bluuebunny/crossref_metadata_2025 huggingface dataset:
Infectious cDNA clones of the DA strain of Theiler's murine encephalomyelitis virus
Authors: Roos R P , Stein S , Ohara Y , Fu J L , Semler B L
Published: January 2020

The DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis viruses cause a persistent demyelinating infection, whereas the GDVII strain and other GDVII subgroup strains cause an acute lethal polioencephalomyelitis. We generated an infectious DA cDNA clone inserted into a transcription vector. Virus derived from transfection of transcripts produced a demyelinating disease indistinguishable from that of wild-type virus. The infectious clone provides a critical reagent for the production of interstrain recombinant viruses to help identify genetic loci responsible for the biological activities of the strains.
Molecular analysis of the transcriptional regulatory region of an early baculovirus gene
Authors: Nissen M S , Friesen P D
Published: January 2020

Transcription of the gene encoding a 35,000-molecular-weight protein (35K protein) from the EcoRI-S region (86.8 to 87.8 map units) of Autographa california nuclear polyhedrosis virus (AcMNPV) occurs early in infection and declines later. The region promoting the gene for the 35K protein, extending from 426 base pairs (bp) upstream to 12 bp downstream from the RNA start site, was linked to the bacterial chloramphenicol acetyltransferase gene (CAT) for analysis. CAT expression was monitored in cells that were transfected with plasmids containing the promoter-CAT fusion as well as cells infected with recombinant viruses containing the chimeric gene inserted into the AcMNPV genome. Mapping of the 5' ends of CAT-specific RNAs indicated that transcription initiated from the proper sites in both assays; moreover, the promoter fragment retained its early activity, despite an alternate location in the viral genome. The 5' boundary of upstream regulatory sequences was determined by constructing deletions of the promoter fragment extending toward the early RNA start site (position +1). In transient assays, a gradual reduction in CAT expression occurred as sequences from positions -426 to -31 were removed. In contrast, promoter deletions from positions -426 to -155 in recombinant viruses exhibited no effect on CAT expression, whereas deletions to position -55 abolished early expression but had no effect on late expression. Late CAT expression was eliminated when deletions to position -4 removed part of the late RNA start site. DNA signals potentiating early transcription were therefore located upstream (between positions -155 and -55) from those involved in late transcription of the gene encoding the 35K protein. Potential consensus sequences for early and late regulatory elements were identified.
Hepatitis B virus (HBV) promoters are regulated by the HBV enhancer in a tissue-specific manner
Authors: Antonucci T K , Rutter W J
Published: January 2020

The activities of the individual hepatitis B virus (HBV) promoters and the effects of the HBV enhancer on these promoters in several human cell types have been compared by measuring the activity and RNA levels of the linked reporter function chloramphenicol acetyltransferase. The relative promoter activities in the human HepG2 (liver), HeLa, and HS27 (fibroblast) cell lines are in the order precore greater than X greater than preS2 greater than preS1; thus, the promoters of the gene producing the largest quantity of viral proteins have relatively low activity. The juxtaposition of the HBV enhancer in either orientation increased the promoter activities only modestly (2- to 5-fold) in the nonliver cell lines, whereas it dramatically increased (20- to 100-fold) the promoter activities in the liver cell line. Thus, the HBV enhancer is especially active in liver cells. This may be one of the causes of hepatotrophicity of the virus.
Expression of intracisternal A-type particles is increased when a B-cell lymphoma differentiates into an immunoglobulin-secreting cell
Authors: Wiest D L , Burkhardt J K , Stockdale A M , Argon Y
Published: January 2020

The murine B-cell lymphoma CH12, like many other murine cells, expresses intracisternal A-type particles (IAPs). When CH12 cells are treated with lipopolysaccharide, the cells differentiate and secrete immunoglobulin M. The expression of IAP genes was also increased, in parallel with the increased expression of immunoglobulin genes. The amount of IAP mRNA increased within 48 h of lipopolysaccharide treatment and reached a level fivefold higher than that in unactivated CH12 cells. The two major IAP transcripts (7.2 and 5.4 kilobases) were induced to similar extents. The increased level of mRNA was reflected in higher levels of the major IAP structural protein p70, whose abundance increased 3.6-fold. The number of IAP particles per cell also increased upon activation of CH12, from 67 in nonsecreting CH12 to 290 in secreting cells. All of the IAPs were confined to the cisternae of the endoplasmic reticulum (ER), regardless of the differentiation state of the cell. Accompanying the induction of p70 was the induction of the related IAP polypeptide p102. A third viral polypeptide, p120, was also made in CH12; its abundance was almost unchanged. Localization of the IAP proteins on ultrathin frozen sections showed that most were assembled into particles in the ER. However, there were small pools of unassembled proteins both in the ER and on the plasma membrane. p70 and p120 could be detected, by iodination, on the surfaces of both secreting and nonsecreting CH12 cells. Unlike p70 and p120, p102 did not seem to be membrane associated. Taken together, these observations show that IAP expression is regulated developmentally in B lymphocytes. Also, these observations point to possible interactions between IAP genes and other cellular genes.
Primate cytomegalovirus assembly protein: genome location and nucleotide sequence
Authors: Robson L , Gibson W
Published: January 2020

