bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphatidylinositol-Dependent Membrane Fusion Induced by a Putative Fusogenic Sequence of Ebola Virus
Authors: Ruiz-Argüello M. Begoña , Goñi Félix M. , Pereira Francisca B. , Nieva José L.
Published: December 2019

The membrane-interacting abilities of three sequences representing the putative fusogenic subdomain of the Ebola virus transmembrane protein have been investigated. In the presence of calcium, the sequence EBO
GE
(GAAIGLAWIPYFGPAAE) efficiently fused unilamellar vesicles composed of phosphatidylcholine, phosphatidylethanolamine, cholesterol, and phosphatidylinositol (molar ratio, 2:1:1:0.5), a mixture that roughly resembles the lipid composition of the hepatocyte plasma membrane. Analysis of the lipid dependence of the process demonstrated that the fusion activity of EBO
GE
was promoted by phosphatidylinositol but not by other acidic phospholipids. In comparison, EBO
EA
(EGAAIGLAWIPYFGPAA) and EBO
EE
(EGAAIGLAWIPYFGPAAE) sequences, which are similar to EBO
GE
except that they bear the negatively charged glutamate residue at the N terminus and at both the N and C termini, respectively, induced fusion to a lesser extent. As revealed by binding experiments, the glutamate residue at the N terminus severely impaired peptide-vesicle interaction. In addition, the fusion-competent EBO
GE
sequence did not associate significantly with vesicles lacking phosphatidylinositol. Tryptophan fluorescence quenching by vesicles containing brominated phospholipids indicated that the EBO
GE
peptide penetrated to the acyl chain level only when the membranes contained phosphatidylinositol. We conclude that binding and further penetration of the Ebola virus putative fusion peptide into membranes might be governed by the nature of the N-terminal residue and by the presence of phosphatidylinositol in the target membrane. Moreover, since insertion of such a peptide leads to membrane destabilization and fusion, the present data would be compatible with the involvement of this sequence in Ebola virus fusion.
Receptor-Binding Properties of a Soluble Form of Human Cytomegalovirus Glycoprotein B
Authors: Boyle Kathleen A. , Compton Teresa
Published: December 2019

The human cytomegalovirus (HCMV) glycoprotein B (gB) (also known as gpUL55) homolog is an important mediator of virus entry and cell-to-cell dissemination of infection. To examine the potential ligand-binding properties of gB, a soluble form of gB (gB-S) was radiolabeled, purified, and tested in cell-binding experiments. Binding of gB-S to human fibroblast cells was found to occur in a dose-dependent, saturable, and specific manner. Scatchard analysis demonstrated a biphasic plot with the following estimated dissociation constants (

K
d

):
K

d
1

, 4.96 × 10
−6
M;
K

d
2

, 3.07 × 10
−7
M. Cell surface heparan sulfate proteoglycans (HSPGs) were determined to serve as one class of receptors able to facilitate gB-S binding. Both HSPG-deficient Chinese hamster ovary (CHO) cells and fibroblast cells with enzymatically removed HSPGs had 40% reductions in gB-S binding, whereas removal of chondroitin sulfate had no effect. However, a significant proportion of gB-S was able to associate with the cell surface in the absence of HSPGs via an undefined nonheparin component. Binding affinity analysis of gB-S binding to wild-type CHO-K1 cells demonstrated biphasic binding kinetics (
K

d
1

, 9.85 × 10
−6
M;

K
d2

, 4.03 × 10
−8
M), whereas gB-S binding to HSPG-deficient CHO-677 cells exhibited single-component binding kinetics (

K
d

, 7.46 × 10
−6
M). Together, these data suggest that gB-S associates with two classes of cellular receptors. The interaction of gB with its receptors is physiologically relevant, as evidenced by an inhibitory effect on HCMV entry when cells were pretreated with purified gB-S. This inhibition was determined to be manifested at the level of virus attachment. We conclude that gB is a ligand for HCMV that mediates an interaction with a cellular receptor(s) during HCMV infection.
Multiple Functions within the Epstein-Barr Virus EBNA-3A Protein
Authors: Cludts Isabelle , Farrell Paul J.
Published: December 2019

