. Nucleotide sequence analysis revealed the presence of an intragenic mutation in each of the ts+ phenotypic revertant viruses, involving a substitution of valine for alanine at amino acid 23 of the NS1 protein. This mutation resulted in acquisition of the ts+ phenotype and also in the large-plaque phenotype on MDCK cells, characteristic of the wild-type A/Alaska/77 parent virus. This amino acid substitution is predicted to generate an area of alpha helix in the secondary structure of the amino-terminal portion of the NS1 protein of the revertant viruses which may compensate for loss of an alpha-helical region due to the deletion of amino acids 66 to 77 in the NS1 protein of the 143-1 virus.
Diversity of hepatitis B virus X gene-related transcripts in hepatocellular carcinoma: a novel polyadenylation site on viral DNA
Authors: Hilger C , Velhagen I , Zentgraf H , Schröder C H
Published: January 2020

RNA was isolated from tissue of two patients with hepatocellular carcinoma developed on the background of a chronic hepatitis B virus infection. For identification and characterization of 3' ends of X gene open reading frame (ORF)-related transcripts, RNA was reverse transcribed into cDNA and subjected to polymerase chain reaction. Cloned amplification products from tumor tissue of one patient represented an approximately even distribution of transcripts terminating at the established poly(A) signal (standard transcripts) and of truncated transcripts terminating at a CATAAA poly(A) signal within the 3' end region of X gene ORF (truncated transcripts). Amplified cDNA from tumor tissue of the second patient could be attributed mainly to the standard type of transcripts, whereas cDNA from the nontumor tissue of the same patient could be assigned to four groups of transcripts: (i) standard transcripts, (ii) transcripts with internal deletions affecting the 3' end of the X gene, (iii) truncated transcripts, and (iv) hybrid transcripts displaying 5' sequences from the X gene ORF fused to cellular sequences.
Independent fluctuation of human immunodeficiency virus type 1 rev and gp41 quasispecies in vivo
Authors: Martins L P , Chenciner N , Asjö B , Meyerhans A , Wain-Hobson S
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) overlapping rev and env coding sequences have been examined from sequential peripheral blood mononuclear cell DNA samples from one individual. These were the same DNA samples from which sequence data for the tat and nef/long terminal repeat loci have been derived and span a 4-year period. The rev/env sequences were established by sequencing cloned polymerase chain reaction products. The structure of the populations of rev protein sequences increased in complexity with disease, while those of the corresponding env sequences remained complex. This suggests that the rev and env populations evolved differently, probably reflecting different selection pressures. No defective rev variants encoded substitutions in residues 76 through 79, indicating that the experimental finding of down regulation of rev activity by competitive inhibition may not necessarily occur in vivo. After having analyzed three HIV loci (15% of the genome) from the same individual over 4 years, it is clear that no two loci evolved similarly, indicating the difficulties in comparing data from different loci.
BK virus and JC virus shed during pregnancy have predominantly archetypal regulatory regions
Authors: Markowitz R B , Eaton B A , Kubik M F , Latorra D , McGregor J A , Dynan W S
Published: January 2020

Twenty-three BK virus and JC virus DNA samples obtained from urine of pregnant women had almost exclusively archetypal transcriptional control regions. Rearrangements characteristic of laboratory strains are apparently not required for reactivation in humans. Unexpectedly, alignment shows that many elements identified previously in the BK virus enhancer are conserved in the JC virus archetype.
Complementary lethal invasion of the central nervous system by nonneuroinvasive herpes simplex virus types 1 and 2
Authors: Nishiyama Y , Kimura H , Daikoku T
Published: January 2020

