bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of simian virus 40 small t antigen-induced progression of rat F111 cells minimally transformed by large T antigen
Authors: Zerrahn J , Deppert W
Published: January 2020

Minimal transformants of rat F111 fibroblasts were established after infection with the large T antigen (large T)-encoding retroviral expression vector pZIPTEX (M. Brown, M. McCormack, K. Zinn, M. Farrell, I. Bikel, and D. Livingston, J. Virol. 60:290-293, 1986). Coexpression of small t antigen (small t) in these cells efficiently led to their progression toward a significantly enhanced transformed phenotype. Small t forms a complex with phosphatase 2A and thereby might influence cellular phosphorylation processes, including the phosphorylation of large T. Since phosphorylation can modulate the transforming activity of large T, we asked whether the phosphorylation status of large T in minimally transformed cells might differ from that of large T in maximally transformed FR(wt648) cells and whether it might be altered by coexpression of small t. We found the phosphate turnover on large T in minimally transformed cells significantly different from that in fully transformed cells. This resulted in underphosphorylation of large T in minimally transformed cells at phosphorylation sites previously shown to be involved in the regulation of the transforming activity of large T. However, coexpression of small t in the minimally transformed cells did not alter the phosphate turnover on large T during progression; i.e., it did not induce a change in the steady-state phosphorylation of large T. This suggests that the helper function of small t during the progression of these cells was not mediated by modulating phosphatase 2A activity toward large T.
A monoclonal antibody which blocks infection with feline immunodeficiency virus identifies a possible non-CD4 receptor
Authors: Hosie M J , Willett B J , Dunsford T H , Jarrett O , Neil J C
Published: January 2020

Monoclonal antibody vpg15 detects a 24-kDa cell surface protein on feline cells permissive for infection with feline immunodeficiency virus (FIV). The antibody blocks infection of FIV-susceptible cells, and expression of the vpg15 marker is decreased in FIV-infected cells in vitro. These results suggest that the antibody may recognize an FIV receptor distinct from CD4.
Efficient analysis of nonviable poliovirus capsid mutants
Authors: Simons J , Rogove A , Moscufo N , Reynolds C , Chow M
Published: January 2020

Nonviable poliovirus capsid mutants were studied by an efficient infection-transfection system. Phenotypically, nonviable poliovirus capsid mutants appear to segregate into three classes: those that form only protomers, those that can form pentamers, and one that can form completed virions.
The human immunodeficiency virus type 1 long terminal repeat specifies two different transcription complexes, only one of which is regulated by Tat
Authors: Lu X , Welsh T M , Peterlin B M
Published: January 2020

The human immunodeficiency virus type 1 long terminal repeat sets up two different transcription complexes, which have been called processive and nonprocessive complexes. By mutating and substituting cis-acting sequences, we mapped elements of the human immunodeficiency virus long terminal repeat that are responsible for creating each transcription complex. Whereas processive complexes are efficiently assembled by upstream promoter elements in the absence of the TATA box, nonprocessive complexes absolutely require the TATA box. Moreover, the TATA box alone can set up these nonprocessive complexes, and nonprocessive but not processive complexes are trans activated by Tat. Finally, a strong DNA-binding site between the TATA box and trans-activation-responsive region interferes with either the assembly or movement of these nonprocessive complexes and diminishes the effects of Tat. Thus, Tat affects a critical step in the formation of elongation-competent transcription complexes.
High-frequency recombination mediated by polyomavirus large T antigen defective in replication
Authors: St-Onge L , Bouchard L , Bastin M
Published: January 2020

We investigated the mechanism by which the large T antigen (T-Ag) of both polyomavirus and simian virus 40 (SV40) promotes homologous recombination in mammalian cells. To this end, we constructed a rat cell line, designated Hy5, that carries two mutated copies of the polyomavirus middle-T-Ag (pmt) oncogene lying as direct repeats on the same chromosome. The structure of the viral insert was devised so that intrachromosomal recombination between the pmt repeats reconstitutes wild-type pmt and yields cell populations amenable to selection for the transformed phenotype. Correction of pmt by gene conversion occurred spontaneously at a rate of ca. 1.7 x 10(-7) per cell generation and was masked by another recombination event that also led to the transformation of the Hy5 cell line. This event was identified as chromosomal inversion and overexpression of the upstream pmt copy as a result of homologous recombination between adjacent pBR322 sequences. Both events were promoted by the polyomavirus large T-Ag by several orders of magnitude, as well as by mutants defective in the initiation of viral DNA synthesis. Large T-Ag also promoted reconstitution of wild-type pmt by unequal exchange between sister chromatids, yielding structures compatible with some of the chromosomal aberrations commonly observed in transformed cells. Our data indicate that large T-Ag has a recombination-promoting activity that can be dissociated from its replicative function.
Protection of sheep against bovine leukemia virus (BLV) infection by vaccination with recombinant vaccinia viruses expressing BLV envelope glycoproteins: correlation of protection with CD4 T-cell response to gp51 peptide 51-70
Authors: Gatei M H , Naif H M , Kumar S , Boyle D B , Daniel R C , Good M F , Lavin M F
Published: January 2020