Two regions of the EBNA-3A protein of Epstein-Barr virus were shown to be capable of binding to the cell protein RBP-Jk (also known as CBF-1), a component of the Notch signaling pathway. Consistent with this binding, EBNA-3A inhibited reporter gene expression from plasmids containing RBP-Jk DNA binding sites within their promoters, including the Cp promoter. When EBNA-3A was linked to a GAL4 DNA binding domain, it repressed the activity of a promoter containing GAL4 binding sites at all plasmid concentrations tested. However, a deletion mutant of EBNA-3A lacking amino acids 100 to 364 showed a biphasic response in the GAL4 assay: it inhibited transcription at low DNA concentrations but activated it at high DNA concentrations. There appears to be a gene activation function within EBNA-3A that is masked in the full-length protein in this assay. Current models for EBNA-3 function have stressed transcription repression through binding to RBP-Jk, but we consider an alternative scheme in which the role of the binding of EBNA-3A, -3B, and -3C to RBP-Jk is to buffer the levels of active EBNA-3 protein. We have also found that the behavior of EBNA-3A in a cell fractionation procedure that distinguishes insoluble matrix from soluble cell fractions is modified by EBNA-LP, indicating a further novel level of interplay between the EBNA proteins.
Sequence-Specific Binding of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein to Short Oligonucleotides
Authors: Fisher Robert J. , Rein Alan , Fivash Matthew , Urbaneja Maria A. , Casas-Finet José R. , Medaglia Maxine , Henderson Louis E.
Published: December 2019

We have analyzed the binding of recombinant human immunodeficiency virus type 1 nucleocapsid protein (NC) to very short oligonucleotides by using surface plasmon resonance (SPR) technology. Our experiments, which were conducted at a moderate salt concentration (0.15 M NaCl), showed that NC binds more stably to runs of d(G) than to other DNA homopolymers. However, it exhibits far more stable binding with the alternating base sequence d(TG)
n
than with any homopolymeric oligodeoxyribonucleotide; thus, it shows a strong sequence preference under our experimental conditions. We found that the minimum length of an alternating d(TG) sequence required for stable binding was five nucleotides. Stable binding to the tetranucleotide d(TG)
2
was observed only under conditions where two tetranucleotide molecules were held in close spatial proximity. The stable, sequence-specific binding to d(TG)
n
required that both zinc fingers be present, each in its proper position in the NC protein, and was quite salt resistant, indicating a large hydrophobic contribution to the binding. Limited tests with RNA oligonucleotides indicated that the preferential sequence-specific binding observed with DNA also occurs with RNA. Evidence was also obtained that NC can bind to nucleic acid molecules in at least two distinct modes. The biological significance of the specific binding we have detected is not known; it may reflect the specificity with which the parent Gag polyprotein packages genomic RNA or may relate to the functions of NC after cleavage of the polyprotein, including its role as a nucleic acid chaperone.
Temporal Gene Regulation of the Channel Catfish Virus (Ictalurid Herpesvirus 1)
Authors: Huang Suming , Hanson Larry A.
Published: December 2019

To identify promoter regions that impart differential temporal regulation of channel catfish virus (CCV) genes, the transcriptional kinetics of an immediate-early gene and prospective early and late genes were characterized. A cDNA clone, designated IE3C, representing a third immediate-early transcript was identified. The 5′ end of the IE3C transcript was mapped to nucleotides 15,368 and 131,043 in the terminal repeat regions of the CCV genome. The full length of the transcript represented by the IE3C clone is 1,412 bp, and it most likely codes for the protein specified by open reading frame (ORF) 12. The putative product of ORF12 contains a consensus RING finger metal binding motif (C
3
HC
4
structure). Temporal expression studies, in conjunction with protein synthesis and DNA replication inhibition, demonstrated that the IE3C transcript belongs to an immediate-early kinetic class, the ORF5 transcript is a member of the early kinetic class, and ORF39 and ORF46 are true late-kinetic-class genes. Additionally, we demonstrated that ORF38 transcription overlaps ORF39 and the products presumably share the same poly(A) signal. The 5′ ends of the transcripts encoding ORF38, ORF39, and ORF46 were mapped to nucleotides 44,862, 45,254, and 59,644, respectively, and potential transcriptional control elements were located.
A Peptide Mimic of a Protective Epitope of Respiratory Syncytial Virus Selected from a Combinatorial Library Induces Virus-Neutralizing Antibodies and Reduces Viral Load In Vivo
Authors: Chargelegue Daniel , Obeid Obeid E. , Hsu Shiou-Chih , Shaw Michael D. , Denbury Andrew N. , Taylor Geraldine , Steward Michael W.
Published: December 2019

