bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Expression in Heterozygous Replicative Form Molecules of φX174
Authors: Merriam V. , Dumas L. B. , Sinsheimer R. L.
Published: January 2020

Heterozygous replicative form molecules of bacteriophage φX174 deoxyribonucleic acid (DNA) have been constructed in vitro. These are composed of viral strands extracted from purified preparations of phage bearing
ts
mutations and complementary strands of either half length or full length synthesized with purified DNA polymerase, in vitro, on DNA from
am
3 phage. In infections with such heterozygous DNA, involving mutations in each of four different cistrons, phage with the genotype of the complementary strand comprised 1 to 20% of the total phage produced by a spheroplast population. From single-burst analysis of the progeny from DNA heterozygous in one cistron (B), it appears that those phage with the genotype of the complementary strand arise as major components in a small proportion of the infected cells rather than comprising a minor component in most cells. The implications of such a pattern of expression are discussed with respect to mechanisms of phage DNA synthesis.
Inhibition of Ribonucleic Acid Bacteriophage Release from Its Host by Rifampin
Authors: Engelberg Hanna , Soudry Esther
Published: January 2020

Rifampin, in addition to interfering with intracellular growth of the ribonucleic acid-containing phage MS2, also inhibits the release of mature phage particles from
Escherichia coli
cells.
The promoter and transcriptional unit of a novel herpes simplex virus 1 alpha gene are contained in, and encode a protein in frame with, the open reading frame of the alpha 22 gene
Authors: Carter K L , Roizman B
Published: January 2020

The herpes simplex virus type 1 genome encodes a set of genes (alpha genes) expressed in the absence of de novo viral protein synthesis. Earlier studies have shown that the product of the alpha 22 gene, a member of this set, is nucleotidylylated by casein kinase II and phosphorylated by viral protein kinases encoded by UL13 and US3. Mutants lacking the carboxyl-terminal domain starting with amino acid 200 exhibit reduced capacity to replicate in primary human cell strains or in cells of rodent derivation and also exhibit reduced expression of a subset of gamma or late genes. We report that the domain of the alpha 22 gene is transcribed by two 3'-coterminal mRNAs. The longer transcript reported encodes the 420-amino-acid alpha 22 protein, whereas the shorter transcript reported here encodes a protein containing the carboxyl-terminal 273 amino acids of the alpha 22 protein. The shorter gene is designated US1.5. The US1.5 mRNA is synthesized in cells infected and maintained in the presence of cycloheximide and under other conditions which restrict viral gene expression to alpha genes. In-frame insertion of linkers encoding 18, 21, or 22 amino acids after codon 200 or 240 of the alpha 22 protein did not affect the known functions or phenotype associated with the wild-type alpha 22 gene or its product. Earlier studies have placed the nucleotidylylated sequences in the amino-terminal portion of the protein. The results of these studies indicate that the US1.5 gene encodes the functions associated with replication in human primary or rodent cells and optimal expression of alpha 0 and gamma genes. This finding brings the number of genes known to map in the unique short region of the herpes simplex virus type 1 DNA to 14 and the total number of different genes to 78.
The region of the herpes simplex virus type 1 LAT gene that is colinear with the ICP34.5 gene is not involved in spontaneous reactivation
Authors: Perng G C , Chokephaibulkit K , Thompson R L , Sawtell N M , Slanina S M , Ghiasi H , Nesburn A B , Wechsler S L
Published: January 2020

The goal of this report was to determine if the region of the LAT gene that is colinear with ICP34.5 (kb 6.2 to 7.1 of LAT) is involved in spontaneous reactivation of herpes simplex virus type 1. We inserted one copy of the ICP34.5 gene into the unique long region of a herpes simplex virus type 1 (strain McKrae) mutant lacking both copies of ICP34.5 (one in each viral long repeat) and the corresponding 917-nucleotide colinear portion of LAT (kb 6.2 to 7.1). Rabbits were ocularly infected with this mutant, and spontaneous reactivation relative to that for the wild-type virus and the original mutant was measured. As we previously reported, the original ICP34.5-deleted virus (d34.5) was significantly impaired for spontaneous reactivation and virulence (G. C. Perng, R. L. Thompson, N. M. Sawtell, W. E. Taylor, S. M. Slanina, H. Ghiasi, R. Kaiwar, A. B. Nesburn, and S. L. Wechsler, J. Virol. 69:3033-3041, 1995). In contrast, we report here that restoration of one copy of ICP34.5 at a distant location completely restored the wild-type level of in vivo spontaneous reactivation, despite retention of the deletion in LAT (spontaneous reactivation rate = 0.3 to 1.4% for the ICP34.5 deletion mutant, 7.7 to 19.6% for the wild type, and 9 to 16.1% for virus with one copy of ICP34.5). Thus, the 917-nucleotide region of LAT from kb 6.2 to 7.1 was not involved in the LAT function required for wild-type spontaneous reactivation. We also found that restoration of a single ICP34.5 gene in a novel location did not restore wild-type virulence (rabbit death rate = 0% [0 of 15] for the original ICP34.5 deletion mutant, 8% [2 of 24] for the single-copy IPC34.5 virus, and 52% [14 of 27] for wild-type virus; P < 0.001 for one versus two copies of ICP34.5). It is likely that either two gene doses of ICP34.5 or its location in the long repeat is essential for full functionality of ICP34.5's virulence function. Furthermore, the ability of the single-copy ICP34.5 virus to reactivate at wild-type levels despite being significantly less virulent than wild-type virus separates the spontaneous reactivation phenotype from the virulence phenotype.
Directed integration of viral DNA mediated by fusion proteins consisting of human immunodeficiency virus type 1 integrase and Escherichia coli LexA protein
Authors: Goulaouic H , Chow S A
Published: January 2020

