bluuebunny/crossref_metadata_2025 huggingface dataset:
Nascent human immunodeficiency virus type 1 reverse transcription occurs within an enveloped particle
Authors: Zhang H , Zhang Y , Spicer T , Henrard D , Poiesz B J
Published: January 2020

Although a small amount of viral DNA has been shown to be enclosed within human immunodeficiency virus type 1 (HIV-1) virions, the majority of full-length viral DNA is formed after this virus infects target cells. Hence, we undertook investigations to identify the physical characteristics of the HIV-1 replication unit during the early events of infection. In these studies, nascent viral DNA synthesis was found to occur between 15 and 30 min after purified, DNase-treated HIV-1 virions were added to HUT 78 cells. At 1 h postinfection, a large amount of strong-stop viral DNA and some first-strand viral DNA had been synthesized. Several lines of evidence, including purification, nuclease digestion, and immunoprecipitation, indicated that these nascent viral DNAs were located within particles containing components such as reverse transcriptase and p24gag and gp120env proteins and having physical characteristics similar to those of intact virions.
The Zif268 cellular transcription factor activates expression of the Epstein-Barr virus immediate-early BRLF1 promoter
Authors: Zalani S , Holley-Guthrie E , Kenney S
Published: January 2020

The Epstein-Barr virus immediate-early protein BZLF1 mediates the switch from latent to lytic infection. BZLF1 transcription can be derived from either the BZLF1 promoter or the BRLF1 promoter (Rp). Productive viral infection of EBV-infected B cells can be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, as well as cross-linking of surface immunoglobulin with antiimmunoglobulin antibody. Both TPA and antiimmunoglobulin antibody are known to activate expression of the cellular transcription factor Zif268 in B cells. In this study, we have examined the regulation of BZLF1 transcription by Zif268. We show that Rp (but not the BZLF1 promoter) is activated by Zif268. Bacterially synthesized Zif268 binds strongly to an upstream sequence in the Rp promoter (located from -131 to -123 relative to the start site) and more weakly to a proximal sequence (-49 to -40). Zif268 activation of Rp requires these two Zif268 binding sites. TPA treatment of B cells induces the expression of Zif268 protein, which binds to Rp. Furthermore, TPA activation of Rp requires the upstream Zif268 site. These findings indicate that Zif268 can activate a critical Epstein-Barr virus immediate-early promoter and, therefore, may play a key role in the regulation of viral latency.
The D1 and D12 subunits are both essential for the transcription termination factor activity of vaccinia virus capping enzyme
Authors: Luo Y , Mao X , Deng L , Cong P , Shuman S
Published: January 2020

Transcription termination by vaccinia virus RNA polymerase during synthesis of early mRNAs requires a virus-encoded termination factor (VTF). VTF is but one of many activities associated with the vaccinia virus mRNA capping enzyme, a heterodimer of 95- and 33-kDa subunits encoded by the D1 and D12 genes, respectively. Although the three catalytic domains involved in cap formation have been assigned to individual subunits or portions thereof, the structural requirements for VTF activity are unknown. We now report that both full-length subunits are required for transcription termination. The 844-amino acid D1 subunit by itself, which is fully active in triphosphatase and guanylyltransferase functions, has no demonstrable VTF activity in vitro. Neither does the D12 subunit by itself. The heterodimeric methyltransferase domain of D1 (residues 498 to 844) and D12 subunits also has no VTF activity. VTF is not affected by a K-to-M mutation of the guanylyltransferase active site at position 260 (K260M) that abolishes enzyme-GMP complex formation or by a H682A/Y683A double mutation of the D1 subunit, which abrogates methyltransferase activity. Thus, the structural requirements for termination are distinct from those for nucleotidyl transfer and methyl transfer.
Domains upstream of the protease (PR) in human immunodeficiency virus type 1 Gag-Pol influence PR autoprocessing
Authors: Zybarth G , Carter C
Published: January 2020

A critical step in the formation of infectious retroviral particles is the activation of the virally encoded protease (PR) and its release from the Gag-Pol precursor polyprotein. To identify factors that influence this step, the maturation of human immunodeficiency virus type 1 PR from various Gag-PR polyproteins was assayed in vitro by a using rabbit reticulocyte lysate as a coupled transcription-translation-autoprocessing system. Highly efficient autoprocessing was detected with polyproteins containing the viral nucleocapsid (NC) domain. In contrast, polyproteins consisting of only p6 and PR domains or containing a truncated NC domain exhibited no autoprocessing activity. Experiments designed to test the dimerization capability of short PR polyproteins revealed that precursors containing the NC domain exhibited very efficient homotypic protein-protein interactions while PR precursors consisting of only p6 and PR did not interact efficiently. The strong correlation between autoprocessing activity and PR polyprotein precursor dimerization suggests that NC and p6* domains play a role in PR activation by influencing the dimerization of the PR domain in the precursor.
The complement control protein homolog of herpesvirus saimiri regulates serum complement by inhibiting C3 convertase activity
Authors: Fodor W L , Rollins S A , Bianco-Caron S , Rother R P , Guilmette E R , Burton W V , Albrecht J C , Fleckenstein B , Squinto S P
Published: January 2020

The herpesvirus saimiri genome encodes a complement control protein homolog (CCPH). Stable mammalian cell transfectants expressing a recombinant transmembrane form of CCPH (mCCPH) or a 5'FLAG epitope-tagged mCCPH (5'FLAGmCCPH) conferred resistance to complement-mediated cell damage by inhibiting the lytic activity of human serum complement. The function of CCPH was further defined by showing that the mCCPH and the 5'FLAGmCCPH transfectants inhibited C3 convertase activity and effectively reduced cell surface deposition of the activated complement component, C3d.
The lef-3 gene of Autographa californica nuclear polyhedrosis virus encodes a single-stranded DNA-binding protein
Authors: Hang X , Dong W , Guarino L A
Published: January 2020