A cDNA encoding the 37-kilodalton (kDa) capsid assembly protein of cytomegalovirus (CMV) strain Colburn was isolated from a lambda gt11 library constructed from CMV Colburn-infected human fibroblast RNA. RNA transcribed in vitro from this cDNA was translated in vitro to give a 40-kDa protein whose electrophoretic mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fragmentation pattern following partial proteolysis were indistinguishable from those of authentic assembly protein precursor. The position of the assembly protein gene was mapped to the EcoRI F, XbaI R, and SalI U restriction fragments, near the middle of the CMV Colburn genome, by Southern hybridizations using the cloned assembly protein cDNA as a probe. Similar sequences were identified by cross-hybridizations in colinear regions of the genomes of human CMV strains Towne and AD169: specifically, in the HindIII H, BamHI V, and EcoRI A fragments of Towne and in the HindIII L and BglII S fragments of AD169. The predominant transcript of the assembly protein gene was determined to be approximately 1 kilobase in size; however, a larger transcript (1.8 kilobases) was also identified. The nucleotide sequence of the assembly protein cDNA was determined and found to contain a single long open reading frame predicted to encode a polypeptide of 36.6 to 37 kDa, close to the 40-kDa size determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the assembly protein precursor. The presence of a single cysteine residue at the carboxy-terminal end of this open reading frame is consistent with data from biochemical studies and indicates that processing of the assembly protein precursor includes a proteolytic cleavage that removes its carboxyl end.
Characterization of partially activated p60c-src in chicken embryo fibroblasts
Authors: Sato M , Kato J , Takeya T
Published: January 2020

Previous studies identified the amino acid changes involved in the activation of p60c-src and revealed that the activation accompanies an alteration of its tyrosine-phosphorylation site. We show here that p60c-src that had been converted to transforming protein by amino acid substitution of the c-src gene either at position 63, 95 and 96, or 338 (J. Kato, T. Takeya, C. Grandori, H. Iba, J. B. Levy, and H. Hanafusa, Mol. Cell. Biol. 6:4155-4160, 1986) and encoded in a Rous sarcoma virus variant was phosphorylated on both Tyr-416 and Tyr-527 in chicken embryo fibroblasts. The results obtained from protease V8 analysis, tryptic peptide mapping, and fractionation with nonionic detergent indicated that the p60 of each variant was present in two forms in the population of the virus-infected cells; one was phosphorylated on Tyr-416, and the other was phosphorylated on Tyr-527. On the other hand, colonies isolated in soft agar contained exclusively p60 of which only Tyr-416 was phosphorylated. These results implied that the limited population of p60 was activated in these Rous sarcoma virus variant-infected chicken embryo fibroblasts and that the activated p60 was concentrated in transformed cells. Furthermore, these two forms of p60 differed in their affinity for the detergent-insoluble cellular matrix in spite of their identical primary amino acid sequences, suggesting that the effect of alteration of the tyrosine phosphorylation site was coupled with the degree of stability of this association.
Human antibodies react with an epitope of the human papillomavirus type 6b L1 open reading frame which is distinct from the type-common epitope
Authors: Jenison S A , Yu X P , Valentine J M , Galloway D A
Published: January 2020

Recombinant proteins encoded by the human papillomavirus type 6b (HPV6b) L1 open reading frame react with sera from patients with condylomata acuminata and also react with rabbit antiserum raised against sodium dodecyl sulfate-disrupted bovine papillomavirus type 1 (BPV1) virions. To map the immunoreactive epitopes, a series of procaryotic expression plasmids was made which contained a nested set of 3' to 5' deletions in the HPV6b L1 open reading frame. The deleted plasmids expressed a set of carboxy to amino terminus truncated fusion proteins. Regions containing the immunoreactive epitopes were mapped by determining which of the deleted fusion proteins retained reactivity with sera in Western immunoblot assays. The coding sequence for a human antibody-reactive linear epitope mapped between HPV6b nucleotide coordinates 7045 and 7087, and the rabbit anti-BPV1-reactive epitope coding sequence mapped between coordinates 6377 and 6454. Synthetic peptides derived from the epitope mapping were reacted with sera in enzyme-linked immunosorbent assay. Human sera reacted with synthetic peptide QSQAITCQKPTPEKEKPDPYK (HPV6b L1 amino acids 417 through 437). Rabbit anti-BPV1 and rabbit antisera raised against HPV16 L1 recombinant proteins reacted with the synthetic peptide DGDMVDTGFGAMNFADLQTNKSDVPIDI (HPV6b L1 amino acids 193 through 220). Human sera which reacted with HPV6b L1 fusion proteins cross-reacted with an HPV11 L1 fusion protein but did not react with fusion proteins encoded by HPV1a, HPV16, or HPV18. Rabbit anti-BPV1 reacted with L1 fusion proteins encoded by all of these HPV types. In contrast to the type-common (rabbit anti-BPV1-reactive) epitope, the human antibody-reactive epitope appears to be relatively HPV type specific.
Transactivation of human immunodeficiency virus promoter by human herpesvirus 6
Authors: Horvat R T , Wood C , Balachandran N
Published: January 2020