We tested whether the selection of target sites can be manipulated by fusing retroviral integrase with a sequence-specific DNA-binding protein. A hybrid protein that has the Escherichia coli LexA protein fused to the C terminus of the human immunodeficiency virus type 1 integrase was constructed. The fusion protein, IN1-288/LA, retained the catalytic activities in vitro of the wild-type human immunodeficiency virus type 1 integrase (WT IN). Using an in vitro integration assay that included multiple DNA fragment as the target DNA, we found that IN1-288/LA preferentially integrated viral DNA into the fragment containing a DNA sequence specifically bound by LexA protein. No bias was observed when the LexA-binding sequence was absent, when the fusion protein was replaced by WT IN, or when LexA protein was added in the reaction containing IN1-288/LA. A majority of the integration events mediated by IN1-288/LA occurred within 30 bp of DNA flanking the LexA-binding sequence. The specificity toward the LexA-binding sequence and the distribution and frequency of target site usage were unchanged when the integrase component of the fusion protein was replaced with a variant containing a truncation at the N or C terminus or both, suggesting that the domain involved in target site selection resides in the central core region of integrase. The integration bias observed with the integrase-LexA hybrid shows that one effective means of altering the selection of DNA sites for integration is by fusing integrase to a sequence-specific DNA-binding protein.
Multiple regulatory events influence human cytomegalovirus DNA polymerase (UL54) expression during viral infection
Authors: Kerry J A , Priddy M A , Jervey T Y , Kohler C P , Staley T L , Vanson C D , Jones T R , Iskenderian A C , Anders D G , Stenberg R M
Published: January 2020

The human cytomegalovirus (HCMV) DNA polymerase gene (UL54; also called pol) is a prototypical early gene in that expression is mandatory for viral DNA replication. Recently, we have identified the major regulatory element in the UL54 promoter responsive to the major immediate early (MIE) proteins (UL122 and UL123) (J.A. Kerry, M.A. Priddy, and R. M. Stenberg, J. Virol. 68:4167-4176, 1994). Mutation of this element, inverted repeat sequence 1 (IR1), abrogates binding of cellular proteins to the UL54 promoter and reduces promoter activity in response to viral proteins in transient-transfection assays. To extend our studies on the UL54 promoter, we aimed to examine the role of IR1 in UL54 regulation throughout the course of infection. These studies show that viral proteins in addition to the MIE proteins can activate the UL54 promoter. Proteins from UL112-113 and IRS1/TRS1, recently identified as essential loci for transient complementation of HCMV oriLyt-dependent DNA replication, were found to function as transactivators of the UL54 promoter in association with MIE proteins. UL112-113 enhanced UL54 promoter activation by MIE proteins three- to fourfold. Constitutive expression of UL112-113 demonstrated that the MIE protein dependence of UL112-113 transactivational activity was not related to activation of cognate promoter sequences, suggesting that UL112-113 proteins function in cooperation with the MIE proteins. Mutation of IR1 was found to abrogate stimulation of the UL54 promoter by UL112-113, suggesting that this element is also involved in UL112-113 stimulatory activity. These results demonstrate that additional viral proteins influence UL54 promoter expression in transient-transfection assays via the IR1 element. To confirm the biological relevance of IR1 in regulating UL54 promoter activity during viral infection, a recombinant virus construct containing the UL54 promoter with a mutated IR1 element regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene (RVIRmCAT) was generated. Analysis of RVIRmCAT revealed that mutation of IR1 dramatically reduces UL54 promoter activity at early times after infection. However, at late times after infection CAT expression by RVIRmCAT, as assessed by RNA and protein levels, was approximately equivalent to expression by wild-type RVpolCAT. These data demonstrate IR1-independent regulation of the UL54 promoter at late times after infection. Together these results show that multiple regulatory events affect UL54 promoter expression during the course of infection.
AKR.H-2b lymphocytes inhibit the secondary in vitro cytotoxic T-lymphocyte response of primed responder cells to AKR/Gross murine leukemia virus-induced tumor cell stimulation
Authors: Rich R F , Green W R
Published: January 2020