The Autographa californica nuclear polyhedrosis virus (AcNPV) replicates in the nuclei of infected cells and encodes several proteins required for viral DNA replication. As a first step in the functional characterization of viral replication proteins, we purified a single-stranded DNA-binding protein (SSB) from AcNPV-infected insect cells. Nuclear extracts were chromatographed on single-stranded DNA agarose columns. An abundant protein with an apparent molecular weight of 43,000 was eluted from the columns at 0.9 to 1.0 M NaCl. This protein was not evident in extracts prepared from control cells, suggesting that the SSB was encoded by the virus. SSB bound to single-stranded DNA in solution, and binding was nonspecific with respect to base sequence, as single-stranded vector DNA competed as efficiently as single-stranded DNA containing the AcNPV origin of DNA replication. Competition binding experiments indicated that SSB showed a preference for single-stranded DNA over double-stranded DNA. To determine whether SSB was encoded by the lef-3 gene of AcNPV, the lef-3 open reading frame was cloned under the control of the bacteriophage T7 promoter. Immunochemical analyses indicated that LEF-3 produced in bacteria or in rabbit reticulocyte lysates specifically reacted with antiserum produced by immunization with purified SSB. Immunoblot analyses of infected cell extracts revealed that SSB/LEF-3 was detected by 4 h postinfection and accumulated through 48 h postinfection.
Brain- and intestine-specific variants of reovirus serotype 3 strain dearing are selected during chronic infection of severe combined immunodeficient mice
Authors: Haller B L , Barkon M L , Li X Y , Hu W M , Wetzel J D , Dermody T S , Virgin H W
Published: January 2020

Mutants of mammalian reoviruses, enteric double-stranded-RNA-containing viruses that spread systemically after primary replication in intestinal tissue, have been extensively studied as models of viral pathogenesis. While reovirus serotype 3 strain Dearing (T3D) causes acute encephalitis in newborn mice, adult severe combined immunodeficient (SCID) mice develop chronic infection with T3D, with some mice living more than 100 days after infection (B. L. Haller, M. L. Barkon, G. P. Vogler, and H. W. Virgin IV, J. Virol. 69:357-364, 1995). To determine whether organ-specific reovirus variants are selected during chronic infection, we characterized the pathogenetic properties of two variants of T3D isolated 87 days after intraperitoneal infection of adult SCID mice. A brain-specific variant (T3DvBr) (i) grew to a higher titer than T3D in SCID mouse brain (but not intestine) after intraperitoneal inoculation, (ii) killed adult SCID mice faster than T3D, and (iii) grew well in neonatal NIH Swiss [NIH(s)] mouse brain tissue after intramuscular but not peroral inoculation. An intestine-specific variant (T3DvInt) (i) grew to a higher titer than T3D in SCID mouse intestine (but not brain) after intraperitoneal inoculation, (ii) killed SCID mice with kinetics equivalent to those of T3D, (iii) was much less virulent than T3D in neonatal NIH(s) mice, (iv) grew better than T3D in intestines after intramuscular or peroral inoculation into neonatal NIH(s) mice, and (v) grew poorly in brain tissue of neonatal NIH(s) mice after intramuscular inoculation. During prolonged infection of SCID mice, organ-specific variants of T3D, which are more efficient than wild-type T3D at one specific stage in reovirus pathogenesis, are selected.
Inactivation of the retinoblastoma susceptibility protein is not sufficient for the transforming function of the conserved region 2-like domain of simian virus 40 large T antigen
Authors: Christensen J B , Imperiale M J
Published: January 2020

Simian virus 40 large T antigen interacts with three cellular proteins, pRb, p107, and p130, through a common binding site on the T antigen protein called the E1A conserved region 2-like (CR2-like) domain. Mutations in this domain inactivate the transforming activity of large T antigen. Since these mutations have been demonstrated to abolish binding to pRb and p107, and presumably therefore affect binding to p130, assessment of the relative roles of these three proteins in transformation of rodent fibroblasts by T antigen has been difficult. We have examined the role of T antigen-pRb interactions in transformation. We have introduced a mutant T antigen, which is unable to bind any of these three proteins, into primary mouse fibroblasts derived from the embryos of mice in which the Rb gene encoding the retinoblastoma protein had been disrupted. This mutant is unable to transform the Rb-negative fibroblasts, indicating that inactivation of pRb is not the sole function of the CR2-like domain in the induction of transformation of mouse fibroblasts by simian virus 40.
The enhancer in the long control region of human papillomavirus type 16 is up-regulated by PEF-1 and down-regulated by Oct-1
Authors: Sibbet G J , Cuthill S , Campo M S
Published: January 2020

The minimal enhancer in the long control region of human papillomavirus type 16 regulates cell type and constitutive expression from the promoter P97. This region contains at least four DNase I footprints (fp4e, fp5e, fp6e, and fp7e). We have shown that fp5e is crucial to enhancer function and have described an apparently novel factor (PEF-1) binding fp5e (S. Cuthill, G. J. Sibbet, and M. S. Campo, Mol. Carcinog. 8:9-104, 1993). Further analyses reveal that Oct-1 or an Oct-related factor binds fp5e at a site overlapping that of PEF-1. The binding of Oct-1 to fp5e has been demonstrated by electrophoretic mobility shift assays, by oligonucleotide competition studies, and by using an Oct-1-specific anti-POU serum. The location of the Oct-1 site has been confirmed by a panel of mutants across fp5e. Mutations that block PEF-1 binding to fp5e also block enhancer/promoter activity of the long control region, whereas mutations that block Oct-1 binding significantly increase enhancer/promoter activity. Thus, although both PEF-1 and Oct-1 interact with fp5e, they appear to regulate human papillomavirus expression in opposite ways.
Pseudotype virions formed between mouse hepatitis virus and lactate dehydrogenase-elevating virus (LDV) mediate LDV replication in cells resistant to infection by LDV virions
Authors: Even C , Plagemann P G
Published: January 2020