We have previously constructed vaccinia virus (VV) recombinants containing a complete or truncated envelope (env) gene of bovine leukemia virus (BLV). Only recombinants carrying the complete env gene (VV-BLV2 and VV-BLV3) expressed env glycoprotein on the surface of virus-infected cells and produced an antibody response in rabbits. In the present study, these VV recombinants were used to immunize sheep prior to challenge with BLV-infected peripheral blood mononuclear cells. Both humoral and cell-mediated immunity were monitored in infected animals. Sheep inoculated with recombinants containing the complete env gene showed a CD4 response to a defined epitope of gp51, but this response was absent 4 months postchallenge. Anti-gp51 antibodies appeared in animals inoculated with complete env 2 weeks after challenge, reached a peak at 4 weeks, and subsequently declined over 16 months. No CD4 response was recorded in animals inoculated with recombinants containing truncated env gene (VV-BLV1). BLV-infected control animals and those animals receiving VV-BLV1 were slower to develop antibodies postchallenge, and the titers of anti-gp51 antibodies continued to increase over 16 months. Proviral DNA was detected by the polymerase chain reaction in the four groups at 6 weeks after challenge. However, it could not be detected 4 months postinfection in the VV groups inoculated with complete env. Provirus was present in the VV-BLV1 and control groups over the 16-month trial period. These results demonstrate that vaccination with VV recombinants containing the complete env gene of BLV protects sheep against infection and that protection correlated with a CD4 T-cell response to a defined epitope.
Bovine leukemia virus RNA sequences involved in dimerization and specific gag protein binding: close relation to the packaging sites of avian, murine, and human retroviruses
Authors: Katoh I , Yasunaga T , Yoshinaka Y
Published: January 2020

In vitro detection of a specific complex of the bovine leukemia virus (BLV) MA(p15) protein and the 5'-terminal RNA dimer led to the hypothesis that the NH2-terminal domain of retrovirus gag protein precursor is involved in the selective viral RNA packaging mechanism. Here we describe mapping of the BLV RNA for dimer-forming and MA(p15)-binding abilities by a simple cDNA probing method followed by mutation analyses with the reactive U5-5' gag RNA. The RNA dimerization is mediated by the region harboring U5, the primer binding site (PBS), and the 30 bases immediately downstream of PBS. This conclusion is supported by computer-assisted RNA secondary-structure analysis which predicted a multibranched stem-loop folding throughout the dimer region determined. Another region from PBS to the 5'-terminal 60 residues of the gag gene, partially overlapping the dimer region, likely provides essential elements for the MA(p15) binding reaction, although the presence of either the 3' or 5' neighboring sequences increases the complex-forming efficiency significantly, and each of the substructures predicted within the core region has, if any, only very weak affinity to MA(p15). These in vitro characterizations of the BLV RNA may reflect general features of the specific protein-RNA interaction in the packaging events of various retroviruses. 5'-terminal folded structures of retroviral RNA molecules and their biological activities are discussed.
Transdominant inhibition of wild-type human immunodeficiency virus type 2 replication by an envelope deletion mutant
Authors: Steffy K R , Wong-Staal F
Published: January 2020

The envelope glycoprotein of human immunodeficiency virus type 2 (HIV-2) is primarily responsible for virus attachment and entry into the target cell population. We constructed an HIV-2 mutant virus containing an in-frame deletion within the putative CD4-binding sequences of the envelope glycoprotein and confirmed that the mutant envelope is unable to bind CD4 and that the mutant virus is noninfectious. To investigate whether this mutant could dominantly interfere with wild-type replication, we coexpressed proviral DNAs of both wild-type and mutant viruses in cells and assayed the production of infectious HIV-2 virions. Interference with virus replication was indeed observed with mutant DNA, and a maximal effect was achieved with 10-fold excess mutant DNA over wild-type DNA in the cotransfection experiments. The transdominant effect on virus replication does not appear to be at the level of wild-type envelope expression or gp120-CD4 interaction. Rather, the interference may be at the level of mixed-oligomer formation during progeny virus assembly and may occur by either destabilizing the multimeric structure of gp120 or forming a defective mixed multimeric gp120 which is unable to complete the receptor binding and/or postbinding events needed for infection.
Susceptibility of AKXD recombinant inbred mouse strains to lymphomas
Authors: Gilbert D J , Neumann P E , Taylor B A , Jenkins N A , Copeland N G
Published: January 2020

We analyzed the susceptibility of 10 AKXD recombinant inbred (RI) mouse strains to lymphomas. These strains were derived from crosses of AKR/J, a highly lymphomatous strain, and DBA/2J, a weakly lymphomatous strain. Of the 10 strains analyzed, nine showed a high incidence of lymphoma development. As with the other 13 AKXD strains analyzed previously (M. L. Mucenski, B. A. Taylor, N. A. Jenkins, and N. G. Copeland, Mol. Cell. Biol. 6:4236-4243, 1986), the mean age at onset of lymphomas and lymphoma types varied among the strains. Whereas some strains were susceptible to T-cell lymphomas, as was the AKR/J parent, other strains were susceptible to B-cell lymphomas or to a combination of T- and B-cell lymphomas. Somatic mink cell focus-forming proviruses appeared causally associated with T-cell lymphomas, whereas somatic ecotropic proviruses appeared causally associated with B-cell lymphomas. Mice with T-cell lymphomas died significantly earlier than mice with other lymphoma types (stem, pre-B, or B cell and myeloid). The numbers of effective loci influencing the mean age at onset of lymphomas, the presence or absence of mink cell focus-forming viruses in tumors, and the frequency of T-cell lymphomas were estimated to be 3.9, 1.8, and 2.7, respectively. Tests of association with marker loci already typed in the AKXD RI strains suggested that two loci, Rmcf and Pmv-25 (or a locus linked to Pmv-25), influence all three trait variables. Finally, D21S16h, a marker locus on distal chromosome 16, showed 50% probability of linkage to a locus that influences the mean age at onset of lymphomas. Additional studies in combination with classical genetic crosses should be helpful in confirming these linkages and in identifying other loci influencing tumor susceptibility in AKXD RI strains.
Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine retroviruses
Authors: Albritton L M , Kim J W , Tseng L , Cunningham J M
Published: January 2020