Patients with acquired immunodeficiency syndrome (AIDS) are often infected with a number of other heterologous viruses in addition to the initial human immunodeficiency virus (HIV) infection, and these agents could act as potential reactivating agents of latent HIV. A new antigenically distinct herpesvirus, designated human herpesvirus 6 (HHV-6), has recently been isolated from patients with AIDS and has been shown to infect a number of different human cells, specifically human T cells, B cells, and glial cells. Since these are some of the same cells that harbor the AIDS virus, it is quite important to determine any interaction between this new herpesvirus and HIV. In this report, we demonstrate that HHV-6 can trans-activate the HIV promoter in human T-cell lines as measured by the expression of the bacterial gene chloramphenicol acetyltransferase. This indicates that stimulation of HIV gene expression by HHV-6 could play a role in HIV pathogenesis.
Maternal transmission of retroviral disease and strategies for preventing infection of the neonate
Authors: Sharpe A H , Hunter J J , Ruprecht R M , Jaenisch R
Published: January 2020

Moloney murine leukemia virus is efficiently transmitted from viremic mothers to offspring, primarily via virus-containing milk. To determine the level in the infectious process at which an antiviral agent can interfere most effectively with perinatal viral transmission, we examined the effect of the drug 3'-azido-3'-deoxythymidine (AZT) on transmission of Moloney murine leukemia virus from viremic mothers to offspring. Although AZT treatment did not affect the titer of virus in milk, it did suppress the development of viremia in all offspring. AZT, however, did not prevent transmission of virus from viremic mothers to 25% of the offspring, but did lead to a marked reduction in virus load in these infected mice. These results provide evidence for effective antiretroviral therapy during gestation and in the perinatal period and are of potential significance for the management of maternal transmission of human retroviruses.
The second-largest subunit of the poxvirus RNA polymerase is similar to the corresponding subunits of procaryotic and eucaryotic RNA polymerases
Authors: Patel D D , Pickup D J
Published: January 2020

We have characterized the poxvirus gene encoding the second-largest subunit of the viral DNA-dependent RNA polymerase. This gene, designated rpo132, is located in the HindIII A fragment of the DNA of the Brighton Red strain of cowpox virus. A similar gene is located in the corresponding position in the HindIII A fragment of the DNA of the Western Reserve strain of vaccinia virus. The rpo132 gene is transcribed throughout the viral multiplication cycle. It has two transcriptional start sites; one is operative at late times only, and the other (80 base pairs downstream) is operative both at early times and at late times. Neither early nor late transcripts originating from the latter RNA start site contain long 5'-terminal poly(A) sequences. The rpo132 gene has the capacity to encode primary gene products of two types. The RNA transcripts whose 5' ends correspond to the early RNA start site can encode a 133-kilodalton (kDa) protein. The RNA transcripts whose 5' ends correspond to the early RNA start site can encode a 132-kDa protein. Transcripts of the latter type are more abundant, suggesting that the 132-kDa protein is the major primary product of this gene. The predicted amino acid sequences of both gene products share extensive similarities with the amino acid sequences of the second-largest subunits of the following enzymes: the RNA polymerase of Escherichia coli, the RNA polymerase II of Saccharomyces cerevisiae, and the RNA polymerase II of Drosophila melanogaster. This result provides further evidence of relatedness between multisubunit DNA-dependent RNA polymerases.
Differences in activities of murine retroviral long terminal repeats in cytotoxic T lymphocytes and T-lymphoma cells
Authors: LoSardo J E , Cupelli L A , Short M K , Berman J W , Lenz J
Published: January 2020

Transcriptional activities of the long terminal repeats (LTRs) of various murine leukemia viruses were tested in the cytotoxic T-cell lines CTLL-1 and CTLL-2. In contrast to T-lymphoma cells, in which the LTRs of T-lymphomagenic virus SL3-3 and Moloney murine leukemia virus are more active than those of other viruses, transcriptional activity in these mature, interleukin-2-dependent cells is not correlated with the specificity of viral leukemogenicity. Several approaches were used to investigate the molecular basis for LTR activity differences in lymphoma cells and mature cytotoxic T cells. Deletion analysis of the Moloney virus LTR showed that the direct repeats associated with enhancer activity have, at most, a slight effect on expression in CTLL-1 cells, whereas they stimulate expression six- to eightfold in T-lymphoma cells. This suggests that the mature T-cell line lacks one or more factors present in T-lymphoma cells that function to augment transcription from the Moloney murine leukemia virus LTR. We also used recombinant viral LTRs to investigate the role of the enhancer core element of SL3-3 in CTLL-1 and CTLL-2 cells. A one-base-pair difference between the core sequences of SL3-3 and nonleukemogenic Akv virus, which is important for SL3-3 activity in T-lymphoma cells, had no effect in these cells. The inability to distinguish the single-base-pair difference in expression assays was correlated with the absence of binding of a cellular factor, S-CBF, to the SL3-3 enhancer core in extracts of CTLL-1 and CTLL-2 nuclei. These studies may have implications for identification of the target cells for viral leukemogenesis, as well as for tracing of changes in the transcriptional machinery during T-lymphocyte differentiation.
Structural defect linked to nonrandom mutations in the matrix gene of biken strain subacute sclerosing panencephalitis virus defined by cDNA cloning and expression of chimeric genes
Authors: Ayata M , Hirano A , Wong T C
Published: January 2020