Infection of cultures of peritoneal macrophages with both lactate dehydrogenase-elevating virus (LDV) and mouse hepatitis virus (MHV) resulted in the formation of pseudotype virions containing LDV RNA which productively infected cells that are resistant to infection by intact LDV virions but not to infection by MHV. These cells were mouse L-2 and 3T3-17Cl-1 cells as well as residual peritoneal macrophages from persistently LDV-infected mice. Productive LDV infection of these cells via pseudotype virions was inhibited by antibodies to the MHV spike protein or to the MHV receptor, indicating that LDV RNA entered the cells via particles containing the MHV envelope. Simultaneous exposure of L-2 cells to both LDV and MHV resulted in infection by MHV but not by LDV. The results indicate that an internal block to LDV replication is not the cause of the LDV nonpermissiveness of many cell types, including the majority of the macrophages in an adult mouse. Instead, LDV permissiveness is restricted to a subpopulation of mouse macrophages because only these cells possess a surface component that acts as an LDV receptor.
Region E3 of subgroup B human adenoviruses encodes a 16-kilodalton membrane protein that may be a distant analog of the E3-6.7K protein of subgroup C adenoviruses
Authors: Hawkins L K , Wilson-Rawls J , Wold W S
Published: January 2020

There is an open reading frame in the E3 transcription unit of adenovirus type 3 (Ad3) and Ad7 that could encode a protein of 16 kDa (16K protein). Ad3 and Ad7 are members of subgroup B of human adenoviruses. Using a rabbit antipeptide antiserum, we show that the 16K protein is expressed in Ad3- and Ad7-infected cells at early and late stages of infection; it is not expressed in cells infected with an Ad7 mutant that deletes the 16K gene. The 16K protein was also transcribed and translated in vitro from DNA containing the open reading frame for the 16K protein. The 16K protein has two hydrophobic domains typical of integral membrane proteins; consistent with this, we detected 16K in the crude membrane but not the cytosol cellular fractions. Although 16K has two potential sites for Asn-linked glycosylation, the protein is not glycosylated. The 16K gene is located in the same position in region E3 as the gene for the 6.7K protein of subgroup C adenoviruses (Ad2 and Ad5). E3-6.7K is an Asn-linked integral membrane glycoprotein, localized in the endoplasmic reticulum, whose function is unknown. The 16K protein has a putative transmembrane domain located in the same place in 16K as is the transmembrane domain in 6.7K, and the C-terminal portion of 16K is partially homologous to the C-terminal cytoplasmic domain of 6.7K; we suggest that these domains in 16K and 6.7K may have a similar function. The N-terminal 102 residues in 16K are not found in 6.7K; these residues may have a function that is unique to the 16K protein. In common with all known E3 proteins, the 16K protein is dispensable for virus replication in cultured cells; this suggests that the 16K protein may function in virus-host interactions.
The C-terminal 25 amino acids of the protease and its substrate ICP35 of herpes simplex virus type 1 are involved in the formation of sealed capsids
Authors: Matusick-Kumar L , Newcomb W W , Brown J C , McCann P J , Hurlburt W , Weinheimer S P , Gao M
Published: January 2020

The herpes simplex virus type 1 protease and its substrate, ICP35, are involved in the assembly of viral capsids. Both proteins are encoded by a single open reading frame from overlapping mRNAs. The protease is autoproteolytically processed at two sites. The protease cleaves itself at the C-terminal site (maturation site) and also cleaves ICP35 at an identical site, releasing a 25-amino-acid (aa) peptide from each protein. To determine whether these 25 aa play a role in capsid assembly, we constructed a mutant virus expressing only Prb, the protease without the C-terminal 25 aa. Phenotypic analysis of the Prb virus in the presence and absence of ICP35 shows the following: (i) Prb retains the functional activity of the wild-type protease which supports virus growth in the presence of ICP35; (ii) in contrast to the ICP35 null mutant delta ICP35 virus, the Prb virus fails to grow in the absence of ICP35; and (iii) trans-complementation experiments indicated that full-length ICP35 (ICP35 c,d), but not the cleaved form (ICP35 e,f), complements the growth of the Prb virus. The most striking phenotype of the Prb virus is that only unsealed aberrant capsid structures are observed by electron microscopy in mutant-infected Vero cells. Our results demonstrate that the growth of herpes simplex virus type 1 requires the C-terminal 25 aa of either the protease or its substrate, ICP35, and that the C-terminal 25 aa are involved in the formation of sealed capsids.
RNA footprint mapping of RNA polymerase II molecules stalled in the intergenic region of polyomavirus DNA
Authors: Brabant F , Acheson N H
Published: January 2020