Infection of rodent cells by ecotropic type C retroviruses requires the expression of a cationic amino acid transporter composed of multiple membrane-spanning domains. By exchanging portions of cDNAs encoding the permissive mouse and nonpermissive human transporters and examining their abilities to specify virus infection upon expression in human 293 cells, we have identified the amino acid residues in the extracellular loop connecting the fifth and sixth membrane-spanning segments of the mouse transporter that are required for both envelope gp70 binding and infection. These findings strongly suggest that the role of the mouse transporter in determining infection is to provide an envelope-binding site. This role is analogous to those of host membrane proteins composed of a single membrane-spanning domain that serve as binding proteins or receptors for other enveloped viruses such as human immunodeficiency virus, Epstein-Barr virus, and murine and human coronaviruses.
A single amino acid substitution in hypervariable region 5 of the envelope protein of feline immunodeficiency virus allows escape from virus neutralization
Authors: Siebelink K H , Rimmelzwaan G F , Bosch M L , Meloen R H , Osterhaus A D
Published: January 2020

We infected a specific-pathogen-free cat (cat 14) with molecularly cloned feline immunodeficiency virus clone 19k1 (FIV19k1 [K. H. J. Siebelink, I. Chu, G. F. Rimmelzwaan, K. Weijer, A. D. M. E. Osterhaus, and M. L. Bosch, J. Virol. 66:1091-1097, 1992]). Serum of this cat obtained 22 weeks postinfection (serum 1422) neutralized FIV19k1 but not FIV19k32, which is 99.3% identical to FIV19k1 in the envelope gene. Serum 1422 also neutralized virus isolated from cat 14 at weeks 2 and 32 postinfection. We then cultured FIV19k1 in the continuous presence of serum 1422, which resulted in a delay in virus replication of 6 weeks. The resulting virus population appeared to be resistant to virus neutralization by serum 1422. Nucleotide sequencing of the env open reading frame of this presumed escape mutant revealed the presence of one silent and two substitution mutations, both of the latter in hypervariable region 5. Through the construction of chimeric viruses and site-directed mutagenesis, we demonstrated that one of these mutations, the substitution of lysine to glutamine at amino acid position 560 in hypervariable region 5, was sufficient to allow the escape of FIV19k1 from neutralization by serum 1422.
RNA-binding activity of hepatitis delta antigen involves two arginine-rich motifs and is required for hepatitis delta virus RNA replication
Authors: Lee C Z , Lin J H , Chao M , McKnight K , Lai M M
Published: January 2020

Hepatitis delta antigen (HDAg) is an RNA-binding protein with binding specificity for hepatitis delta virus (HDV) RNA (J. H. Lin, M. F. Chang, S. C. Baker, S. Govindarajan, and M. M. C. Lai, J. Virol. 64:4051-4058, 1990). By amino acid sequence homology search, we have identified within its RNA-binding domain two stretches of an arginine-rich motif (ARM), which is present in many prokaryotic and eukaryotic RNA-binding proteins. The first one is KERQDHRRRKA and the second is EDEKRERRIAG, and they are separated by 29 amino acids. Deletion of either one of these ARM sequences resulted in the total loss of the in vitro RNA-binding activity of HDAg. Thus, HDAg is different from other RNA-binding proteins in that it requires two ARM-like sequences for its RNA-binding activity. Replacement of the spacer sequence between the two ARMs with a shorter stretch of sequence also reduced RNA binding in vitro. Furthermore, site-specific mutations of the basic amino acid residues in both ARMs resulted in the total loss or reduction of RNA-binding activity. The biological significance of the RNA-binding activity was studied by examining the trans-activating activity of the RNA-binding mutants. The plasmids expressing HDAgs with various mutations in the RNA-binding motifs were cotransfected with a replication-defective HDV dimer cDNA construct into COS cells. It was found that all the HDAg mutants which had lost the in vitro RNA-binding activity also lost the ability to complement the defect of HDV RNA replication. We conclude that the trans-activating function of HDAg requires its binding to HDV RNA.
Quantitation of latent varicella-zoster virus DNA in human trigeminal ganglia by polymerase chain reaction
Authors: Mahalingam R , Wellish M , Lederer D , Forghani B , Cohrs R , Gilden D
Published: January 2020

Competitive polymerase chain reaction was used to quantitate latent varicella-zoster virus (VZV) DNA in human trigeminal ganglia. Ganglionic DNA from five subjects was amplified with oligonucleotide primers specific for VZV gene 28. Two of the samples were also analyzed with primers specific for VZV gene 62. Our results indicated that there are 6 to 31 copies of the VZV genome in every 100,000 ganglionic cells.
Minus-strand DNA is present within murine type C ecotropic retroviruses prior to infection
Authors: Zhu J , Cunningham J M
Published: January 2020