It is well-known that viral thymidine kinase (TK) expression is important for the maximum demonstration of virulence of herpes simplex virus (HSV). In this study, we have investigated interactions of a TK- mutant of virulent HSV type 2 (HSV-2) (syn+) and a nonneuroinvasive HSV-1 (syn) in mice. When the mice were inoculated with each virus alone in their rear footpads, no mice were killed even after infection with high doses of viruses (greater than 10(6) PFU per mouse), whereas 100% of the mice died when inoculated with 10(5) PFU of a 1:1 mixture of HSV-2 TK- mutant and nonneuroinvasive HSV-1. The 1:1 mixture exhibited even more virulence than the parental HSV-2; the mean survival time of coinfected mice was significantly shorter than that of mice inoculated with 10(5) PFU of the virulent HSV-2. We have also examined the genotypes and phenotypes of viruses isolated from the central nervous system of coinfected mice. Of 50 isolates, 7 were judged to be recombinants from their restriction endonuclease cleavage patterns, but all were nonneuroinvasive. In addition, all syn+ viruses (23 clones) tested were found to have TK- phenotypes, indicating that the majority of viruses present in the central nervous system were TK- viruses, since about 90% of viruses detected in spinal cords and brains exhibited syn+ phenotypes. These results strongly suggest that the lethal invasion of the central nervous system by HSV-2 TK- and nonneuroinvasive HSV-1 was the consequence of in vivo complementation between the two viruses.
Characterization of murine monoclonal antibodies directed against the core proteins of human immunodeficiency virus types 1 and 2
Authors: Niedrig M , Hinkula J , Harthus H P , Bröker M , Hopp L , Pauli G , Wahren B
Published: January 2020

Monoclonal antibodies (MAbs) raised against the core proteins of human immunodeficiency virus type 1 (HIV-1; laboratory strain HTLV-IIIB) and HIV-2 (strain ROD) were investigated in a variety of tests, e.g., enzyme-linked immunosorbent assay (ELISA), immunostaining of Western immunoblots, immunofluorescence, immunoprecipitation, and alkaline phosphatase anti-alkaline phosphatase assay. The MAbs were grouped according to their cross-reactions. Seven HIV-1-specific MAbs reacted exclusively with HIV-1, and five showed cross-reactivity with HIV-2 and simian immunodeficiency virus of macaques in ELISA. Four of the 15 MAbs against HIV-2 reacted only with the HIV-2 protein p26. Six showed cross-reactivity with HIV-1, and five showed a broad reaction with all three viruses. Overlapping 30-amino-acid-long peptides derived from the p24 protein sequence of HIV-1 were used in an epitope-mapping system. Three different immunogenic regions (A, B, and C) could be defined. Specific regions where anti-HIV-1 and -HIV-2 MAbs cross-reacted were mapped with shorter oligopeptides.
Deletions in the tRNA(Lys) primer-binding site of human immunodeficiency virus type 1 identify essential regions for reverse transcription
Authors: Rhim H , Park J , Morrow C D
Published: January 2020

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA primer bound near the 5' end of the genomic RNA at a position termed the primer-binding site (PBS). The PBS is an 18-nucleotide region of the HIV-1 genome complementary to cellular tRNA(Lys). To further investigate the sequence requirements for the PBS in reverse transcription, deletions in the PBS were created and subcloned into a plasmid containing the infectious HIV-1 proviral genome. The mutations deleted the entire PBS (delta PBS) or the first 9 (delta 1-9), the second 9 (delta 10-18), or 12 (delta 7-18) nucleotides of the PBS. An additional mutation in the PBS was created in which the second nine nucleotides were deleted and nine additional nucleotides were substituted [Lys(1-9)]. The transfection of plasmids containing the wild-type or mutant proviral genomes into tissue culture cells resulted in expression of the HIV-1 gag and env gene products, as determined by immunoprecipitation using sera from AIDS patients. HIV-1 virus was released from the transfected cells, as determined by analysis of the supernatants for reverse transcriptase activity. The infectivity of the viruses derived from the transfection was examined by coculture experiments with SupT1 cells, which support high-level replication of HIV-1. The transfection of plasmids containing HIV-1 proviral genomes with the delta PBS and PBS (delta 1-9) mutations did not produce infectious virus. In contrast, the HIV-1 proviral genomes with the delta 10-18, delta 7-18, and Lys(1-9) mutations in the PBS produced infectious virus upon transfection, although the kinetics of appearance was significantly delayed for the mutant viruses compared with the wild type. To further explore the nature of this defect, the PBS region from integrated proviral genomes was amplified by polymerase chain reaction and individual DNA products were subcloned into M13mp19, followed by a sequence analysis of the PBS region from individual M13 phage clones. In each of the PBS regions examined, the 18-nucleotide PBS complementary to tRNA(Lys) was present. However, nucleotide deletions and insertions were found 3' to the PBS from the samples derived from the transfection of plasmids containing mutant proviral genomes. Upon reinfection, the revertant viruses maintained the deletions 3' to the PBS and had kinetics of replication similar to that of the wild-type virus.(ABSTRACT TRUNCATED AT 400 WORDS)
Utilization of DNA recombination for the two-step replacement of growth factor sequences in the vaccinia virus genome
Authors: Spyropoulos D D , Stallard V , Roberts B E , Cohen L K
Published: January 2020