We have previously shown that AKR.H-2b congenic mice, though carrying the responder H-2b major histocompatibility complex haplotype, are unable to generate secondary cytolytic T-lymphocyte (CTL) responses specific for AKR/Gross murine leukemia virus (MuLV). Our published work has shown that this nonresponsive state is specific and not due to clonal deletion or irreversible functional inactivation of antiviral CTL precursors. In the present study, an alternative mechanism based on the presence of inhibitory AKR.H-2b cells was examined. Irradiated or mitomycin C-treated AKR.H-2b spleen cells function as in vitro stimulator cells in the generation of C57BL/6 (B6) anti-AKR/Gross virus CTL, consistent with their expression of viral antigens. In contrast, untreated viable AKR.H-2b spleen cells functioned very poorly as stimulators in vitro. Viable AKR.H-2b spleen cells were also able to cause dramatic (up to > or = 25-fold) inhibition of antiviral CTL responses stimulated in vitro by standard AKR/Gross MuLV-induced tumor cells. This inhibition was specific: AKR.H-2b modulator spleen cells did not inhibit allogeneic major histocompatibility complex-specific CTL production, even when a concurrent antiviral CTL response in the same culture well was inhibited by the modulator cells. These results and those of experiments in which either semipermeable membranes were used to separate AKR.H-2b modulator spleen cells from AKR/Gross MuLV-primed responder cells or the direct transfer of supernatants from wells where inhibition was demonstrated to wells where there was antiviral CTL responsiveness argued against a role for soluble factors as the cause of the inhibition. Rather, the inhibition was dependent on direct contact of AKR.H-2b cells in a dose-dependent manner with the responder cell population. Inhibition was shown not to be due to the ability of AKR.H-2b cells to function as unlabeled competitive target cells. Exogenous interleukin-2 added at the onset of the in vitro CTL-generating cultures partially restored the antiviral response that was decreased by AKR.H-2b spleen cells. Positive and negative cell selection studies and the development of inhibitory cell lines indicated that B lymphocytes and both CD4- CD8+ and CD4+ CD8- T lymphocytes from AKR.H-2b mice could inhibit the generation of AKR/Gross virus-specific CTL in vitro. AKR.H-2b macrophages were shown not to be required to demonstrate AKR/Gross MuLV-specific inhibition, however, confirming that the inhibition by T-cell (or B-cell)-depleted spleen populations was dependent on the enriched B-cell (T-cell) population per se.(ABSTRACT TRUNCATED AT 250 WORDS)
Role of the mu 1 protein in reovirus stability and capacity to cause chromium release from host cells
Authors: Hooper J W , Fields B N
Published: January 2020

The reovirus M2 gene is associated with the capacity of type 3 strain Abney (T3A) intermediate subviral particles (ISVPs) to permeabilize cell membranes as measured by chromium (51Cr) release (P. Lucia-Jandris, J. W. Hooper, and B. N. Fields, J. Virol. 67:5339-5345, 1993). In addition, reovirus mutants with lesions in the M2 gene can be selected by heating virus at 37 degrees C for 20 min in 33% ethanol (D. R. Wessner and B. N. Fields, J. Virol. 67:2442-2447, 1993). In this report we investigated the mechanism by which the reovirus M2 gene product (the mu 1 protein) influences the capacity of reovirus ISVPs to permeabilize membranes, using ethanol-selected T3A mutants. Each of three T3A ethanol-resistant mutants isolated (JH2, JH3, and JH4) exhibited a decreased capacity to cause 51Cr release relative to that of wild-type T3A. Sequence analysis of the M2 genes of wild-type T3A and the T3A mutants indicated that each mutant possesses a single amino acid substitution in a central region of the 708-amino-acid mu 1 protein: JH2 (residue 466, Tyr to Cys), JH3 (residue 459, Lys to Glu), and JH4 (residue 497 Pro to Ser). Assays performed with reovirus natural isolates, reassortants, and a set of previously characterized type 3 strain Dearing (T3D) ethanol-resistant mutants revealed a strong correlation between ethanol sensitivity and the capacity to cause 51Cr release. We found that ISVPs generated from the T3A and T3D mutants were stable when heated to 50 degrees C, whereas wild-type T3A ISVPs are inactivated under these conditions. Together, these data suggest that amino acid substitutions in a central region of the mu 1 protein affect the capacity of the ISVP to permeabilize L-cell membranes by altering the stability of the virus particle.
Human immunodeficiency virus type 1 envelope protein endocytosis mediated by a highly conserved intrinsic internalization signal in the cytoplasmic domain of gp41 is suppressed in the presence of the Pr55gag precursor protein
Authors: Egan M A , Carruth L M , Rowell J F , Yu X , Siliciano R F
Published: January 2020

The mechanisms involved in the incorporation of viral glycoproteins into virions are incompletely understood. For retroviruses, incorporation may involve interactions between the Gag proteins of these viruses and the cytoplasmic domains of the relevant envelope (Env) glycoproteins. Recent studies have identified within the cytoplasmic tail of the human immunodeficiency virus type 1 (HIV-1) Env protein a tyrosine-containing internalization motif similar to those found in the cytoplasmic domains of certain cell surface proteins that undergo rapid constitutive endocytosis in clathrin-coated pits. Given that surface expression of the HIV-1 Env protein is essential for the production of infectious virus, the presence of this internalization motif is surprising. We show here that in contrast to the rapid rate of Env protein internalization observed in cells expressing the Env protein in the absence of other HIV-1 proteins, the rate of internalization of Env protein from the surfaces of HIV-1-infected cells is extremely slow. The presence of the Pr55gag precursor protein is necessary and sufficient for inhibition of Env protein internalization, while a mutant Pr55-gag that is incapable of mediating Env incorporation into virions is also unable to inhibit endocytosis of the Env protein. The failure of the Env protein to undergo endocytosis from the surface of an HIV-1-infected cell may reflect the fact that the proposed interaction of the matrix domain of the Gag protein with Env during assembly prevents the interaction of Env with host adaptin molecules that recruit plasma membrane molecules such as the transferrin receptor into clathrin-coated pits. When the normal ratio of Gag and Env proteins in the infected cells is altered by overexpression of Env protein, this mechanism allows removal of excess Env protein from the cell surface. Taken together, these results suggest that a highly conserved system to reduce surface levels of the Env protein functions to remove Env protein that is not associated with Gag and that is therefore not destined for incorporation into virions. This mechanism for the regulation of surface levels of Env protein may protect infected cells from Env-dependent cytopathic effects or Env-specific immune responses.
Proline-rich tandem repeats of antibody complementarity-determining regions bind and neutralize human immunodeficiency virus type 1 particles
Authors: Fontenot J D , Zacharopoulos V R , Phillips D M
Published: January 2020