Viral minus-strand DNA has been identified within ecotropic murine retroviruses prior to infection. The abundance of minus-strand DNA is inversely proportional to the distance from the primer binding site, suggesting that viral DNA is synthesized by reverse transcriptase with the genomic RNA as template. These findings demonstrate that replication of the retroviral genome is not initiated by infection and may begin after activation of reverse transcriptase by gag-pol cleavage during virus assembly.
The human papillomavirus E7 oncoprotein and the cellular transcription factor E2F bind to separate sites on the retinoblastoma tumor suppressor protein
Authors: Wu E W , Clemens K E , Heck D V , Münger K
Published: January 2020

The ability of the high-risk and low-risk human papillomavirus E7 oncoproteins to disrupt complexes of the retinoblastoma tumor suppressor protein pRB and the cellular transcription factor E2F was studied. The ability of E7 to disrupt this transcription factor complex correlated with the different pRB binding efficiencies of the high-risk and low-risk human papillomavirus-encoded E7 proteins. The pRB binding site was the sole determinant for these observed differences. The phosphorylation status of the casein kinase II site that is immediately adjacent to the pRB binding site in E7 had no marked effect on this biochemical property of E7. Peptides consisting of the pRB binding site of E7, however, were not able to disrupt the pRB/E2F complex. These data suggest that additional carboxy-terminal sequences in E7 are also required for the efficient disruption of the pRB/E2F complex and that E7 and E2F may interact with nonidentical sites of pRB.
Human immunodeficiency virus type 1 envelope gp120 is cleaved after incubation with recombinant soluble CD4
Authors: Werner A , Levy J A
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) infects human CD4+ cells by a high-affinity interaction between its envelope glycoprotein gp120 and the CD4 molecule on the cell surface. Subsequent virus entry into the cells involves other steps, one of which could be cleavage of the gp120 followed by virus-cell fusion. The envelope gp120 is highly variable among different HIV-1 isolates, but conserved amino acid sequence motifs that contain potential proteolytic cleavage sites can be found. Following incubation with a soluble form of CD4, we demonstrate that gp120 of highly purified HIV-1 preparations is, without addition of exogenous proteinase, cleaved most likely in the V3 loop, yielding two proteins of 50 and 70 kDa. The extent of gp120 proteolysis is HIV-1 strain dependent and correlates with the recombinant soluble CD4 sensitivity to neutralization of the particular strain. The origin of the proteolytic activity in the virus preparations remains unclear. The results support the hypothesis that cleavage of gp120 is required for HIV infection of cells.
Identification and baculovirus expression of the VP4 protein of the human group B rotavirus ADRV
Authors: Mackow E R , Werner-Eckert R , Fay M E , Tao H , Chen G
Published: January 2020

A complete cDNA copy of the fourth RNA segment of the human group B rotavirus adult diarrheal rotavirus (ADRV) has been cloned into lambda phage and excised into plasmid pSK Bluescript. Gene segment 4 contains 2,303 bases and encodes one long open reading frame beginning at base 16 and terminating at base 2263. The encoded protein contains 749 amino acids, with a calculated molecular mass of 84.4 kDa and a pI of 6.1. Gene 4 cDNA was inserted into a recombinant baculovirus via homologous recombination. The gene 4 polypeptide migrates at 84 kDa when expressed either by a recombinant baculovirus or in vitro in a rabbit reticulocyte lysate. The gene 4 protein is immunoprecipitable by hyperimmune serum to ADRV, human ADRV convalescent-phase serum, a porcine group B rotavirus infection serum, and a monoclonal antibody made to ADRV virion. Guinea pig hyperimmune serum to the baculovirus-expressed ADRV VP4 protein recognizes virus and immunoprecipitates an 84-kDa protein from in vitro translations of total ADRV mRNA. In addition, the gene 4-encoded protein shares significant amino acid identity and similarity with the group A rotavirus VP4 protein. This information, together with our previous identification of an 84-kDa protein present on iodinated intact virion but not EDTA-treated ADRV, suggests that gene 4 encodes the VP4 protein equivalent present on the outer capsid of ADRV. The ADRV VP4 protein is also 58% identical to the IDIR rat group B rotavirus gene segment 3 protein. The substantial differences between these two group B VP4 proteins suggests that they are distantly related and likely to define two different group B rotavirus VP4 serotypes. The baculovirus-expressed VP4 protein should be useful for developing serotyping reagents and tests for human and animal group B rotaviruses as well as for addressing the role of VP4 in ADRV neutralization.
Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites
Authors: Grakoui A , McCourt D W , Wychowski C , Feinstone S M , Rice C M
Published: January 2020