RNA polymerase II molecules that transcribe the late strand of the 5.3-kb circular polyomavirus genome stall just upstream of the DNA replication origin, in a region containing multiple binding sites for polyomavirus large T antigen. Stalling of RNA polymerases depends on the presence of functional large T antigen and on the integrity of large T antigen binding site A. To gain insight into the interaction between DNA-bound large T antigen and RNA polymerase II, we mapped the position of stalled RNA polymerases by analyzing nascent RNA chains associated with these polymerases. Elongation of RNA in vitro, followed by hybridization with a nested set of DNA fragments extending progressively farther into the stalling region, allowed localization of the 3' end of the nascent RNA to a position 5 to 10 nucleotides upstream of binding site A. Ribonuclease treatment of nascent RNAs on viral transcription complexes, followed by in vitro elongation and hybridization, allowed localization of the distal end of stalled RNA polymerases to a position 40 nucleotides upstream of binding site A. This RNA footprint shows that elongating RNA polymerases stall at a site very close to the position of DNA-bound large T antigen and that they protect approximately 30 nucleotides of nascent RNA against ribonuclease digestion.
Processing and evolution of the N-terminal region of the arterivirus replicase ORF1a protein: identification of two papainlike cysteine proteases
Authors: den Boon J A , Faaberg K S , Meulenberg J J , Wassenaar A L , Plagemann P G , Gorbalenya A E , Snijder E J
Published: January 2020

Two adjacent papainlike cysteine protease (PCP) domains, PCP alpha and PCP beta, were identified in the N-terminal region of the open reading frame 1a replicase proteins of the arteriviruses porcine reproductive and respiratory syndrome virus and lactate dehydrogenase-elevating virus. The replicase of the related virus equine arteritis virus contains only one active PCP in the corresponding region. Sequence comparison revealed that the equine arteritis virus PCP alpha counterpart probably was inactivated by loss of its catalytic Cys residue. For both porcine reproductive and respiratory syndrome virus and lactate dehydrogenase-elevating virus, the generation of two processing products, nsp1 alpha and nsp1 beta, was demonstrated by in vitro translation. Site-directed mutagenesis and sequence comparison were used to identify the putative active-site residues of the PCP alpha and PCP beta protease domains and to show that they mediate the nsp1 alpha/1 beta and nsp1 beta/2 cleavages, respectively.
Enhanced virus resistance of transgenic mice expressing the human MxA protein
Authors: Pavlovic J , Arzet H A , Hefti H P , Frese M , Rost D , Ernst B , Kolb E , Staeheli P , Haller O
Published: January 2020

MxA is a GTPase that accumulates to high levels in the cytoplasm of interferon-treated human cells. Expression of MxA cDNA confers to transfected cell lines a high degree of resistance against several RNA viruses, including influenza, measles, vesicular stomatitis, and Thogoto viruses. We have now generated transgenic mice that express MxA cDNA in the brain and other organs under the control of a constitutive promoter. Embryonic fibroblasts derived from the transgenic mice were nonpermissive for Thogoto virus and showed reduced susceptibility for influenza A and vesicular stomatitis viruses. The transgenic animals survived challenges with high doses of Thogoto virus by the intracerebral or intraperitoneal route. Furthermore, the transgenic mice were more resistant than their nontransgenic littermates to intracerebral infections with influenza A and vesicular stomatitis viruses. These results demonstrate that MxA is a powerful antiviral agent in vivo, indicating that it may protect humans from the deleterious effects of infections with certain viral pathogens.
Sequences derived from the highly antigenic VP1 region 140 to 160 of foot-and-mouth disease virus do not prime for a bovine T-cell response against intact virus
Authors: van Lierop M J , Wagenaar J P , van Noort J M , Hensen E J
Published: January 2020

Although VP1 region 140 to 160 of foot-and-mouth disease virus (FMDV) is able to elicit neutralizing antibody in cattle, the protection against virus challenge that is conferred by peptide immunization is often poor. Here, we show that bovine T cells primed with peptides derived from this region generally show no reactivity to intact FMDV. In contrast, T-cell epitope VP4[20-34] is able to prime for a virus-specific response.
Proteins and cis-acting elements associated with transactivation of the varicella-zoster virus (VZV) immediate-early gene 62 promoter by VZV open reading frame 10 protein
Authors: Moriuchi H , Moriuchi M , Cohen J I
Published: January 2020

Varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein, the homolog of herpes simplex virus type 1 (HSV-1) VP16, is a virion-associated transactivator of the VZV immediate-early (IE) gene 62 (IE62) promoter. VP16 forms a complex with cellular factors (Oct1 and host cell factor [HCF]) and TAATGARAT elements (found in all HSV-1 IE promoter/enhancer sequences) to mediate stimulation of IE transcription. The VZV IE62 promoter also contains three TAATGARAT-like elements. Mutagenesis studies of the VZV IE62 promoter indicated that TAATGARAT-like elements contribute to transactivation of the VZV IE62 promoter by ORF10 protein. Other cis-acting elements such as GA-rich and cyclic AMP-responsive elements were also needed for full transactivation by ORF10 protein. In mobility shift assays, ORF10 protein formed a complex with either of two TAATGARAT-like elements that lack an overlapping octamer-binding motif (octa-/TAATGARAT) but not with a TAATGARAT element with an overlapping octamer-binding motif (octa+/TAATGARAT). In contrast, VP16 formed a high-affinity ternary complex with an octa+/TAATGARAT element and a low-affinity complex with octa-/TAATGARAT elements. Addition of antibodies to ORF10 protein, Oct1, or HCF disrupted the complexes, demonstrating that ORF10 protein interacts with Oct1 and HCF. These results suggest that transactivation of the VZV IE62 gene by ORF10 protein and HSV IE genes by VP16 require similar cellular proteins but distinct cis-acting elements.
Transgenic mice support replication of hepatitis delta virus RNA in multiple tissues, particularly in skeletal muscle
Authors: Polo J M , Jeng K S , Lim B , Govindarajan S , Hofman F , Sangiorgi F , Lai M M
Published: January 2020