Biken strain, a nonproductive measles viruslike agent isolated from a subacute sclerosing panencephalitis (SSPE) patient, contains a posttranscriptional defect affecting matrix (M) protein. A putative M protein was translated in vitro with RNA from Biken strain-infected cells. A similar protein was detected in vivo by an antiserum against a peptide synthesized from the cloned M gene of Edmonston strain measles virus. By using a novel method, full-length cDNAs of the Biken M gene were selectively cloned. The cloned Biken M gene contained an open reading frame which encoded 8 extra carboxy-terminal amino acid residues and 20 amino acid substitutions predicted to affect both the hydrophobicity and secondary structure of the gene product. The cloned gene was expressed in vitro and in vivo into a 37,500 Mr protein electrophoretically and antigenically distinct from the M protein of Edmonston strain but identical to the M protein in Biken strain-infected cells. Chimeric M proteins synthesized in vitro and in vivo showed that the mutations in the carboxy-proximal region altered the local antigenicity and those in the amino region affected the overall protein conformation. The protein expressed from the Biken M gene was unstable in vivo. Instability was attributed to multiple mutations in both the amino and carboxy regions. A surprising number of mutations in both the coding and noncoding regions of the Biken M gene were identical to those in an independently isolated SSPE virus strain with a similar defect. These results offer insights into the basis of the defect in Biken strain and pose intriguing questions about the evolutionary origins of SSPE viruses in general.
Growth and survival of reovirus in intestinal tissue: role of the L2 and S1 genes
Authors: Bodkin D K , Fields B N
Published: January 2020

Reovirus serotype 1 Lang can be recovered in high titer from the intestines of neonatal mice up to day 8 after peroral inoculation. By contrast, reovirus serotype 3 Dearing cannot be recovered from intestinal tissue past day 4 after peroral inoculation. This difference between the two reoviruses was mapped by using reassortants generated from nonmutagenized laboratory stocks. When the L2 and S1 genes of reovirus serotype 3 Dearing were present in reassortants, the reassortants behaved like serotype 3 Dearing in exhibiting a decreased capacity to be recovered from intestinal tissue. Likewise, viruses which contained the L2 and S2 genes from serotype 1 Lang exhibited an enhanced capacity to grow and survive, which is characteristic of serotype 1 Lang. Thus, the capacity of reovirus to survive in intestinal tissue was determined by the L2 and S1 genes.
Regulated expression of the human cytomegalovirus pp65 gene: octamer sequence in the promoter is required for activation by viral gene products
Authors: Depto A S , Stenberg R M
Published: January 2020

To better understand the regulation of late gene expression in human cytomegalovirus (CMV)-infected cells, we examined expression of the gene that codes for the 65-kilodalton lower-matrix phosphoprotein (pp65). Analysis of RNA isolated at 72 h from cells infected with CMV Towne or ts66, a DNA-negative temperature-sensitive mutant, supported the fact that pp65 is expressed at low levels prior to viral DNA replication but maximally expressed after the initiation of viral DNA replication. To investigate promoter activation in a transient expression assay, the pp65 promoter was cloned into the indicator plasmid containing the gene for chloramphenicol acetyltransferase (CAT). Transfection of the promoter-CAT construct and subsequent superinfection with CMV resulted in activation of the promoter at early times after infection. Cotransfection with plasmids capable of expressing immediate-early (IE) proteins demonstrated that the promoter was activated by IE proteins and that both IE regions 1 and 2 were necessary. Analysis of promoter deletion mutants indicated that the 5' minimal sequence required for activation is -61 from the CAP site (+1) and that an 8-base-pair sequence located at -51 to -58 is necessary for activation of the pp65 promoter. This sequence is repeated once at +93 and is found as an inverted repeat at +67. These studies suggest that interactions between IE proteins and this octamer sequence may be important for the regulation and expression of this CMV gene.
The p53 complex from monkey cells modulates the biochemical activities of simian virus 40 large T antigen
Authors: Tack L C , Wright J H , Deb S P , Tegtmeyer P
Published: January 2020

We have compared the ATPase, DNA-binding, and helicase activities of free simian virus 40 (SV40) large T antigen (To) and T antigen complexed with cellular p53 (T+p53). Each activity is essential for productive viral infection. The T+p53 and To fractions were prepared by sequential immunosorption of infected monkey cells with monoclonal antibodies specific for p53 and T antigen. The immune-complexed T fractions were then assayed in parallel. For ATP hydrolysis, the Vmax for T+p53 was 143 nmol of ADP per min per mg of protein, or 18-fold greater than for To. ATP had no effect on the stability of the T+p53 complex. The T+p53 complex was significantly more active than To in hydrolyzing dATP, dGTP, GTP, and UTP. Of the nucleotide substrates tested, the greatest relative increase (T+p53/To) was in hydrolyzing dGTP and GTP. In DNase footprinting assays performed under replication conditions, the T+p53 complex protected regions I, II, and III of origin DNA while equivalent amounts of To protected only regions I and II. Region III is known to contribute to the efficiency of DNA replication and contains the SP1-binding sites of the early viral promoter. The T+p53 fraction was also a more efficient helicase than To, especially with a GC-rich primer and template. Thus, the T+p53 complex has enhanced ATPase, GTPase, DNA-binding, and helicase activities. These findings imply that complex formation between cellular monkey p53 and SV40 T antigen modulates a number of essential activities of T in SV40 productive infection.
Recombinant BALB and Harvey sarcoma viruses with normal proto-ras-coding regions transform embryo cells in culture and cause tumors in mice
Authors: Cichutek K , Duesberg P H
Published: January 2020