The proline-rich tandem repeat domain of human mucin MUC1 forms an extended structure containing large repeating loops that are crested by a turn. We show that the repeating-loop structure of MUC1 can be replaced by an antibody complementarity-determining region loop of a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing antibody to create a chimeric, multivalent, mucin-like, anti-HIV-1 compound. We used 8 residues of an antibody molecule to replace 8 of 20 residues of the MUC1 tandem-repeat sequence. The antiviral peptide discussed here contains three copies of a 20-residue tandem repeat, (IYYDYEEDPAPGSTAPPAHG)3, for a total of 60 residues. We demonstrate that the mucin-antibody chimera retains the binding specificity of the parent antibody (monoclonal antibody F58), GPGR of the HIV-1 gp120 V3 neutralizing epitope, and the ability to neutralize virus particles. In inhibition enzyme-linked immunosorbent assay, the mucin-antibody chimeric peptide could inhibit 71 to 84% of binding to a V3 loop peptide by monoclonal antibodies known to be specific for GPGR in the V3 loop. The mucin-antibody chimeric peptide could also inhibit monoclonal antibody binding to native gp120 captured from virus particles. In addition, the chimeric peptide neutralized the homologous HIV-IIIB virus in a standard neutralization assay. The methods of antiviral peptide design and construction presented here are general and theoretically limited only by the size of the antibody repertoire. This approach could be used to synthesize peptides for a variety of therapeutic applications.
Charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in RNA packaging and infectivity
Authors: Poon D T , Wu J , Aldovini A
Published: January 2020

Interaction of the human immunodeficiency virus type 1 (HIV-1) Gag precursor polyprotein (Pr55Gag) with the viral genomic RNA is required for retroviral replication. Mutations that reduce RNA packaging efficiency have been localized to the highly basic nucleocapsid (NC) p7 domain of Pr55Gag, but the importance of the basic amino acid residues in specific viral RNA encapsidation and infectivity has not been thoroughly investigated in vivo. We have systematically substituted the positively charged residues of the NC domain of Pr55Gag in an HIV-1 viral clone by using alanine scanning mutagenesis and have assayed the effects of these mutations on virus replication, particle formation, and RNA packaging in vivo. Analysis of viral clones with single substitutions revealed that certain charged amino acid residues are more critical for RNA packaging efficiency and infectivity than others. Analysis of viral clones with multiple substitutions indicates that the presence of positive charge in each of three independent domains--the zinc-binding domains, the basic region that links them, and the residues that Hank the two zinc-binding domains--is necessary for efficient HIV-1 RNA packaging. Finally, we note that some mutations affect virus replication more drastically than RNA incorporation, providing in vivo evidence for the hypothesis that NC p7 may be involved in aspects of the HIV life cycle in addition to RNA packaging.
Processing of the equine arteritis virus replicase ORF1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains
Authors: van Dinten L C , Wassenaar A L , Gorbalenya A E , Spaan W J , Snijder E J
Published: January 2020

The replicase open reading frame lb (ORF1b) protein of equine arteritis virus (EAV) is expressed from the viral genome as an ORF1ab fusion protein (345 kDa) by ribosomal frameshifting. Processing of the ORF1b polyprotein was predicted to be mediated by the nsp4 serine protease, the main EAV protease. Several putative cleavage sites for this protease were detected in the ORF1b polyprotein. On the basis of this tentative processing scheme, peptides were selected to raise rabbit antisera that were used to study the processing of the EAV replicase ORF1b polyprotein (158 kDa). In immunoprecipitation and immunoblotting experiments, processing products of 80, 50, 26, and 12 kDa were detected. Of these, the 80-kDa and the 50-kDa proteins contain the putative viral polymerase and helicase domains, respectively. Together, the four cleavage products probably cover the entire ORF1b-encoded region of the EAV replicase, thereby representing the first complete processing scheme of a coronaviruslike ORF1b polyprotein. Pulse-chase analysis revealed that processing of the ORF1b polyprotein is slow and that several large precursor proteins containing both ORF1a- and ORF1b-encoded regions are generated. The localization of ORF1b-specific proteins in the infected cell was studied by immunofluorescence. A perinuclear staining was observed, which suggests association with a membranous compartment.
Human immunodeficiency virus type 1 Nef binds directly to Lck and mitogen-activated protein kinase, inhibiting kinase activity
Authors: Greenway A , Azad A , Mills J , McPhee D
Published: January 2020