Processing of the hepatitis C virus (HCV) H strain polyprotein yields at least nine distinct cleavage products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. As described in this report, site-directed mutagenesis and transient expression analyses were used to study the role of a putative serine proteinase domain, located in the N-terminal one-third of the NS3 protein, in proteolytic processing of HCV polyproteins. All four cleavages which occur C terminal to the proteinase domain (3/4A, 4A/4B, 4B/5A, and 5A/5B) were abolished by substitution of alanine for either of two predicted residues (His-1083 and Ser-1165) in the proteinase catalytic triad. However, such substitutions have no observable effect on cleavages in the structural region or at the 2/3 site. Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity. NS3 proteinase-dependent cleavage sites were localized by N-terminal sequence analysis of NS4A, NS4B, NS5A, and NS5B. Sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV NS3 proteinase substrate specificity. These features include an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position.
The RRE of human immunodeficiency virus type 1 contributes to cell-type-specific viral tropism
Authors: Dayton E T , Konings D A , Lim S Y , Hsu R K , Butini L , Pantaleo G , Dayton A I
Published: January 2020

As part of a general program investigating the mechanism of the Rev axis of human immunodeficiency virus type 1 (HIV-1) autoregulation, a series of proviral HIV-1 mutants which differ from the parental HXB2 strain at selected positions within the RRE were constructed. All of the mutations were designed to perturb the RRE by introducing local helix disruptions without altering the coding potential of the overlapping envelope open reading frame. Viral replication in various cell types was monitored by a cell supernatant reverse transcriptase assay and Northern (RNA blot) analysis. All proviral RRE mutants displayed at least some impairment in replication. However, the relative impairment varied drastically among the various cell types tested. This suggests that the RRE may contribute to cell-type-specific viral tropism.
The NF-kappa B binding site is necessary for efficient replication of simian immunodeficiency virus of macaques in primary macrophages but not in T cells in vitro
Authors: Bellas R E , Hopkins N , Li Y
Published: January 2020

We demonstrate here that the nuclear factor-kappa B (NF-kappa B) binding site in the simian immunodeficiency virus (SIVmac) long terminal repeat is essential for efficient virus replication in primary alveolar macrophages but dispensable for efficient replication in primary T cells. Mutation of the NF-kappa B site does not seriously impair replication of a T-cell-tropic SIVmac239 or a macrophagetropic SIVmacEm* in peripheral blood lymphocytes or established CD4+ cell lines; however, mutation of the NF-kappa B site prevents efficient SIVmacEm* replication in primary alveolar macrophages. These data suggest that efficient replication in primary macrophages requires both envelope and long terminal repeat determinants.
Precise missense and silent point mutations are fixed in the genomes of poliovirus mutants from persistently infected cells
Authors: Borzakian S , Pelletier I , Calvez V , Colbere-Garapin F
Published: January 2020

Poliovirus mutants selected in persistently infected human neuroblastoma cells have a modified cell tropism and can establish a secondary persistent infection in nonneural cells, such as HEp-2c cells. Nucleotide sequence analysis revealed that the genome of a persistent mutant, S11, differed from that of the parental lytic Sabin 1 poliovirus strain by 31 point mutations. Three mutations occurred in the noncoding regions. The other mutations resulted in 12 amino acid substitutions; 1 substitution occurred in a nonstructural protein (3A), while the other 11 substitutions were clustered in the capsid proteins VP2 and VP1. The same missense mutations, as well as many of the silent mutations that we observed in mutant S11, also accumulated in the genome of two other persistent viruses isolated from independent infections. This finding indicates that both missense and silent mutations are selected during the persistent infection of neuroblastoma cells and suggests that the secondary structure of RNA in the coding region may play a role in viral infection.
Prolonged replication in the mouse central nervous system of reoviruses isolated from persistently infected cell cultures
Authors: Morrison L A , Fields B N , Dermody T S
Published: January 2020

We examined pathogenic characteristics of plaque-purified reoviruses isolated from persistently infected L-cell cultures (PI viruses) after intracranial inoculation into newborn mice. The PI viruses were isolated from independent cultures initiated with high-passage stocks of the wild-type (wt) strain, type 3 Dearing. The virulence of most PI viruses was equivalent to that of the wt strain. However, replication of PI viruses in the central nervous system of infected mice was prolonged to 25 (but not 50) days postinoculation. Thirty-eight percent (n = 186) of mice inoculated with the PI viruses had residual virus detectable in brain tissue 25 days after inoculation, in contrast to only 16% (n = 57) of mice inoculated with wt virus (P = 0.009). Mean residual brain titers were more than 20-fold higher in mice inoculated with PI viruses compared with wt virus (4.3 x 10(4) versus 2.1 x 10(3); P = 0.006). Tropism of PI virus within the brain resembled that of wt virus, and the distribution of PI virus antigen in the brain did not change over time. The extent of necrosis in the brains of mice harboring PI virus 25 days after inoculation was minimal, despite continued presence of high titers of infectious virus. The latter observation resembles the absence of cytopathicity seen in L-cell cultures persistently infected with reovirus. These observations suggest that the interaction of PI viruses with cells can be altered in vivo as well as in cell culture, but virus is eventually cleared from the infected animal.
Alteration of location of dimer linkage sequence in retroviral RNA: little effect on replication or homologous recombination
Authors: Jones J S , Allan R W , Temin H M
Published: January 2020