The ras genes of BALB and Harvey sarcoma viruses contain point mutations in codon 12 or codons 12 and 59, relative to proto-ras from normal animal and human cells. By in vitro recombination between cloned rat proto-ras and cloned BALB and Harvey sarcoma proviruses, we constructed recombinant proviruses with normal proto-ras-coding regions. These recombinant proviruses transformed mouse 3T3 cells upon transfection. However, when the transforming efficiencies of proviral DNAs were compared after transfection with helper provirus, recombinant proviruses were 2 to 30 times less efficient than the corresponding wild-type proviruses. Recombinant sarcoma viruses isolated from cells transformed by cloned proviral DNA contained the expected normal ras-coding region. They transformed rat embryo cells and induced erythroblastosis and sarcomas in newborn mice as efficiently as wild-type viruses did. We conclude that conversion of normal proto-ras genes to viral ras genes depends on truncation of normal proto-ras regulatory elements and substitution by retroviral (long terminal repeat) promoters and that the transforming function of long terminal repeat-ras genes is enhanced by point mutations.
Modulation of acute and latent herpes simplex virus infection in C57BL/6 mice by adoptive transfer of immune lymphocytes with cytolytic activity
Authors: Bonneau R H , Jennings S R
Published: January 2020

The ability of highly lytic herpes simplex virus (HSV) cytolytic T lymphocytes to modulate the interaction between the murine host (adult C57BL/6 [H-2b] mice) and HSV type 1 Patton resulting in acute infection in the footpad and latent infection in the sensory lumbosacral dorsal root ganglia (L6, L5, L4, and L3) innervating the footpad was investigated. Results indicated that a critical threshold level of infectious HSV was required to establish infection. The adoptive transfer of cytolytic T lymphocytes derived from in vitro cultures after restimulation with HSV-infected, syngeneic stimulator cells exhibiting class I H-2-restricted, L3T4- Lyt-2+ HSV-specific cytolytic activity immediately before infection with a high dose of HSV reduced the levels of infectious HSV recovered from the footpad tissue during acute infection and the levels of latent HSV reactivated from the dorsal root ganglia to levels expected from mice infected with a low dose. Depletion of Lyt-2+ cells from the transferred population abrogated the protective ability, while depletion of L3T4+ cells had little effect. These results suggest that functionally lytic HSV-specific cytolytic T lymphocytes present at the time of HSV infection have the potential to participate in the control of the acute infection and in the subsequent establishment of latent infection.
Resolution of vaccinia virus DNA concatemer junctions requires late-gene expression
Authors: Merchlinsky M , Moss B
Published: January 2020

Vaccinia virus replicates in the cytoplasm of infected cells, generating transient replicative intermediates containing the DNA for the terminal sequences as concatemeric junctions. The processing of the terminal sequences for a series of vaccinia virus conditional lethal mutants at the nonpermissive temperature was analyzed by restriction enzyme digestion and Southern blot hybridization of DNA isolated from infected cells. Three phenotypes were observed: DNA replication negative (Rep-), DNA replication positive but concatemer resolution negative (Rep+ Res-), and DNA replication positive and concatemer resolution positive (Rep+ Res+). Interestingly, all six Rep+ Res- mutants from separate complementation groups were defective in late protein synthesis. Isatin beta-thiosemicarbazone, a drug that blocks late protein synthesis, also prevented resolution of concatemers. Orthogonal field gel electrophoresis of the DNA generated by the late defective mutants revealed a distribution of linear genome multimers. The multimers were processed into mature monomers after a shift to the permissive temperature in the presence of cytosine arabinoside for all the Rep+ Res- mutants except ts22, an irreversible mutant which cleaves RNA late in infection (R.F. Pacha and R.C. Condit, J. Virol. 56:395-403, 1985). Genome formation can be divided into two stages: DNA replication, which generates concatemers, and resolution, which processes concatemers into monomers with hairpin termini. Early viral genes are required for the former, and late viral genes are required for the latter.
Genetic analysis of endogenous xenotropic murine leukemia viruses: association with two common mouse mutations and the viral restriction locus Fv-1
Authors: Frankel W N , Stoye J P , Taylor B A , Coffin J M
Published: January 2020

We have defined 40 endogenous xenotropic virus (Xmv) loci from several common inbred strains of mice by examining provirus-cell DNA junction fragments in recombinant inbred mice. Some inbred strains carried unique proviruses, but most Xmv loci were present in several strains, indicating that many Xmv integration events preexisted modern inbreeding. It was also clear that most Xmv junction fragment variation between inbred strains resulted from independent integration events and not modification or restriction site polymorphism following integration. Chromosomal assignments were determined for 32 Xmv loci by comparing their recombinant inbred strain distribution patterns to those of known genetic markers. The Xmv loci were generally dispersed throughout the genome, but several chromosomal regions contained more than one provirus. Furthermore, several close genetic associations with cellular genes were discovered. Four Xmv loci were closely linked to Fv-1b, a dominant viral resistance gene present in C57BL/6J, BALB/cJ, A/J, and several other strains. Xmv-28 was closely linked to rd (retinal degeneration), and Xmv-10 was closely linked to a (non-agouti), both of which are old mutations as inferred from their broad distribution in mice. We suggest that Xmv integration contributed to genetic diversity in the past and that much of this diversity exists today in common laboratory strains.
Modulation of lentivirus replication by antibodies: Fc portion of immunoglobulin molecule is essential for enhancement of binding, internalization, and neutralization of visna virus in macrophages
Authors: Jolly P E , Huso D L , Sheffer D , Narayan O
Published: January 2020