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children worldwide. As yet, there is no effective vaccine against RSV infection, and previous attempts to develop a formalin-inactivated vaccine resulted in exacerbated disease in recipients subsequently exposed to the virus. In the work described here, a combinatorial solid-phase peptide library was screened with a protective monoclonal antibody (MAb 19) to identify peptide mimics (mimotopes) of a conserved and conformationally-determined epitope of RSV fusion (F) protein. Two sequences identified (S1 [HWYISKPQ] and S2 [HWYDAEVL]) reacted specifically with MAb 19 when they were presented as solid-phase peptides. Furthermore, after amino acid substitution analyses, three sequences derived from S1 (S1S [HWSISKPQ], S1K [KWYISKPQ], and S1P [HPYISKPQ]), presented as multiple antigen peptides (MAPs), also showed strong reactivity with MAb 19. The affinity constants of the binding of MAb 19, determined by surface plasmon resonance analyses, were 1.19 × 10
9
and 4.93 × 10
9
M
−1
for S1 and S1S, respectively. Immunization of BALB/c mice with these mimotopes, presented as MAPs, resulted in the induction of anti-peptide antibodies that inhibited the binding of MAb 19 to RSV and neutralized viral infection in vitro, with titers equivalent to those in sera from RSV-infected animals. Following RSV challenge of S1S mimotope-immunized mice, a 98.7% reduction in the titer of virus in the lungs was observed. Furthermore, there was a greatly reduced cell infiltration in the lungs of immunized mice compared to that in controls. These results indicate the potential of peptide mimotopes to protect against RSV infection without exacerbating pulmonary pathology.
Activation of Heterologous Gene Expression by the Large Isoform of Hepatitis Delta Antigen
Authors: Wei Yu , Ganem Don
Published: December 2019

Hepatitis delta virus (HDV) encodes two isoforms of its principal gene product, hepatitis delta antigen (HDAg). These two forms play distinctive and complementary roles in viral replication. Here we report that the large (LHDAg), but not the small (SHDAg), isoform of HDAg has the capacity to activate the expression of cotransfected genes driven by a variety of promoters, including the pre-S, S, and C promoters of hepatitis B virus. Mutational analysis of the C-terminal 19 amino acids unique to LHDAg shows that changing prolines to alanines in the two PXXP motifs in this region specifically ablates the activation function without abolishing another activity of LHDAg, namely, its ability to inhibit HDV RNA synthesis. However, C-terminal truncations that also disrupt these PXXP motifs only slightly diminished the activation function, indicating that the proline mutations were not acting by inactivating potential SH3 interactions that could be mediated by these motifs. Mutation of the isoprenylated cysteine to serine decreases but does not abolish the activation activity, and overexpression of SHDAg does not interfere with the transactivation function of LHDAg. Although the mechanism and biological significance of this activity of LHDAg remain unknown, the presence of this activity serves as yet another marker that functionally distinguishes this protein from the closely related isoform SHDAg.
Modulation of Feline Immunodeficiency Virus Infection by Stromal Cell-Derived Factor
Authors: Hosie Margaret J. , Broere Nelleke , Hesselgesser Joseph , Turner Julie D. , Hoxie James A. , Neil James C. , Willett Brian J.
Published: December 2019