Hepatitis delta virus (HDV) is hepatotropic and frequently causes fulminant hepatitis in both human and nonhuman primate hosts. To understand the molecular basis of HDV tissue tropism and the mechanism of pathogenesis, transgenic mice in which replication-competent HDV dimeric RNA is expressed under the control of either liver-specific or universal transcriptional promoters were developed. The expressed RNA replicated efficiently in the liver and several tissues of nonhepatic origin. Surprisingly, maximal replication of HDV RNA occurred in skeletal muscle and was almost 100-fold greater than in the liver. These findings suggest that the hepatotropism of HDV is most likely a receptor-mediated restriction and that muscle-specific factors may facilitate HDV RNA replication. No evidence of cytopathology was apparent in most of the tissues examined, including the liver, supporting the contention that hepatocellular disease is not mediated by direct cytopathological effects associated with HDV RNA replication and gene expression. However, mild muscle atrophy in some of the transgenic mice was noted. Delta antigen was detected in the nuclei of myocytes. Only the small form, not the large form, of delta antigen was detected, suggesting that the RNA editing event which causes the conversion of delta antigen did not occur in transgenic mice. Furthermore, the 0.8-kb antigenomic RNA species, which is postulated to be the mRNA for delta antigen, was not detected in mice. The preferential replication of HDV RNA in skeletal muscle suggests that HDV RNA replication can be facilitated by certain muscle-specific factors.
Sequential interactions of structural proteins in phage phi 29 procapsid assembly
Authors: Lee C S , Guo P
Published: January 2020

The mechanism of viral capsid assembly is an intriguing problem because of its fundamental importance to research on synthetic viral particle vaccines, gene delivery systems, antiviral drugs, chimeric viruses displaying antigens or ligands, and the study of macromolecular interactions. The genes coding for the scaffolding (gp7), capsid (gp8), and portal vertex (gp10) proteins of the procapsid of bacteriophage phi 29 of Bacillus subtilis were expressed in Escherichia coli individually or in combination to study the mechanism of phi 29 procapsid assembly. When expressed alone, gp7 existed as a soluble monomer, gp8 aggregated into inclusion bodies, and gp10 formed the portal vertex. Circular dichroisin spectrum analysis indicated that gp7 is mainly composed of alpha helices. When two of the proteins were coexpressed, gp7 and gp8 assembled into procapsid-like particles with variable sizes and shapes, gp7 and gp10 formed unstable complexes, and gp8 and gp10 did not interact. These results suggested that gp7 served as a bridge for gp8 and gp10. When gp7, gp8, and gp10 were coexpressed, active procapsids were produced. Complementation of extracts containing one or two structural components could not produce active procapsids, indicating that no stable intermediates were formed. A dimeric gp7 concatemer promoted the solubility of gp8 but was inactive in the assembly of procapsid or procapsid-like particles. Mutation at the C terminus of gp7 prevented it from interacting with gp8, indicating that this part of gp7 may be important for interaction with gp8. Coexpression of the portal protein (gp20) of phage T4 with phi 29 gp7 and gp8 revealed the lack of interaction between T4 gp20 and phi 29 gp7 and/or gp8. Perturbing the ratio of the three structural proteins by duplicating one or another gene did not reduce the yield of potentially infectious particles. Changing of the order of gene arrangement in plasmids did not affect the formation of active procapsids significantly. These results indicate that phi 29 procapsid assembly deviated from the single-assembly pathway and that coexistence of all three components with a threshold concentration was required for procapsid assembly. The trimolecular interaction was so rapid that no true intermediates could be isolated. This finding is in accord with the result of capsid assembly obtained by the equilibrium model proposed by A. Zlotnick (J. Mol. Biol. 241:59-67, 1994).
Association of mouse fibrinogen-like protein with murine hepatitis virus-induced prothrombinase activity
Authors: Parr R L , Fung L , Reneker J , Myers-Mason N , Leibowitz J L , Levy G
Published: January 2020

Previously, we demonstrated induction of a unique macrophage prothrombinase during infection of BALB/cJ mice by mouse hepatitis virus strain 3 (MHV-3). By immunologic screening, a clone representing this prothrombinase was isolated from a cDNA library and sequenced. The sequence identified this clone as representing part of a gene, musfiblp, that encodes a fibrinogen-like protein. Six additional clones were isolated, and one clone, p11-3-1, encompassed the entire coding region of musfiblp. Murine macrophages did not constitutively express musfiblp but, when infected with MHV-3, synthesized musfiblp-specific mRNA. musfiblp mRNA induction was earlier and significantly greater in BALB/cJ than A/J macrophages. Prothrombinase activity was demonstrated when musfiblp was expressed from p11-3-1 in RAW 264.7 cells. These data suggest that musfiblp encodes the MHV-induced prothrombinase.
Transactivation of the human T-cell lymphotropic virus type 1 Tax1-responsive 21-base-pair repeats requires Holo-TFIID and TFIIA
Authors: Duvall J F , Kashanchi F , Cvekl A , Radonovich M F , Piras G , Brady J N
Published: January 2020