Antibodies to visna virus neutralized the virus in fibroblasts and macrophages but specifically enhanced the binding, penetration, and uncoating of the virus in the latter cells. F(ab')2 fragments of the immune antibody neutralized the virus in fibroblasts but did not enhance the early stages of the virus life cycle in macrophages. Furthermore, these fragments did not neutralize infectivity in macrophages but delayed the appearance of infectious virus in cells after the inoculation of preincubated virus-F(ab')2 complexes.
The Sendai virus nucleocapsid exists in at least four different helical states
Authors: Egelman E H , Wu S S , Amrein M , Portner A , Murti G
Published: January 2020

Sendai virus nucleocapsids have been observed by electron microscopy to coexist in three different helical pitch conformations, 5.3, 6.8, and 37.5 nm. The 5.3- and 6.8-nm conformations are present both in uranyl acetate negatively stained preparations and in tantalum-tungsten metal-shadowed preparations, whereas the 37.5-nm conformation, which has not been previously reported, is present only in the shadowed preparations. The 5.3-nm pitch conformation appears to be a mixture of two discrete structural states, with a small difference in the twist of the structure between the two. We have used image reconstruction techniques on an averaged data set from eight negatively stained nucleocapsids to produce a three-dimensional reconstruction at 2.4-nm resolution of the structure in one of the 5.3-nm pitch states. There are 13.07 nucleocapsid protein (NP) subunits in each turn of the helix in this state. The helical repeat is 79.5 nm, containing 196 subunits in 15 turns of the left-handed 5.3-nm helix. The arrangement of subunits produces a 5.0-nm-diameter hollow core which forms an internal helical groove. The RNA accounts for about 3% of the mass of the nucleocapsid, and so its location is not conspicuous in the reconstruction. Because of the RNA remains associated with the NP subunits during mRNA transcription and genome replication, structural transitions in the nucleocapsid may determine the accessibility of the genome to polymerases. Alternatively, the large hollow core and internal helical groove we have reconstructed may allow access to the RNA even in the tightly coiled 5.3-nm pitch conformation.
The cellular 68,000-Mr protein kinase is highly autophosphorylated and activated yet significantly degraded during poliovirus infection: implications for translational regulation
Authors: Black T L , Safer B , Hovanessian A , Katze M G
Published: January 2020

We investigated the possible translational regulatory roles played by the interferon-induced, double-stranded-RNA-activated protein kinase (P68) and its natural substrate, eucaryotic initiation factor 2 (eIF-2), in poliovirus-infected cells. We demonstrated that protein kinase P68 was both highly autophosphorylated and activated during poliovirus infection. In accordance with these results, immunoprecipitation analysis revealed that phosphorylation of the endogenous eIF-2 alpha subunit also increased in poliovirus-infected cells. We found that double-stranded RNA synthesized during infection likely induced the high levels of P68 autophosphorylation. To determine whether the increase in kinase activity also could be attributed to induction of P68 synthesis, physical levels of protein kinase were measured. It was unexpectedly found that P68 protein levels did not increase but rather dramatically declined in poliovirus-infected cells. Pulse-chase experiments confirmed that the protein kinase was significantly degraded during virus infection. We corroborated our in vivo observations by developing an in vitro assay for P68 degradation using cell extracts. The possible consequences of P68 degradation and increased eIF-2 alpha phosphorylation for protein synthesis regulation in poliovirus-infected cells are discussed.
Molecular localization of abortive infection of resident peritoneal macrophages by herpes simplex virus type 1
Authors: Morahan P S , Mama S , Anaraki F , Leary K
Published: January 2020

Mononuclear phagocytes exhibit different patterns of intrinsic resistance to herpes simplex virus type 1 (HSV-1) that are related to the heterogeneity of macrophage populations and may reflect the particular differentiation or maturation state of the macrophages. In this study, we characterized the molecular basis for the block in HSV-1 replication in resident peritoneal macrophages from B6C3F1 mice. Infected resident peritoneal macrophages were analyzed for the presence of virus-specific mRNA by Northern (RNA) blotting and in situ hybridization and for proteins by immunofluorescence. The data were compared with those obtained in HSV-1-infected permissive Vero cells. The immediate-early genes ICP4, ICP0, ICP22, and ICP27 were transcribed in resident peritoneal macrophages, as was the early gene tk. Virus-specific mRNA for the major DNA-binding protein ICP8 was barely detectable, and that for another early gene, the viral DNA polymerase, was not detected. In addition, transcripts for the delayed-early gene glycoprotein D and the true late gene glycoprotein C (gC) were not detectable in resident peritoneal macrophages. In situ hybridization and immunofluorescence studies confirmed that transcripts and proteins for the immediate-early and some early HSV-1 genes were present. These data also established that 14% of the resident peritoneal macrophages were positive for RNA and polypeptide specific for the immediate-early gene ICP4 and that 7 to 11% were positive for RNA or polypeptides specific for the early genes tk and ICP8. The fact that only a few cells expressed viral products emphasizes the heterogeneity that exists even in this relatively homogeneous resident peritoneal macrophage population. Consistent with the Northern blot analysis, no RNA specific for the early DNA polymerase gene or the late gC gene was detected by in situ hybridization nor could the polypeptide for the gC gene be seen by immunofluorescence. Thus, while early transcriptional events were initiated in some resident peritoneal macrophages, there was a block in replication localized at the level of expression of the early to delayed-early viral genes.
Evidence for the role of the P2 protein of human rhinovirus in its host range change
Authors: Lomax N B , Yin F H
Published: January 2020