The α-chemokine receptor CXCR4 has recently been shown to support syncytium formation mediated by strains of feline immunodeficiency virus (FIV) that have been selected for growth in the Crandell feline kidney cell line (CrFK-tropic virus). Given that both human and feline CXCR4 support syncytium formation mediated by FIV, we investigated whether human stromal cell-derived factor (SDF-1) would inhibit infection with FIV. Human SDF-1α and SDF-1β bound with a high affinity (KDs of 12.0 and 10.4 nM, respectively) to human cells stably expressing feline CXCR4, and treatment of CrFK cells with human SDF-1α resulted in a dose-dependent inhibition of infection by FIVPET. No inhibitory activity was detected when the interleukin-2 (IL-2)-dependent feline T-cell line Mya-1 was used in place of CrFK cells, suggesting the existence of a CXCR4-independent mechanism of infection. Furthermore, neither the human β-chemokines RANTES, MIP-1α, MIP-1β, and MCP-1 nor the α-chemokine IL-8 had an effect on infection of either CrFK or Mya-1 cells with CrFK-tropic virus. Envelope glycoprotein purified from CrFK-tropic virus competed specifically for binding of SDF-1α to feline CXCR4 and CXCR4 expression was reduced in FIV-infected cells, suggesting that the inhibitory activity of SDF-1α in CrFK cells may be the result of steric hindrance of the virus-receptor interaction following the interaction between SDF and CXCR4. Prolonged incubation of CrFK cells with SDF-1α led to an enhancement rather than an inhibition of infection. Flow cytometric analysis revealed that this effect may be due largely to up-regulation of CXCR4 expression by SDF-1α on CrFK cells, an effect mimicked by treatment of the cells with phorbol myristate acetate. The data suggest that infection of feline cells with FIV can be mediated by CXCR4 and that, depending on the assay conditions, infection can be either inhibited or enhanced by SDF-1α. Infection with FIV may therefore prove a valuable model in which to study the development of novel therapeutic interventions for the treatment of AIDS.
T Cells Expressing Activated LFA-1 Are More Susceptible to Infection with Human Immunodeficiency Virus Type 1 Particles Bearing Host-Encoded ICAM-1
Authors: Fortin Jean-François , Cantin Réjean , Tremblay Michel J.
Published: December 2019

The incorporation of host-derived proteins in nascent human immunodeficiency virus type 1 (HIV-1) particles is a well-established phenomenon. We recently demonstrated that the physical presence of host-encoded ICAM-1 glycoproteins on HIV-1 leads to a significant increase in virus infectivity in an ICAM-1/LFA-1-dependent fashion (J.-F. Fortin, R. Cantin, G. Lamontagne, and M. Tremblay, J. Virol. 71:3588–3596, 1997). We show here that conversion of LFA-1 to high affinity for ICAM-1 with the use of anti-LFA-1 antibodies (clones NKI-L16 and MEM83) markedly enhances the susceptibility of different target T-lymphoid cell lines, as well as of primary peripheral blood mononuclear cells, to infection by ICAM-1-bearing HIV-1 particles (6- to 95-fold). It is known that T-cell receptor (TCR) cross-linking induces a transient increase in LFA-1 affinity for ICAM-1. Treatment of peripheral blood mononuclear cells with anti-TCR antibodies (clone OKT3) resulted in a transient increase in susceptibility to infection by ICAM-1-positive virions that parallels the previously reported kinetics of the LFA-1/ICAM-1 adhesion mechanism. Our results led us to postulate that the strong interaction taking place between virally incorporated ICAM-1 and cell surface-activated LFA-1 markedly enhances the efficiency of virus binding and entry, thus favoring greater infection by ICAM-1-bearing HIV-1 particles. In view of the knowledge that primary HIV-1 isolates harbor host-derived ICAM-1 on their surfaces, these results provide new information about the role of host-derived ICAM-1 in the life cycle of HIV-1 and how it could positively modulate the dynamics of the viral infection, mainly in cellular compartments, such as the lymphoid tissues, where the level of cellular activation is high and where the probability of encountering a T cell expressing the activated LFA-1 form is also elevated.
Chimeric Measles Viruses with a Foreign Envelope
Authors: Spielhofer Pius , Bächi Thomas , Fehr Thomas , Christiansen Gudrun , Cattaneo Roberto , Kaelin Karin , Billeter Martin A. , Naim Hussein Y.
Published: December 2019