Retrovirus particles contain a dimer of retroviral genomic RNA. A defined region of the retrovirus genome has previously been shown to be important for both dimerization and encapsidation. To study the importance of the position of this encapsidation and dimerization signal for retroviral replication and homologous recombination, we used a previously described spleen necrosis virus-based helper cell system. This system allows retroviral vectors with multiple genetic markers to be studied after a single cycle of retroviral replication. The sequence responsible for dimerization, the encapsidation/dimer linkage sequence (E/DLS), was moved from its normal location near the 5' end of the retroviral genome to a location near the 3' end of the genome. We characterized four pairs of retroviral vectors: (i) with both E/DLSs at the 5' ends of the genomes, (ii) with both E/DLSs at the 3' ends of the genomes, and (iii) two with one E/DLS at the 5' end of the genome and one at the 3' end of the genome. We found that moving the E/DLS to the 3' end of the genome resulted in at most an approximately factor of 5 reduction in virus titer in a single cycle of retroviral replication. Furthermore, we found no changes that were attributable to the alteration of the position of the E/DLS in the minus-strand DNA primer transfers or the plus-strand DNA primer transfers, the rate of homologous recombination, or the number of internal template switches in recombinant proviruses. These results indicate that any alignment or conformation necessary for retroviral replication or recombination is not the result of the position of the E/DLS.
Comparison of trans-dominant inhibitory mutant human immunodeficiency virus type 1 genes expressed by retroviral vectors in human T lymphocytes
Authors: Bahner I , Zhou C , Yu X J , Hao Q L , Guatelli J C , Kohn D B
Published: January 2020

trans-Dominant inhibitory mutant versions of the human immunodeficiency virus type 1 (HIV-1) regulatory genes tat and rev have previously been described. We have constructed a series of retroviral vectors to transduce these genes and compare their inhibitory activities. The inhibitory activities were measured with transient transfection assays by using a reporter which expresses an HIV-1 gag-Escherichia coli lacZ fusion protein with strict dependence on coexpression of both tat and rev. Additionally, the vectors were packaged as amphotropic virions and used to stably transduce human CEM T lymphocytes. The transduced CEM cells were challenged with HIV-1, and the effects of the mutant HIV-1 genes were determined by measuring the levels of HIV-1 p24gag produced. A tat gene substituted at amino acid 41 (tatk41a) retained partial trans-activating activity and lacked inhibitory activity. A tat gene with a premature stop codon at amino acid 54 (tat54ter) showed moderate trans-dominant inhibition of the reporter plasmid but failed to significantly inhibit HIV-1 replication. The M10 rev mutant, with a 2-amino-acid substitution, showed strong trans-dominant inhibitory activity both in the reporter plasmid and in the HIV-1 infection assay. The greatest inhibition of HIV-1 growth was seen when M10 was expressed under the transcriptional control of a human cytomegalovirus promoter; slightly less inhibition was achieved when expression of M10 was controlled by the Moloney murine leukemia virus long terminal repeat, and minimal inhibition was seen when the HIV-1 long terminal repeat controlled the M10 gene. These results demonstrate the potential utility of retroviral vectors expressing trans-dominant inhibitory mutant HIV-1 genes for gene therapy approaches to AIDS.
Complex nature of the major viral polyadenylated transcripts in Epstein-Barr virus-associated tumors
Authors: Smith P R , Gao Y , Karran L , Jones M D , Snudden D , Griffin B E
Published: January 2020

The most abundant polyadenylated viral transcripts in the Epstein-Barr virus (EBV)-associated tumor nasopharyngeal carcinoma are a family (apparent sizes, 4.8, 5.2, 6.2, and 7.0 kb) of highly spliced cytoplasmic RNAs expressed from the BamHI-I and -A regions of the viral genome in an antisense direction with respect to several viral lytic functions encoded within the same region and concerned with the lytic cycle of the virus. We have called these complementary-strand transcripts. They are also expressed in B cells, including Burkitt's lymphoma and EBV-immortalized marmoset cell lines, and tumors generated in cottontop tamarins in response to EBV infection, but at a lower level. The complete structure of the major 4.8-kb RNAs (seven or eight exons) was determined in this study; the larger, but related, transcripts appear to be produced by differential splicing. The transcriptional promoter for the major complementary-strand transcripts, located in BamHI-I, contains several well-characterized transcriptional control elements (E2A, SP1, and AP1) and is functionally active in both B lymphocytes and epithelial cells. It appears to be a bifunctional viral promoter, as it also contains the initiation codon for a gene (BILF2) that encodes a glycoprotein that is expressed off the other strand. Splicing events create a number of small AUG-initiated open reading frames, one of which has homology to functionally significant regions of the EBV-encoded nuclear antigen 2 and to E2 (in papillomavirus). The complex nature of these transcripts and their potential role in the virus association with malignancy are considered.
Effect of intergenic consensus sequence flanking sequences on coronavirus transcription
Authors: Makino S , Joo M
Published: January 2020