Human rhinovirus 39 (HRV39) is blocked in nonpermissive L cells at both adsorption and intracellular replication steps. We have selected a host range variant of HRV39 capable of bypassing the intracellular replication block and found it to have altered nonstructural P2 proteins. These alterations are similar to those reported earlier for host range variants of HRV2 (F. H. Yin and N. B. Lomax, J. Virol. 48:410-418, 1983). This observation suggests that the intracellular replication block for both HRV2 and HRV39 in mouse L cells is at the same step. We propose that the P2 protein is an essential viral component that cannot function in mouse L cells unless altered. This alteration occurs spontaneously in stocks of HRV39 during growth in permissive HeLa cells.
Translational readthrough of the murine leukemia virus gag gene amber codon does not require virus-induced alteration of tRNA
Authors: Feng Y X , Hatfield D L , Rein A , Levin J G
Published: January 2020

An in vitro system to assay translational readthrough of the UAG termination codon at the murine leukemia virus (MuLV) gag-pol junction was developed by using rabbit reticulocyte lysates programmed by SP6-generated Moloney MuLV gag-pol mRNA. Under conditions in which the suppressor activity of the lysate was dependent on addition of tRNA, it could be shown that readthrough synthesis was stimulated to approximately the same extent by equivalent amounts of tRNA from MuLV-infected and uninfected NIH 3T3 cells. Analysis of glutamine tRNA, which mediates suppression in vivo, showed that the level of glutamine acceptor activity and the chromatographic profile of glutamine isoacceptors were unchanged following virus infection. On the basis of these results, we conclude that the suppressor tRNA occurs normally within the tRNA population of uninfected cells and need not be induced in response to virus infection.
Epitope mapping of neutralizing monoclonal antibodies against duck hepatitis B virus
Authors: Cheung R C , Robinson W S , Marion P L , Greenberg H B
Published: January 2020

In this article we report the first topological mapping of neutralizing epitopes of a hepadnavirus. Duck hepatitis B virus is the only hepadnavirus that can replicate and spread from cell to cell in tissue culture. As a result, it is possible to study hepadnaviral neutralization in vitro with this system. To accomplish this goal, we produced a library of monoclonal antibodies against duck hepatitis B virus and identified 12 neutralizing monoclonal antibodies by using an in vitro neutralization assay. The characteristics of six of the neutralizing monoclonal antibodies were further studied by epitope mapping. From the results of competitive binding studies, three distinct neutralizing epitopes were identified on the pre-S polypeptides and one was identified on the S polypeptide. Our findings suggest that antibodies to both the pre-S and S gene products of duck hepatitis B virus can neutralize viral infection in vitro. The pre-S gene product is at least as important as the S gene product in eliciting neutralizing antibodies.
Artificial cleavage site recognized by plum pox potyvirus protease in Escherichia coli
Authors: García J A , Riechmann J L , Laín S
Published: January 2020

A synthetic plum pox virus (PPV) NIb-CP cleavage site was recognized by a PPV protease in an in vivo Escherichia coli expression system. The presence of the natural NIb-CP cleavage site did not affect processing at the artificial one. However, although both the proteases and the cleavage sites of PPV and tobacco etch virus show high sequence homology, a similar cassette from the tobacco etch virus NIb-CP junction was not efficiently recognized by the PPV protease.
Cell-associated West Nile flavivirus is covered with E+pre-M protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release
Authors: Wengler G , Wengler G
Published: January 2020

Flaviviruses are enveloped viruses which accumulate in cellular vacuoles prior to release. The membrane of cell-associated virus contains the proteins pre-M and E. During release of virus the pre-M protein is cleaved, and only its carboxy-terminal segment remains associated with the virus as M protein. Studies of the association of membrane proteins of intracellular and extracellular particles of West Nile virus show that in cell-associated virus the pre-M and E proteins are present as E+pre-M heterodimers. Cleavage of pre-M during release leads to dissociation of the heterodimers: the amino-terminal region of the pre-M protein is lost from the virus, whereas the proteins M and E remain associated with the viral membrane as separate molecules. The E protein of extracellular virus has a tendency to oligomerize into trimers, and both E-protein monomers and trimers are present on extracellular virions. We have prepared partially purified extracellular virus without loss of viral infectivity. These preparations contain approximately 600 physical particles for each PFU. Since purification of cell-associated virus results in significant loss of PFU, an inactivation of virus may occur during this procedure. Preparations of cell-associated virus contained approximately 40,000 physical particles for each PFU.
Induction of immune response to influenza virus with anti-idiotypic antibodies
Authors: Anders E M , Kapaklis-Deliyannis G P , White D O
Published: January 2020