It is now well established that human immunodeficiency virus type I (HIV-1) Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T-cell function. The effect of Nef on host cell activation could be explained in part by its interaction with specific cellular proteins involved in signal transduction, including at least a member of the src family kinase, Lck, and the serine/threonine kinase, mitogen-activated protein kinase (MAPK). Recombinant Nef directly interacted with purified Lck and MAPK in coprecipitation experiments and binding assays. A proline-rich repeat sequence [(Pxx)4] in Nef occurring between amino acid residues 69 to 78 is highly conserved and bears strong resemblance to a defined consensus sequence identified as an SH3 binding domain present in several proteins which can interact with the SH3 domain of various signalling and cytoskeletal proteins. Binding and coprecipitation assays with short synthetic peptides corresponding to the proline-rich repeat sequence [(Pxx)4] of Nef and the SH2, SH3, or SH2 and SH3 domains of Lck revealed that the interaction between these two proteins is at least in part mediated by the proline repeat sequence of Nef and the SH3 domain of Lck. In addition to direct binding to full-length Nef, MAPK was also shown to bind the same proline repeat motif. Nef protein significantly decreased the in vitro kinase activity of Lck and MAPK. Inhibition of key members of signalling cascades, including those emanating from the T-cell receptor, by the HIV-1 Nef protein undoubtedly alters the ability of the infected T cell to respond to antigens or cytokines, facilitating HIV-1 replication and contributing to HIV-1-induced disease pathogenesis.
A replication-competent promoter-trap retrovirus
Authors: Coulombe J , Avis Y , Gray D A
Published: January 2020

A promoter-trap retrovirus has been constructed in which a promoterless polyomavirus middle T antigen gene was inserted in the U3 region of the long terminal repeat of a replication-competent Moloney murine leukemia virus. The resulting virus, designated PyT, was used to infect mouse mammary glands in situ. As expected, mammary tumors appeared in some infected animals. These tumors were found to contain PyT proviruses of the predicted structure. From one such tumor, the PyT provirus and surrounding sequences from the integration site were cloned. The provirus was found to have integrated adjacent to the promoter of a novel mouse gene (TRAP1) that was expressed at low levels in various mouse tissues. These data show that the PyT retrovirus provides a sensitive means of detecting active promoters in vivo.
Mutational analysis of the encephalomyocarditis virus primary cleavage
Authors: Hahn H , Palmenberg A C
Published: January 2020

Sixteen substitution mutations of the conserved DvExNPGP sequence, implicated in cardiovirus and aphthovirus primary polyprotein cleavage, were created in encephalomyocarditis virus cDNA, expressed, and characterized for processing activity. Nearly all the mutations severely decreased the efficiency of the primary cleavage reaction during cell-free synthesis of viral precursors, indicating a stringent requirement for the natural sequence in this processing event. When representative mutations were tested in full-length genomic contexts, they were lethal and no revertants were observed. Not only were the primary cleavage reactions deficient in these polyproteins, but subsequent cleavage of P1 by endogenous or exogenous 3C pro was also impaired. This indicates that primary cleavage has a role in the proper processing of the viral capsid precursor.
A vaccinia virus core protein, p39, is membrane associated
Authors: Cudmore S , Blasco R , Vincentelli R , Esteban M , Sodeik B , Griffiths G , Krijnse Locker J
Published: January 2020

We describe herein the characterization of p39, the product of the A4L gene of vaccinia virus. By immunolabelling of thawed cryosections from infected HeLa cells, we show that this protein is initially located in the central region, or viroplasm, of the viral factories, as well as in the immature virions, with very small amounts of labelling observed on the surrounding membranes. The localization of p39 changes dramatically during the transition of the immature virion to the intracellular mature virus (IMV), coincident with the appearance of the core structure in the center of the IMV, with p39 located between this core and the surrounding membranes. Complementary biochemical data, such as partitioning into the Triton X-114 detergent phase and stripping of the viral membranes with Nonidet P-40 and dithiothreitol, suggest that p39 is associated with the innermost of the two membranes surrounding the core. Sodium carbonate treatment also indicates that p39 is associated with membranes, even at the early stages of viral assembly. However, following in vitro translation of p39 in the presence of microsomal membranes, we failed to detect any association of the independently expressed protein with membranes. We also failed to detect any posttranslational acylation of p39 with myristate or palmitate, suggesting that p39 does not achieve its membrane association through lipid anchors. Therefore, p39 is most likely membrane associated through an interaction with an integral membrane protein(s) present in the innermost of the two membranes surrounding the IMV. These data, together with our recent data showing that p39 colocalizes with the spike-like protrusions on the IMV core (N. Roos, M. Cyrklaff, S. Cudmore, R. Blasco, J. Krijnse-Locker, and G. Griffiths, EMBO J. 15:2343-2355, 1996), suggest that p39 may form part of this spike and that it possibly functions as a matrix-like linker protein between the core and the innermost of the two membranes surrounding the IMV.
A hydrophobic heptad repeat of the core protein of woodchuck hepatitis virus is required for capsid assembly
Authors: Yu M , Miller R H , Emerson S , Purcell R H
Published: January 2020