Insertion of a region, including the 18-nucleotide-long intergenic sequence between genes 6 and 7 of mouse hepatitis virus (MHV) genomic RNA, into an MHV defective interfering (DI) RNA leads to transcription of subgenomic DI RNA in helper virus-infected cells (S. Makino, M. Joo, and J. K. Makino, J. Virol. 66:6031-6041, 1991). In this study, the subgenomic DI RNA system was used to determine how sequences flanking the intergenic region affect MHV RNA transcription and to identify the minimum intergenic sequence required for MHV transcription. DI cDNAs containing the intergenic region between genes 6 and 7, but with different lengths of upstream or downstream flanking sequences, were constructed. All DI cDNAs had an 18-nucleotide-long intergenic region that was identical to the 3' region of the genomic leader sequence, which contains two UCUAA repeat sequences. These constructs included 0 to 1,440 nucleotides of upstream flanking sequence and 0 to 1,671 nucleotides of downstream flanking sequence. An analysis of intracellular genomic DI RNA and subgenomic DI RNA species revealed that there were no significant differences in the ratios of subgenomic to genomic DI RNA for any of the DI RNA constructs. DI cDNAs which lacked the intergenic region flanking sequences and contained a series of deletions within the 18-nucleotide-long intergenic sequence were constructed to determine the minimum sequence necessary for subgenomic DI RNA transcription. Small amounts of subgenomic DI RNA were synthesized from genomic DI RNAs with the intergenic consensus sequences UCUAAAC and GCUAAAC, whereas no subgenomic DI RNA transcription was observed from DI RNAs containing UCUAAAG and GCTAAAG sequences. These analyses demonstrated that the sequences flanking the intergenic sequence between genes 6 and 7 did not play a role in subgenomic DI RNA transcription regulation and that the UCUAAAC consensus sequence was sufficient for subgenomic DI RNA transcription.
Identification of the cleavage site and determinants required for poliovirus 3CPro-catalyzed cleavage of human TATA-binding transcription factor TBP
Authors: Das S , Dasgupta A
Published: January 2020

Host cell RNA polymerase II-mediated transcription is inhibited by poliovirus infection. We have shown previously that the human TATA-binding protein (TBP), a general transcription factor required for transcription of all RNA polymerase II genes, is directly cleaved both in vitro and in vivo by the virus-coded protease 3CPro. 3CPro specifically cleaves glutamine-glycine bonds in the viral polyprotein. Cellular transcription factor TBP contains three glutamine-glycine sites, at amino acids 12, 18, and 108. By using site-directed mutagenesis, we determined that the glutamine-glycine bond at amino acid 18, but not that at amino acid 12 or 108, is cleaved by the viral protease. Both the glutamine and the glycine appear to be important for the cleavage. Further mutations around the glutamine-glycine site at position 18 suggest that determinants other than the glutamine-glycine bond in TBP are also required for 3CPro-induced cleavage. An alanine at position P4 and a proline at position P2, proximal to the scissile glutamine-glycine pair, appear to be important for 3CPro-mediated cleavage of TBP. Our results suggest that the cleavage specificity of 3CPro for a cellular transcription factor is very similar to its mode of cleavage of viral polyproteins.
Spontaneous and differentiation-dependent regulation of measles virus gene expression in human glial cells
Authors: Schneider-Schaulies S , Schneider-Schaulies J , Bayer M , Löffler S , ter Meulen V
Published: January 2020

The expression of measles virus (MV) in six different permanent human glioma cell lines (D-54, U-251, U-138, U-105, U-373, and D-32) was analyzed. Although all cell lines were permissive for productive replication of all MV strains tested, U-251, D-54, and D-32 cells spontaneously revealed restrictions of MV transcription similar to those observed for primary rat astroglial cells and brain tissue. In vitro differentiation of D-54 and U-251 cells by substances affecting the intracellular cyclic AMP level caused a significant reduction of the expression of the viral proteins after 18, 72, and 144 h of infection. This pronounced restriction was not paralleled to a comparable level by an inhibition of the synthesis and biological activity in vitro of virus-specific mRNAs as shown by quantitative Northern (RNA) blot analyses and in vitro translation. The block in viral protein synthesis could not be attributed to the induction of type I interferon by any of the substances tested. Our findings indicate that down-regulation of MV gene expression in human brain cells can occur by a cell type-dependent regulation of the viral mRNA transcription and a differentiation-dependent regulation of translation, both of which may be crucial for the establishment of persistent MV infections in the central nervous system.
Juxtaposition between activation and basic domains of human immunodeficiency virus type 1 Tat is required for optimal interactions between Tat and TAR
Authors: Luo Y , Peterlin B M
Published: January 2020

trans activation of the human immunodeficiency virus type 1 long terminal repeat requires that the viral trans activator Tat interact with the trans-acting responsive region (TAR) RNA. Although the N-terminal 47 amino acids represent an independent activation domain that functions via heterologous nucleic acid-binding proteins, sequences of Tat that are required for interactions between Tat and TAR in cells have not been defined. Although in vitro binding studies suggested that the nine basic amino acids from positions 48 to 57 in Tat bind efficiently to the 5' bulge in the TAR RNA stem-loop, by creating several mutants of Tat and new hybrid proteins between Tat and the coat protein of bacteriophage R17, we determined that this arginine-rich domain is not sufficient for interactions between Tat and TAR in vivo. Rather, the activation domain is also required and must be juxtaposed to the basic domain. Thus, in vitro TAR RNA binding does not translate to function in vivo, which suggests that other proteins are important for specific and productive interactions between Tat and TAR.
Legitimate and illegitimate cleavage of human immunodeficiency virus glycoproteins by furin
Authors: Morikawa Y , Barsov E , Jones I
Published: January 2020

Coexpression of human immunodeficiency virus type 1 glycoproteins with the subtilisin-like protease furin leads to processing of gp160 and gp140, a truncated form of gp160, to gp120. In addition, we show that gp120 itself is further cleaved by furin at sites near the primary cleavage site and within the V3 loop.
A second nonstructural protein functions in the regulation of alphavirus negative-strand RNA synthesis
Authors: Sawicki D L , Sawicki S G
Published: January 2020