Measles virus (MV) and vesicular stomatitis virus (VSV) are both members of the
Mononegavirales
but are only distantly related. We generated two genetically stable chimeric viruses. In MGV, the reading frames of the MV envelope glycoproteins H and F were substituted by a single reading frame encoding the VSV G glycoprotein; MG/FV is similar but encodes a G/F hybrid in which the VSV G cytoplasmic tail was replaced by that of MV F. In contrast to MG/FV, MGV virions do not contain the MV matrix (M) protein. This demonstrates that virus assembly is possible in the absence of M; conversely, the cytoplasmic domain of F allows incorporation of M and enhances assembly. The formation of chimeric viruses was substantially delayed and the titers obtained were reduced about 50-fold in comparison to standard MV. In the novel chimeras, transcription and replication are mediated by the MV ribonucleoproteins but the envelope glycoproteins dictate the host range. Mice immunized with the chimeric viruses were protected against lethal doses of wild-type VSV. These findings suggest that it is feasible to construct MV variants bearing a variety of different envelopes for use as vaccines or for gene therapeutic purposes.
Autologous and Heterologous Neutralization Analyses of Primary Feline Immunodeficiency Virus Isolates
Authors: del Mauro Daniela , Matteucci Donatella , Giannecchini Simone , Maggi Fabrizio , Pistello Mauro , Bendinelli Mauro
Published: December 2019

Feline immunodeficiency virus (FIV) provides a model system with which the significance of neutralizing antibody (NA) in immunosuppressive lentivirus infections may be studied. To date, no detailed analysis of the neutralization properties of primary FIV isolates has been reported. In this study, we have conducted the first comprehensive study of the sensitivity to autologous and heterologous neutralization in a lymphoid cell-based assay of 15 primary FIV isolates and, for comparison, of one tissue culture-adapted strain. Primary isolates in general proved highly NA resistant, although there was considerable individual variation. Variation was also observed in the capacity of immune sera to neutralize heterologous FIV isolates. The ability of sera to neutralize isolates or for isolates to be neutralized by sera did not correlate with epidemiological and genetic relatedness or with the quasispecies complexity of the isolates. From the study of specific-pathogen-free cats experimentally infected with viral isolates associated with NA of different breadths, it appears that the development of FIV vaccines cannot rely on the existence of viral strains inherently capable of inducing especially broad NA responses.
Requirement for an Aromatic Amino Acid or Histidine at the N Terminus of Sindbis Virus RNA Polymerase
Authors: Shirako Yukio , Strauss James H.
Published: December 2019