The capsid particle of hepadnaviruses is assembled from its dimer precursors. However, the mechanism of the protein-protein interaction is still poorly understood. A small region in the capsid protein of woodchuck hepatitis virus (WHV) contains four hydrophobic residues, including leucine 101, leucine 108, valine 115, and phenylalanine 122, that are conserved and spaced every seventh residue in the primary sequence to form a hydrophobic heptad repeat (hhr). A hydrophobic force often plays an important role in the interaction of proteins. Therefore, to investigate the role of this region in capsid assembly, we individually changed the codons specifying these four hydrophobic amino acids to codons specifying alanine or proline. In addition, we examined the in vivo infectivity of a WHV genome bearing a naturally occurring single amino acid change (histidine 104-->proline) in the hhr region. The phenotype of each altered genome was determined in both eukaryotic and prokaryotic systems by a capsid protein assay and electron microscopic examination. We show that replacement of any one of the four hydrophobic residues with alanine did not prevent capsid assembly. However, assembled capsid particles were not detected if combinations of any two of the four residues were substituted with alanines or if the spacing of these four hydrophobic residues was changed. An individual introduction of a proline (which dramatically changes the secondary structure of proteins) into different positions of this small region also abolished capsid assembly in vitro or viral replication in vivo. These results suggested that the hhr region of the core protein of WHV was critical for capsid assembly.
N-butyldeoxynojirimycin-mediated inhibition of human immunodeficiency virus entry correlates with impaired gp120 shedding and gp41 exposure
Authors: Fischer P B , Karlsson G B , Dwek R A , Platt F M
Published: January 2020

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is an inhibitor of human immunodeficiency virus (HIV) replication and HIV-induced syncytium formation in vitro. Although an NB-DNJ-mediated change in viral envelope N-glycan composition inhibits HIV entry at the level of post-CD4 binding, the exact mechanism of inhibition remains to be established. In this study we have examined the effects of NB-DNJ on virion envelope composition and CD4-induced gp120 shedding and gp41 exposure. Virion composition analysis revealed an NB-DNJ-mediated reduction of 15% in overall virion envelope glycoprotein content and a reduction of 26% in the proteolytic maturation of virion gp160. Taken together, these two effects resulted in a reduction of approximately 40% in virion gp120 content. CD4-induced shedding of gp120 from the surfaces of envelope-transfected Cos cells was undetectable when gp120 was expressed in the presence of NB-DNJ. Similarly, the shedding of virion-associated gp120 was reduced 7.4-fold. CD4-induced exposure of cryptic gp41 epitopes on the surfaces of HIV-expressing ACH-2 cells was also greatly impaired, and the exposure of virion-associated gp41 epitopes was reduced 4.0-fold. Finally, CD4-induced increases in the binding of antibodies to the V3 loop of ACH-2-cell-expressed envelope glycoproteins were reduced 25-fold when the glycoproteins were expressed in the presence of NB-DNJ. These results suggest that the NB-DNJ-mediated retention of glycosylated N-glycans inhibits HIV entry by a combined effect of a reduction in virion gp120 content and a qualitative defect within the remaining gp120, preventing it from undergoing conformational changes after CD4 binding.
Novel DNA binding specificities of a putative herpesvirus bZIP oncoprotein
Authors: Qian Z , Brunovskis P , Lee L , Vogt P K , Kung H J
Published: January 2020

Marek's disease virus is a highly oncogenic herpesvirus that can cause T lymphomas and peripheral nerve demyelination in chickens. meq, a candidate oncogene of Marek's disease virus, encodes a basic leucine zipper (bZIP) transcription factor which contains a large proline-rich domain in its C terminus. On the basis of its bZIP structural homology, meq is perhaps the only member of the jun-fos gene family completely viral in origin. We previously showed that Meq's C-terminal domain has potent transactivation activity and that its bZIP domain can dimerize with itself and with c-Jun also. In an effort to identify viral and cellular targets of Meq, we have determined the optimal binding sites for Meq-Jun heterodimers and Meq-Meq homodimers. By a PCR-based approach using cyclic amplification of selected targets, Meq-Jun heterodimers were found to optimally bind tetradecanoylphorbol acetate response element (TRE) and cyclic AMP response element (CRE) consensus sequences. This result was consistent with the results of our previous functional analysis implicating Meq-Jun heterodimers in the transactivation of the Meq promoter through a TRE- or CRE-like sequence. Interestingly, Meq-Meq homodimers were found to bind two distinct motif elements. The first [GAGTGATG AC(G)TCATC] has a consensus which includes a TRE or CRE core flanked by additional nucleotides critical for tight binding. Methylation interference and mutational analyses confirmed the importance of the flanking residues. The sequences of a subset of TRE and CRE sites selected by Meq-Meq are closely related to the binding motif of Maf, another bZIP oncoprotein. The second putative Meq binding site (RACACACAY) bears a completely different consensus not shared by other bZIP proteins. Binding to this consensus sequence also requires secondary structure characteristics associated with DNA bending. CACA motifs are known to promote DNA curvature and function in a number of special biological processes. Our results lend further weight to the increasing importance of DNA bending in transcriptional regulation and provide a baseline for the identification of Meq-responsive targets.
Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells
Authors: Fraefel C , Song S , Lim F , Lang P , Yu L , Wang Y , Wild P , Geller A I
Published: January 2020