Previous studies (D.L. Sawicki, D. B. Barkhimer, S. G. Sawicki, C. M. Rice, and S. Schlesinger, Virology 174:43-52, 1990) identified a temperature-sensitive (ts) defect in Sindbis virus nonstructural protein 4 (nsP4) that reactivated negative-strand synthesis after its normal cessation at the end of the early phase of replication. We now report identification of two different ts alterations in nsP2 of Ala-517 to Thr in ts17 or Asn-700 to Lys in ts133 that also reactivated negative-strand synthesis. These same mutations caused severely reduced protease processing by nsP2 and recognition of the internal promoter for subgenomic mRNA synthesis and were responsible for the conditional lethality and RNA negativity of these mutants. Reactivation of negative-strand synthesis by mutations in nsP2 resembled that in nsP4: it was a reversible property of stable replication complexes and did not require continuation of viral protein synthesis. Recombinant viruses expressing both mutant nsP2 and nsP4 reactivated negative-strand synthesis more efficiently than did either mutant protein alone, consistent with the hypothesis that both nsP2 and nsP4 participate in template recognition. We propose that these alterations cause nsP2 and nsP4 to switch from their normal preference to recognize negative strands as templates to recognize positive strands and thereby mimic the initial formation of a replication complex.
Increased hematopoiesis in mice soon after infection by Friend murine leukemia virus
Authors: Mitchell T
Published: January 2020

Friend murine leukemia virus (F-MuLV), an erythroleukemogenic replication-competent retrovirus, induces leukemia in its host after a long latency. However, the early effects of infection may determine the pathway that eventually leads to malignant transformation. To determine how F-MuLV affects host cell proliferation soon after infection, BALB/c mice were inoculated with virus and then were assayed for susceptibility to appropriately pseudotyped spleen focus-forming virus (SFFV) as an indicator of erythropoietic activity. Twelve-week-old mice exposed to F-MuLV for 9 days were more susceptible (by a factor of 30) to superinfection by SFFV than were nonviremic mice. To test whether increased susceptibility was the result of increased hematopoietic activity, hematopoietic progenitors from the spleens of F-MuLV-infected mice were enumerated with a clonal culture assay. Nine days after inoculation with F-MuLV, the numbers of colony-forming progenitors increased by a factor of 4. Morphological analysis of the cultured colonies showed that erythroid, granulocytic, monocytic, and mixed granulocytic-monocytic progenitors all had increased. Thus, F-MuLV more rapidly induced a generalized increase in hematopoiesis than has previously been reported. The splenic hyperplasia induced by F-MuLV soon after infection may explain its ability to accelerate leukemogenesis in mice also infected by the polytropic Friend mink cell focus-forming virus.
Chicken adenovirus (CELO virus) particles augment receptor-mediated DNA delivery to mammalian cells and yield exceptional levels of stable transformants
Authors: Cotten M , Wagner E , Zatloukal K , Birnstiel M L
Published: January 2020

Delivery of genes via receptor-mediated endocytosis is severely limited by the poor exit of endocytosed DNA from the endosome. A large enhancement in delivery efficiency has been obtained by including human adenovirus particles in the delivery system. This enhancement is probably a function of the natural adenovirus entry mechanism, which must include passage through or disruption of the endosomal membrane. In an effort to identify safer virus particles useful in this application, we have tested the chicken adenovirus CELO virus for its ability to augment receptor-mediated gene delivery. We report here that CELO virus possesses pH-dependent, liposome disruption activity similar to that of human adenovirus type 5. Furthermore, the chicken adenovirus can be used to augment receptor-mediated gene delivery to levels comparable to those found for the human adenovirus when it is physically linked to polylysine ligand-condensed DNA particles. The chicken adenovirus has the advantage of being produced inexpensively in embryonated eggs, and the virus is naturally replication defective in mammalian cells, even in the presence of wild-type human adenovirus.
La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate
Authors: Meerovitch K , Svitkin Y V , Lee H S , Lejbkowicz F , Kenan D J , Chan E K , Agol V I , Keene J D , Sonenberg N
Published: January 2020

Translation initiation on poliovirus RNA occurs by internal binding of ribosomes to a sequence within the 5' untranslated region. We have previously characterized a HeLa cell protein, p52, that binds to a fragment of the poliovirus 5' untranslated region (K. Meerovitch, J. Pelletier, and N. Sonenberg, Genes Dev. 3:1026-1034, 1989). Here we report the purification of the HeLa p52. Protein microsequencing identified p52 as La autoantigen. The La protein is a human antigen that is recognized by antibodies from patients with autoimmune disorders such as systemic lupus erythematosus and Sjögren's syndrome. We show that the La protein stimulates translation of poliovirus RNA, but not brome mosaic virus, tobacco mosaic virus, and alfalfa mosaic virus 4 RNA, translation in a reticulocyte lysate. In addition, La corrects aberrant translation of poliovirus RNA in a reticulocyte lysate. Subcellular immunolocalization showed that La protein is mainly nuclear, but after poliovirus infection, La is redistributed to the cytoplasm. Our results suggest that La protein is involved in poliovirus internal initiation of translation and might function through a similar mechanism in the translation of cellular mRNAs.