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against five monoclonal antibodies (MAbs) specific for different antigenic sites on the hemagglutinin (HA) of influenza virus Mem71H-BelN (H3N1) [A/Memphis/1/71 (H3N2) x A/Bel/42 (H1N1)]. Each of the anti-Id sera was directed predominantly towards a unique (private) idiotype of the immunizing MAb, none of the five idiotypes being detectable in pooled BALB/c antisera against Mem71H-BelN virus or on most other anti-HA MAbs tested. Partial idiotypic sharing was observed, however, between certain MAbs, from different mice, having the same or similar epitope specificity for HA. When used as immunogens in BALB/c mice, two of the five anti-Id preparations induced antibodies that reacted with Mem71H-BelN virus and displayed neutralizing activity. Mice of other inbred strains responded similarly, indicating that the response was not genetically restricted by the Igh locus. From their pattern of reactivity with mutants of Mem71H-BelN virus with known single amino acid substitutions in the HA molecule, the antiviral antibodies elicited by anti-Id antibodies were shown to be directed to the same antigenic site on A/Memphis/1/71 HA as the original immunizing MAb (site A or site E, respectively). However, several of these antisera were shown to contain additional distinct subpopulations of antibodies specific for heterologous influenza A virus strains, either of the H3 subtype or of a different HA subtype (H1 or H2). Since the induction of antibodies to HA of different subtypes is not a feature of the antibody response to influenza virus itself, their induction by anti-Id antibodies merits further investigation.
Transcription inhibition site on the M protein of vesicular stomatitis virus located by marker rescue of mutant tsO23(III) with M-gene expression vectors
Authors: Li Y , Luo L Z , Wagner R R
Published: January 2020

The matrix (M) protein of vesicular stomatitis virus serves as an endogenous inhibitor of viral transcription, a function missing or deficient in M proteins of temperature-sensitive (ts) mutants assigned to complementation group III. Previous studies with mutant tsO23(III) and vaccinia virus M-gene expression vectors revealed that the temperature-sensitive phenotype is due to a mutation leading to substitution of phenylalanine for leucine at amino acid III, whereas loss of the major antigenic determinant (epitope 1) of the mutant M protein results from the substitution of glutamic acid for the wild-type amino acid glycine at position 21 (Y. Li, L. Luo, R. M. Snyder, and R. R. Wagner, J. Virol. 62:3729-3737, 1988). We demonstrate here that transcription inhibition activity is restored to rescued tsO23 virus only when the rescuing vaccinia virus recombinant expresses M protein with glycine and not glutamic acid at amino acid 21. These experiments indicate the importance of the conformational integrity of the amino-terminal domain in determining the capacity of the vesicular stomatitis virus M protein to down regulate endogenous transcription.
Sequences in the visna virus long terminal repeat that control transcriptional activity and respond to viral trans-activation: involvement of AP-1 sites in basal activity and trans-activation
Authors: Hess J L , Small J A , Clements J E
Published: January 2020

Visna virus is a pathogenic lentivirus of sheep whose gene expression is developmentally regulated in cells of the monocyte-macrophage lineage. Gene expression directed by the visna virus long terminal repeat (LTR) is increased in infected cells by a virus-encoded trans-acting protein. trans-Activation is mediated in part by increases in the steady-state level of mRNA. Deletion and linker-scanner mutants were constructed to locate sequences in the LTR that regulate transcription and are responsive to viral trans-activation. The activities of these mutants were tested by using them to drive transcription of the bacterial gene for chloramphenicol acetyltransferase in transient expression assays. Three regions located between-140 and the cap site were found to be important for basal transcriptional activity, and the importance of each region was found to be dependent on the cell type. Sequences responsive to viral trans-activation were found to be the same sequences required for basal transcriptional activity. The visna virus LTR contains six sequences that are homologous to the recognition site for cellular transcriptional factor AP-1 and a single sequence homologous to the recognition site for transcriptional factor AP-4. Both of these classes of binding sites appear to be important for regulating the basal level of transcription of visna virus. The AP-1-binding site most proximal to the TATA box was found to be one target for viral trans-activation. The visna virus promoter was found to be activated by serum; this serum response has also been mapped to the AP-1-related sequences in the LTR.
Expression and purification of biologically active v-sis/platelet-derived growth factor B protein by using a baculovirus vector system
Authors: Giese N , May-Siroff M , LaRochelle W J , van Wyke Coelingh K , Aaronson S A
Published: January 2020

Malignant transformation induced by simian sarcoma virus is mediated by its v-sis protein, the monkey homolog of the platelet-derived growth factor (PDGF) B chain. By use of an appropriately engineered baculovirus expression vector, the v-sis protein was expressed in the insect cell line Spodoptera frugiperda (Sf9) at a level 50- to 100-fold higher than that observed with overexpression in mammalian-cell transfectants. The sis protein produced by Sf9 cells underwent processing similar to that observed in mammalian cells, including efficient disulfide-linked dimer formation. Moreover, the recombinant sis protein was capable of binding PDGF receptors and inducing DNA synthesis as efficiently as PDGF-B synthesized by mammalian cells. A significant fraction of sis protein was released from Sf9 cells, which made possible a one-step immunoaffinity purification to near homogeneity with a 40% recovery of biological activity. These results demonstrate that a protein whose normal processing requires both intrachain and interchain disulfide-bridge formation can be efficiently expressed in a biologically active form in insect cells by using a baculovirus vector system.