Herpes simplex virus type 1 (HSV-1) plasmid vectors have promise for genetic intervention in the brain, but several problems caused by the helper virus have compromised their utility. To develop a helper virus-free packaging system for these vectors, the DNA cleavage/packaging signals were deleted from a set of cosmids that represents the HSV-1 genome. Following cotransfection into cells, this modified cosmid set supported replication and packaging of vector DNA. However, in the absence of the DNA cleavage/packaging signals, the HSV-1 genome was not packaged, and consequently vector stocks were free of detectable helper virus. In the absence of helper virus, the vectors efficiently infected rat neural cells in culture or in the brain with minimal cytopathic effects. beta-galactosidase-positive cells were observed for at least 1 month in vivo, and vector DNA persisted for this period. This system may facilitate studies on neuronal physiology and potential therapeutic applications.
The human cytomegalovirus UL37 immediate-early regulatory protein is an integral membrane N-glycoprotein which traffics through the endoplasmic reticulum and Golgi apparatus
Authors: Al-Barazi H O , Colberg-Poley A M
Published: January 2020

The human cytomegalovirus (HCMV) UL37 immediate-early gene is predicted to encode a type I membrane-bound glycoprotein, gpUL37. Following expression of the UL37 open reading frame in vitro, its signals for translocation and N-glycosylation were recognized by microsomal enzymes. Its orientation in the microsomes is that of a type I protein. gpUL37 produced in HCMV-infected human cells was selectively immunoprecipitated by rabbit polyvalent antiserum generated against the predicted unique domains of the UL37 open reading frame and migrated as an 83- to 85-kDa protein. Tunicamycin treatment, which inhibits N-glycosylation, increased the rate of migration of the UL37 protein to 68 kDa, verifying its modification by N-glycosylation in HCMV-infected cells. Consistent with this observation, gpUL37 was found to be resistant to digestion with either endoglycosidase F or H but sensitive to peptide N-glycosidase F digestion. These results suggested that gpUL37 is N-glycosylated and processed in both the endoplasmic reticulum (ER) and the Golgi apparatus. Direct demonstration of passage of gpUL37 through the ER and the Golgi was obtained by confocal microscopy. gpUL37 colocalized with protein disulfide isomerase, a protein resident in the ER, and with a Golgi protein. Subcellular fractionation of HCMV-infected cells demonstrated that gpUL37 is an integral membrane protein. Taken together, our results demonstrate that the HCMV gpUL37 immediate-early regulatory protein is a type I integral membrane N-glycoprotein which traffics through the ER and the Golgi network.
Analysis of the serotype-specific epitopes of avian infectious bronchitis virus strains Ark99 and Mass41
Authors: Jia W , Wang X , Parrish C R , Naqi S A
Published: January 2020

The Ark and Mass serotype-specific epitopes of infectious bronchitis virus were studied by immunofluorescence and immunoprecipitation of mutant and recombinant spike glycoproteins (S protein) expressed in mouse L cells. Serotype-specific monoclonal antibodies could bind to the recombinant proteins of Ark99 and Mass41 expressed from the chimeras in which the N-terminal thirds of the S1 sequences were reciprocally exchanged. Therefore, it appears that the respective serotype-specific epitopes of both strains were localized within the C-terminal two-thirds of the S1 proteins. Deletion and insertion of a five-amino-acid fragment on the S1 proteins of Ark99 and Mass41, altered the serotype-specific epitopes. This result implies that the five-amino-acid insertion on the S1 protein of the Ark serotype was involved in determining the conformation of the protein, probably acting as a spacer. In addition, it appears that an interaction between sequences of the N-terminal third and the remaining portion of the S1 protein determines the tertiary structure of the protein as well as the conformation of the serotype-specific epitope.
Transcriptional activation function is not required for stimulation of DNA replication by bovine papillomavirus type 1 E2
Authors: Grossel M J , Sverdrup F , Breiding D E , Androphy E J
Published: January 2020

Bovine papillomavirus type 1 replication was previously shown to require both the E1 initiator protein and the E2 transactivator protein. We show here that E1, in the absence of E2, is sufficient for low-level bovine papillomavirus type 1 DNA replication in C-33A cells. In addition, studies of genetically isolated E2 point mutants demonstrate that enhancement of replication by E2 does not require its transcriptional activation function. The uncoupling of the E2 functions suggests that stimulation of transcription and replication by enhancer proteins occurs via divergent mechanisms.
The herpes simplex virus type 1 latency-associated transcript promoter is activated through Ras and Raf by nerve growth factor and sodium butyrate in PC12 cells
Authors: Frazier D P , Cox D , Godshalk E M , Schaffer P A
Published: January 2020

Herpes simplex virus establishes latent infections in the nuclei of sensory neurons. These infections are characterized by the abundant expression of a series of 5' coterminal transcripts termed the latency-associated transcripts (LATs). Available evidence indicates that LAT expression is specifically regulated in latently infected neurons. Although previous studies have examined the regulation of LAT expression in neuronal and nonneuronal cells, the mechanism of regulation of LAT expression in neuronal cells in response to external factors has not been investigated. To address this question, we characterized the activity of LAT promoter fusion constructs in PC12 cells following treatment with nerve growth factor (NGF) and/or sodium butyrate (NaB), agents that affect expression of cell cycle-associated genes. Expression from the LAT promoter was induced 8- to 12-fold by either NGF or NaB alone and 40- to 60-fold when the two agents were added simultaneously. Fibroblast growth factor also induced expression from the LAT promoter but to a lesser extent than NGF. Treatment with factors such as epidermal growth factor, phorbol myristate acetate, cyclic AMP, or KCI had no significant effect on LAT promoter activity. Notably, promoter-reporter constructs containing immediate-early (ICP0 and ICP4), early (ICP8 and UL9), and late (UL10 and UL30) viral promoters were induced only two- to fourfold by NGF, suggesting that the LAT promoter may be unusual among herpes simplex virus genes in the magnitude of its response to this factor. To identify pathways leading to LAT activation in vitro, we characterized the response of the LAT promoter to NGF and/or NaB in PC12-derived cell lines containing mutations in specific signal transduction pathways. We found that activation of the LAT promoter requires Ras activation and that activation of the serine/threonine kinase, Raf, is sufficient to induce LAT expression. Together, these results indicate that the LAT promoter is regulated via the Ras/Raf signal transduction pathway in response to external factors such as NGF and NaB and that LAT expression may be regulated by NGF in latently infected neurons.
Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy
Authors: Mittereder N , March K L , Trapnell B C
Published: January 2020