An efficient procedure for the generation of sequence-specific alterations of the vaccinia virus genome was demonstrated. Homologous DNA recombination within cells infected with vaccinia virus was used for the deletion or replacement of promoter sequences of the viral growth factor gene by a procedure comparable to transplacement in Saccharomyces cerevisiae. This DNA replacement procedure can potentially be used to generate any sequence alteration within the vaccinia virus genome. Deletion of growth factor promoter sequences resulted in a dramatic reduction in growth factor gene transcription and protein synthesis. Replacement of growth factor promoter sequences with promoter sequences of the strong constitutive 40-kDa gene resulted in an increase in gene transcription and protein synthesis and an altered temporal pattern of expression. Virus containing mutations in the growth factor gene demonstrated different plaque morphologies on cell culture monolayers.
Characterization of endogenous and recombinant proviral elements of a highly tumorigenic AKR cell line
Authors: Lamont C , Culp P , Talbott R L , Phillips T R , Trauger R J , Frankel W N , Wilson M C , Coffin J M , Elder J H
Published: January 2020

As an approach to evaluating the contribution of classes of endogenous viral sequences to leukemogenesis, a genomic library was prepared from the highly tumorigenic AKR SL12.3 cell line and screened for env-containing proviruses. An extensive battery of virus-derived probes and specific oligonucleotide probes were used to segregate 83 positive clones into related groups. The nonecotropic endogenous retroviruses were identified as members of the polytropic, modified polytropic, or xenotropic groups. At least three unique xenotropic proviruses were detected that differed from the published xenotropic sequence within a variable region of the 5' portion of env. Changes among the xenotropic proviruses included relative insertions and/or deletions that maintain an open reading frame and hence the potential to encode viable envelope gene products. Several recombinant viruses were also detected. Recombination was not random and primarily involved the formation of mink cell focus-inducing class I retroviruses via recombination between polytropic elements and ecotropic virus. One other recombinant was detected which contained ecotropic virus sequences in the 5' region encoding p15 of an otherwise xenotropic provirus. An interesting observation was the finding that certain clones contained more than one provirus within the average 20-kb cloned insert. This would not be expected if integration were totally random. The de novo recombinant proviruses identified here provide a series of potential candidates to be evaluated for their contribution to the tumorigencity of the SL12.3 cell line.
The vpx gene of simian immunodeficiency virus facilitates efficient viral replication in fresh lymphocytes and macrophage
Authors: Yu X F , Yu Q C , Essex M , Lee T H
Published: January 2020

vpx is a unique open reading frame found in simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) but not in HIV-1. It encodes a 12- to 16-kDa virion-associated protein. Although vpx is dispensable for viral replication in several established human lymphocyte cell lines, there is no consensus regarding whether this gene is required for efficient viral replication in freshly isolated lymphocytes. We report here that the vpx mutant of SIVmac exhibits different degrees of impairment from wild-type SIVmac in freshly isolated lymphocytes. This defect is more pronounced in macrophages from the same donors. Our findings suggest that vpx is required for efficient viral replication in fresh lymphocytes and macrophages.
Anti-idiotype antibodies that mimic gp86 of human cytomegalovirus inhibit viral fusion but not attachment
Authors: Keay S , Baldwin B
Published: January 2020

Human cytomegalovirus (CMV) infects cells by sequential processes involving attachment, fusion with the cell membrane, and penetration of the capsid. We used two monoclonal anti-idiotype that mimic one of the CMV envelope glycoproteins, gp86, to study its role in the early phases of CMV infection. Neither of two such antibodies inhibited virus binding to human embryonic lung (HEL) fibroblasts; however, both antibodies inhibited the fusion of CMV with HEL cells, as measured by an assay in which viral envelope is labeled with a fluorescent amphiphile (octadecyl rhodamine B chloride, or R18), resulting in increased fluorescence during fusion of virus with the cell membrane. Because these anti-idiotype antibodies were shown previously to bind to specific receptors on HEL cell membranes, these findings suggest that both gp86 and its cell membrane receptor may function in the fusion of human CMV with HEL cells.