The human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent for adult T-cell leukemia and tropical spastic paraparesis/HTLV-1-associated myelopathy. The HTLV-1 Tax1 gene product has been shown to transactivate transcription of viral and cellular promoters. To examine the biochemical mechanism of Tax1 transactivation, we have developed an in vitro transactivation assay in which wild-type Tax1 is able to specifically transactivate a polymerase II promoter through upstream Tax1-responsive elements. The in vitro system utilizes the HTLV-1 21-bp repeats cloned upstream of the ovalbumin promoter and G-free cassette. Purified Tax1 specifically transactivates this template 5- to 10-fold in a concentration-dependent manner. No transactivation of the ovalbumin promoter (pLovTATA) template control was observed. Tax1 transactivation was inhibited by low concentrations of alpha-amanitin and was effectively neutralized by anti-Tax1 but not control sera. Consistent with in vivo transactivating activity, Tax1 NF-kappa B mutant M22, but not cyclic AMP-responsive element-binding protein mutant M47, transactivated the template containing the tandem 21-bp repeat. In a reconstituted in vitro transcription assay, Tax1 transactivation was dependent upon basal transcription factors TFIIB, TFIIF, Pol II, TFIID, and TFIIA. TATA-binding protein did not functionally substitute for TFIID in the transactivation assay by Tax1 but was sufficient for basal transcription. Finally, we have used anti-TFIIA antibody (p55) to ask if Tax1 transactivation required TFIIA activity. Addition of TFIIA antibody to in vitro transcription reactions, as well as depletion of TFIIA by preclearing with antibody, showed that TFIIA was required for Tax1 transactivation. Only a slight (twofold) drop of basal transcription was observed. Tax1 transactivation was restored when purified HeLa TFIIA was added back into the reconstituted system. We propose that Tax1 utilizes a transactivation pathway involving the activator regulated basal transcription factors TFIID and TFIIA.
Viral determinants of the variable sensitivity of herpes simplex virus strains to gD-mediated interference
Authors: Dean H J , Warner M S , Terhune S S , Johnson R M , Spear P G
Published: January 2020

Cells that express glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) resist infection by HSV-1 and HSV-2 because of interference with viral penetration. The results presented here show that both HSV-1 and HSV-2 gD can mediate interference and that various HSV-1 and HSV-2 strains differ in sensitivity to this interference. The relative degree of sensitivity was not necessarily dependent on whether the cell expressed the heterologous or homologous form of gD but rather on the properties of the virus. Marker transfer experiments revealed that the allele of gD expressed by the virus was a major determinant of sensitivity to interference. Amino acid substitutions in the most distal part of the gD ectodomain had a major effect, but substitutions solely in the cytoplasmic domain also influenced sensitivity to interference. In addition, evidence was obtained that another viral gene(s) in addition to the one encoding gD can influence sensitivity to interference. The results indicate that HSV-1 and HSV-2 gD share determinants required to mediate interference with infection by HSV of either serotype and that the pathway of HSV entry that is blocked by expression of cell-associated gD can be cleared or bypassed through subtle alterations in virion-associated proteins, particularly gD.
Human cytomegalovirus upregulates NF-kappa B activity by transactivating the NF-kappa B p105/p50 and p65 promoters
Authors: Yurochko A D , Kowalik T F , Huong S M , Huang E S
Published: January 2020

During human cytomegalovirus (HCMV) infection, a series of regulated events take place following virus binding and entry into the cell, including the upregulation of cellular transcription factors, such as NF-kappa B, which play an essential role in the viral life cycle. We show here that NF-kappa B message is induced during HCMV infection and that the induction is biphasic, suggesting an initial induction at immediate-early (IE) times and a second round of induction at early times. This hypothesis is supported by experiments using cyclohexamide, which showed that the first tier of induction was drug insensitive, while the second tier was drug sensitive. We then show that virus binding alone is sufficient to stimulate NF-kappa DNA binding activity, supporting its role in the initial induction of NF-kappa B. To begin to elucidate the mechanism(s) for the second tier of NF-kappa B regulation, we examined promoter constructs from the NF-kappa B subunits (p105/p50 and p65) for responsiveness following HCMV infection. HCMV infection transactivated the p105/p50 and p65 promoters. The viral IE proteins (IE1-72, IE2-55, and IE2-86) are expressed during the time we see NF-kappa B induction, so we examined their role in NF-kappa B induction. The IE1-72, IE2-55, and IE2-86 proteins transactivated the p65 promoter, while only the IE2-55 protein transactivated the p105/p50 promoter. The p105/p50 promoter has NF-kappa B sites; therefore, upregulation could also be caused by an autoregulatory mechanism. The p65 promoter, however, has been demonstrated to contain only Sp1 sites. To investigate the potential role of SP1, we examined nuclear extracts from HCMV-infected cells. Here, we show that there is a biphasic increase in SP1 activity during viral infection and that there is apparently an absolute requirement for SP1 in the transactivation of the p65 promoter. In conclusion, we suggest a model in which the initial induction of NF-kappa B occurs through viral modulation of cellular factors and the sustained levels of NF-kappa B induction are regulated by a combination of cellular and viral factors.
Upstream CREs participate in the basal activity of minute virus of mice promoter P4 and in its stimulation in ras-transformed cells
Authors: Perros M , Deleu L , Vanacker J M , Kherrouche Z , Spruyt N , Faisst S , Rommelaere J
Published: January 2020

The activity of the P4 promoter of the parvovirus minute virus of mice (prototype strain MVMp) is stimulated in ras-transformed FREJ4 cells compared with the parental FR3T3 line. This activation may participate in the oncolytic effect of parvoviruses, given that P4 drives a transcriptional unit encoding cytotoxic nonstructural proteins. Our results suggest that the higher transcriptional activity of promoter P4 in FREJ4 cells is mediated at least in part by upstream CRE elements. Accordingly, mutations in the CRE motifs impair P4 function more strongly in the FREJ4 derivative than in its FR3T3 parent. Further evidence that these elements contribute to hyperactivity of the P4 promoter in the ras transformant is the fact that they form distinct complexes with proteins from FREJ4 and FR3T3 cell extracts. This difference can be abolished by treating the FREJ4 cell extracts with cyclic AMP-dependent protein kinase (PKA) or treating original cultures with a PKA activator. These findings can be linked with two previously reported features of ras-transformed cells: the activation of a PKA-inhibited protein kinase cascade and the reduction of PKA-induced protein phosphorylation. In keeping with these facts, P4-directed gene expression can be up- or downmodulated in vivo by exposing cells to known inhibitors or activators of PKA, respectively.
Differential antigen burden modulates the gamma interferon but not the immunoglobulin response in mice that vary in susceptibility to Sendai virus pneumonia
Authors: Mo X Y , Sangster M , Sarawar S , Coleclough C , Doherty P C
Published: January 2020