Development of adenovirus vectors as potential therapeutic agents for multiple applications of in vivo human gene therapy has resulted in numerous preclinical and clinical studies. However, lack of standardization of the methods for quantifying the physical concentration and functionally active fraction of virions in these studies has often made comparison between various studies difficult or impossible. This study was therefore carried out to define the variables for quantification of the concentration of adenovirus vectors. The methods for evaluation of total virion concentration included electron microscopy and optical absorbance. The methods for evaluation of the concentration of functional virions included detection of gene transfer (transgene transfer and expression) and the plaque assay on 293 cells. Enumeration of total virion concentration by optical absorbance was found to be a precise procedure, but accuracy was dependent on physical disruption of the virion to eliminate artifacts from light scattering and also on a correct value for the extinction coefficient. Both biological assays for enumerating functional virions were highly dependent on the assay conditions and in particular the time of virion adsorption and adsorption volume. Under optimal conditions, the bioactivity of the vector, defined as the fraction of total virions which leads to detected target cell infection, was determined to be 0.10 in the plaque assay and 0.29 in the gene transfer assay. This difference is most likely due to the fact that detection by gene transfer requires only measurement of levels of transgene expression in the infected cell whereas plaque formation is dependent on a series of biological events of much greater complexity. These results show that the exact conditions for determination of infectious virion concentration and bioactivity of recombinant adenovirus vectors are critical and must be standardized for comparability. These observations may be very useful in comparison of data from different preclinical and clinical studies and may also have important implications for how adenovirus vectors can optimally be used in human gene therapy.
PCR-based detection and partial characterization of a retrovirus associated with contagious intranasal tumors of sheep and goats
Authors: Cousens C , Minguijon E , Garcia M , Ferrer L M , Dalziel R G , Palmarini M , De las Heras M , Sharp J M
Published: January 2020

A type D-related retrovirus has been demonstrated in enzootic nasal tumors (ENTs) of sheep and goats. This retrovirus, ENT virus (ENTV), has antigenic cross-reactivity with the jaagsiekte sheep retrovirus (JSRV), which is associated with a contagious lung tumor of sheep (sheep pulmonary adenomatosis). Here, we present the first report of nucleic acid sequence from ENTV which confirms, at the nucleic acid level, that this retrovirus is related to JSRV yet apparently distinct from it. Reverse transcription-PCR followed by restriction enzyme digestion specifically identified ENTV. By this technique, ENTV was demonstrated exclusively in tumor tissues and exudates of animals with ENT. Thus, there is a unique and consistent association between ENT and the retrovirus, just as there is between JSRV and sheep pulmonary adenomatosis. This gives further weight to the hypothesis that these retroviruses are the etiologic agents of the tumors.
Inducible expression of the vaccinia virus A17L gene provides a synchronized system to monitor sorting of viral proteins during morphogenesis
Authors: Rodríguez D , Risco C , Rodríguez J R , Carrascosa J L , Esteban M
Published: January 2020

The vaccinia virus (VV) A17L gene encodes a 21- to 23-kDa virion component that forms a stable complex with the 14-kDa envelope protein (A27L gene). In a previous report, we described the construction of a VV recombinant, VVindA17L, in which the expression of the A17L gene is inducibly regulated by isopropyl-beta-D-thiogalactoside (IPTG). We demonstrated that shutoff of the A17L gene results in a blockade of virion morphogenesis at a very early stage (D. Rodríguez, M. Esteban, and J. R. Rodríguez, J. Virol. 69:4640-4648, 1995). In the present study, we show that virus growth is restored if the inducer is provided not later than 6 h postinfection. Immunofluorescence and immunoelectron microscopy analysis of VVindA17L-infected cells revealed that in the absence of the 21- to 23-kDa protein, the 14-kDa protein is distributed throughout the cytoplasm. After IPTG addition, the 14-kDa protein can be detected around viral factories and immature virions; at later times, it localizes in the external membranes of intracellular mature virions. Immunoelectron microscopy with anti-21- to 23-kDa antibodies showed that soon after induction, the protein accumulates in membranes of the rough endoplasmic reticulum and in the nuclear envelope. With time, the protein localizes in viral crescents and subsequently associates to the membranes of immature and intracellular mature virions. These results are consistent with a model in which the 21- to 23-kDa protein would be synthesized at the endoplasmic reticulum, from where the protein could be translocated to the membranes of the intermediate compartment to generate the precursors of the viral membranes. Also, these results argue that 14-kDa envelope protein becomes posttranslationally associated to viral membranes through its interaction with the 21-kDa protein.