Sendai virus, a paramyxovirus which causes murine pneumonia, grew to approximately 10-fold higher titers and was cleared less rapidly from the lungs of 129/J (129) than H-2b-compatible C57BL/6J (B6) mice. The more susceptible 129 mice also made higher titers of gamma interferon (IFN-gamma) and immunoglobulin G2a (IgG2a) virus-specific antibody. Analysis with acutely irradiated (950 rads) mice and immunologically reconstituted bone marrow (BM) radiation chimeras indicated that the enhanced virus growth was a function of the radiation-resistant respiratory epithelium. Prolonged exposure to more virus in turn influenced the magnitude of IFN-gamma production, most of which was made by CD4+ T lymphocytes. Somewhat surprisingly, however, the 129 pattern of a higher virus-specific serum Ig response skewed towards IgG2a mapped to the reconstituting BM. Thus, the characteristics of the humoral response are at least partly dissociated from both the antigen load, resulting from viral replication, and the level of IFN-gamma production. Further analysis of double chimeras (B6+129 BM-->B6 recipients) confirmed that the divergent humoral immune response to Sendai virus in B6 and 129 mice is largely determined by the inherent characteristics of the lymphoid cells.
Synthesis and secretion of recombinant tick-borne encephalitis virus protein E in soluble and particulate form
Authors: Allison S L , Stadler K , Mandl C W , Kunz C , Heinz F X
Published: January 2020

A quantitative study was performed to investigate the requirements for secretion of recombinant soluble and particulate forms of the envelope glycoprotein E of tick-borne encephalitis (TBE) virus. Full-length E and a carboxy terminally truncated anchor-free form were expressed in COS cells in the presence and absence of prM, the precursor of the viral membrane protein M. Formation of a heteromeric complex with prM was found to be necessary for efficient secretion of both forms of E, whereas only low levels of anchor-free E were secreted in the absence of prM. The prM-mediated transport function could also be provided by coexpression of prM and E from separate constructs, but a prM-to-E ratio of greater than 1:1 did not further enhance secretion. Full-length E formed stable intracellular heterodimers with prM and was secreted as a subviral particle, whereas anchor-free E was not associated with particles and formed a less stable complex with prM, suggesting that prM interacts with both the ectodomain and anchor region of E.
Forms of Deoxyribonucleic Acid Produced by Virions of the Ribonucleic Acid Tumor Viruses
Authors: Manly Kenneth F. , Smoler Donna F. , Bromfeld Esther , Baltimore David
Published: January 2020

The in vitro product of mouse leukemia virus deoxyribonucleic acid (DNA) polymerase can be separated into two fractions by sedimentation in sucrose gradients. These two fractions were analyzed for their content of single-stranded DNA, double-stranded DNA, and DNA-ribonucleic acid (RNA) hybrid by (i) digestion with enzymes of known specificity and (ii) equilibrium centrifugation in Cs
2
SO
4
gradients. The major fraction early in the reaction contained equal amounts of single-stranded DNA and DNA-RNA hybrid and little double-stranded DNA. The major fraction after extensive synthesis contained equal amounts of single-and double-stranded DNA and little hybrid. In the presence of actinomycin D, the predominant product was single-stranded DNA. To account for these various forms of DNA, we postulate the following model: the first DNA synthesis occurs in a replicative complex containing growing DNA molecules attached to an RNA molecule. Each DNA molecule is displaced as single-stranded DNA by the synthesis of the following DNA strand, and the single-stranded DNA is copied to form double-stranded DNA either before or after release of the single strand from the RNA. Actinomycin blocks this conversion of single-to double-stranded DNA.
Biophysical and Biochemical Studies on Rhinovirus and Poliovirus II. Chemical and Hydrodynamic Analysis of the Rhinovirion
Authors: McGregor Sandy , Mayor Heather D.
Published: January 2020

Chemical analysis of rhinovirus 14 revealed a ribonucleic acid (RNA) content of 29.8% and a high adenylic acid content (35%). A partial specific volume of 0.682 cm
3
/g was obtained for the rhinovirion. Rhinovirus and poliovirus had identical sedimentation coefficients of 158
S.
A diffusion coefficient of 1.71 × 10
−7
cm
2
/sec was consistent with a hydrated diameter of 25 nm for the rhinovirion. The calculated molecular weights of the rhinovirion and its genome were 7.1 × 10
6
and 2.1 × 10
6
daltons, respectively. Sedimentation analysis of infectious RNA confirmed the similarity of the molecular size of the poliovirus and rhinovirus genomes.
Lipid Composition of Purified Vesicular Stomatitis Viruses
Authors: McSharry James J. , Wagner Robert R.
Published: January 2020

Methods are described for the production of vesicular stomatitis (VS) virus of sufficient purity for reliable chemical analysis. VS virions released from infected cells were concentrated and purified at least 150-fold by sequential steps of precipitation with polyethylene glycol, column chromatography, rate zonal centrifugation, and equilibrium centrifugation. The Indiana serotype (VS
Ind
virus) propagated in L-cells was found to contain 3% ribonucleic acid, 64% protein, 13% carbohydrate, and 20% lipid; the molar ratio of cholesterol to phospholipid was 0.6 or greater. Thin-layer chromatography revealed no unusual neutral lipids or phospholipids and gas-liquid chromatography revealed no unusual fatty acids incorporated into VS virions. The antigenically distinct New Jersey serotype (VS
NJ
virus) grown in L-cells showed a similar lipid profile except that the proportion of neutral lipids was larger than in VS
Ind
virus also grown in L-cells. This differences was less pronounced when the lipid composition of VS
Ind
and VS
NJ
viruses grown in chick embryo cells was compared, but VS
NJ
virus grown in either cell type always contained larger amounts of neutral lipids other than cholesterol than did VS
Ind
virus. The lipid composition of both VS
Ind
and VS
NJ
viruses grown in L-cells or chick embryo cells more closely resembled that of plasma membrane than of whole cells. A consistent finding was the relatively large amounts of phosphatidylethanolamine and sphingomyelin and the relatively small amounts of phosphatidylcholine in both VS viruses compared with uninfected whole L-cells and chick embryo cells or their plasma membranes. The methods available for isolation of plasma membranes were inadequate for conclusive comparison of the lipids of VS virions with the lipids of the plasma membranes of their host cells. Nevertheless, the data obtained are consistent with two hypotheses: (i) the lipid composition of VS viruses primarily reflects their membrane site of maturation, and (ii) the newly synthesized viral proteins inserted into cell membranes influence the proportions of phospholipids and neutral lipids selected for incorporation into the viral membrane.
Analysis of the Molecular Forms of Simian Virus 40 Deoxyribonucleic Acid Synthesized in Cycloheximide-Treated Cell Cultures
Authors: Kit Saul , Nakajima K.
Published: January 2020

Cycloheximide addition at various times from 24 to 36 hr after virus infection markedly inhibits the rate of simian virus 40 (SV40) deoxyribonucleic acid (DNA) synthesis in monkey kidney (CV-1) cultures. To determine whether superhelical (form I) SV40 DNA was synthesized in the cycloheximide-inhibited cultures, extracts were prepared by the method of Hirt from cultures labeled with
3
H-thymidine (
3
H-dT) and were analyzed by cesium chloride-ethidium bromide (CsCl-EtBr) equilibrium centrifugation and by velocity sedimentation in neutral sucrose gradients. When control or cycloheximide-treated cultures were labeled for 2 or 4 hr with
3
H-dT at 36 or 37 hr after infection, 71 to 83% of the radioactivity soluble in 1
m
NaCl was detected in closed-circular SV40 DNA (form I). Cycloheximide treatment did not generate an increase of higher multiple circular forms of SV40 DNA. In pulse-chase experiments with or without cycloheximide treatment, radioactivity first appeared in nicked molecular forms sedimenting faster than open-circular SV40 DNA (form II), and then was chased into superhelical form I SV40 DNA. These results suggest that in cycloheximide-treated SV40-infected cultures: (i) polynucleotide ligase concentrations are adequate, and (ii) duplication errors causing formation of circular oligomers of SV40 DNA are not enhanced.
Studies on Nondefective Adenovirus-Simian Virus 40 Hybrid Viruses I. A Newly Characterized Simian Virus 40 Antigen Induced by the Ad2
+
ND
1
Virus
Authors: Lewis Andrew M. , Rowe Wallace P.
Published: January 2020

The nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2
+
ND
1
, does not induce heat-labile SV40 T antigen but does induce a previously uncharacterized heat-stable SV40 antigen—the SV40 “U” antigen. This antigen is detectable by both immunofluorescence and complement fixation by using sera from hamsters with SV40 tumors. Sera from hamsters bearing SV40 tumors can be divided into two groups, those that react with both SV40 T and U antigens (T
+
U
+
sera) and those that react with SV40 T antigen only (T
+
U
−
sera). SV40 U-specific sera from monkeys immunized with Ad2
+
ND
1
-infected cells do not react with SV40 T antigen by immunofluorescence but do react with an antigen in the nucleus of SV40-transformed cells and with an early, cytosine arabinoside-resistant antigen present in the nucleus of SV40-infected cells. A heat-stable SV40 antigen detectable by complement fixation with T
+
U
+
hamster sera is present in extracts of SV40-induced hamster tumors and in cell packs of SV40-infected or -transformed cells. SV40 U-antigen synthesis by Ad2
+
ND
1
virus is partially sensitive to inhibitors of deoxyribonucleic acid synthesis, whereas U-antigen synthesis by SV40 virus is an early cytosine arabinoside-resistant event. As an early SV40 antigen differing from SV40 T antigen, U antigen may play a role in malignant transformation mediated by SV40.
Deoxyribonucleic Acid Polymerases of Rous Sarcoma Virus: Kinetics of Deoxyribonucleic Acid Synthesis and Specificity of the Products
Authors: Garapin Axel-Claude , Fanshier Lois , Leong Jo-Ann , Jackson Jean , Levinson Warren , Bishop J. Michael
Published: January 2020

The deoxyribonucleic acid (DNA) polymerase(s) of Rous sarcoma virus synthesizes two principal products—single-stranded DNA in the form of a DNA:ribonucleic acid (RNA) hybrid and double-stranded DNA. All of the single-stranded product and 50% of the double-stranded product can be hybridized to 70
S
viral RNA. These results, in combination with data obtained by analysis of the kinetics of double-stranded DNA synthesis, indicate that the synthesis of double-stranded DNA is a sequel to the synthesis of single-stranded DNA and is dependent upon the latter for the provision of initial template.