The N terminal amino acid of nonstructural protein nsP4, the viral RNA polymerase, is a tyrosine in all sequenced alphaviruses; this is a destabilizing amino acid for the N-end rule pathway and results in rapid degradation of nsP4 produced in infected cells or in reticulocyte lysates. We have constructed 11 mutants of Sindbis virus bearing Phe, Ala, Thr, Cys, Leu, Met, Asn, Gln, Glu, Arg, or Pro at the N terminus of nsP4. Translation of RNAs in reticulocyte lysates showed that cleavage at the nsP3/nsP4 site occurred efficiently for all mutants except for Glu-nsP4, which was cleaved inefficiently, and Pro-nsP4, which was not detectably cleaved, and that Tyr, Cys, Leu, Arg, and Phe destabilized nsP4 but Ala, Met, Thr, Asn, Gln, and Glu stabilized nsP4 to various extents. The viability of the mutants was examined by transfection of chicken cells at 30 or 40°C. The Phe-nsP4 mutant formed large plaques at both temperatures. The Met-nsP4 mutant was also viable but formed small plaques at 30°C and minute plaques at 40°C. The remaining mutants did not form plaques at either temperature. However, after prolonged incubation at 30°C, all the mutants except Glu-nsP4 and Pro-nsP4 produced viable viruses. In the case of Cys-, Leu-, Asn-, Gln-, or Arg-nsP4, revertants that were indistinguishable in plaque phenotype from the wild-type virus arose by same-site reversion to Tyr, Trp, Phe, or His by a single nucleotide substitution in the original mutant codon. Viable viruses also arose from the Ala-, Leu-, Cys-, Thr-, Asn-, Gln-, and Arg-nsP4 mutants that retained the original mutations at the N terminus of nsP4, but these viruses formed smaller plaques than the wild-type virus and many were temperature sensitive. Our results indicate that only nsP4s bearing N-terminal Tyr, Phe, Trp, or His have wild-type or near-wild-type activity for RNA replication and that rapid degradation of nsP4 is not a prerequisite for its function. nsP4s bearing other N-terminal residues, with the exception of Met-nsP4, have only very low or negligible activity, so that no detectable infectious virus can be produced. However, suppressor mutations can arise that enable most such nsP4s to regain significant but still suboptimal activity.
Analysis of Host Range Restriction Determinants in the Rabbit Model: Comparison of Homologous and Heterologous Rotavirus Infections
Authors: Ciarlet Max , Estes Mary K. , Barone Christopher , Ramig Robert F. , Conner Margaret E.
Published: December 2019

The main limitation of both the rabbit and mouse models of rotavirus infection is that human rotavirus (HRV) strains do not replicate efficiently in either animal. The identification of individual genes necessary for conferring replication competence in a heterologous host is important to an understanding of the host range restriction of rotavirus infections. We recently reported the identification of the P type of the spike protein VP4 of four lapine rotavirus strains as being P[14]. To determine whether VP4 is involved in host range restriction in rabbits, we evaluated infection in rotavirus antibody-free rabbits inoculated orally with two P[14] HRVs, PA169 (G6) and HAL1166 (G8), and with several other HRV strains and animal rotavirus strains of different P and G types. We also evaluated whether the parental rhesus rotavirus (RRV) (P5B[3], G3) and the derived RRV-HRV reassortant candidate vaccine strains RRV × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4) would productively infect rabbits. Based on virus shedding, limited replication was observed with the P[14] HRV strains and with the SA11 Cl3 (P[2], G3) and SA11 4F (P6[1], G3) animal rotavirus strains, compared to the homologous ALA strain (P[14], G3). However, even limited infection provided complete protection from rotavirus infection when rabbits were challenged orally 28 days postinoculation (DPI) with 10
3
50% infective doses of ALA rabbit rotavirus. Other HRVs did not productively infect rabbits and provided no significant protection from challenge, in spite of occasional seroconversion. Simian RRV replicated as efficiently as lapine ALA rotavirus in rabbits and provided complete protection from ALA challenge. Live attenuated RRV reassortant vaccine strains resulted in no, limited, or productive infection of rabbits, but all rabbits were completely protected from heterotypic ALA challenge. The altered replication efficiency of the reassortants in rabbits suggests a role for VP7 in host range restriction. Also, our results suggest that VP4 may be involved in, but is not exclusively responsible for, host range restriction in the rabbit model. The replication efficiency of rotavirus in rabbits also is not controlled by the product of gene 5 (NSP1) alone, since a reassortant rotavirus with ALA gene 5 and all other genes from SA11 was more severely replication restricted than either parental rotavirus strain.
Human Immunodeficiency Virus Replication and Genotypic Resistance in Blood and Lymph Nodes after a Year of Potent Antiretroviral Therapy
Authors: Günthard Huldrych F. , Wong Joseph K. , Ignacio Caroline C. , Guatelli John C. , Riggs Nanette L. , Havlir Diane V. , Richman Douglas D.
Published: December 2019

Potent antiretroviral therapy can reduce human immunodeficiency virus (HIV) in plasma to levels below the limit of detection for up to 2 years, but the extent to which viral replication is suppressed is unknown. To search for ongoing viral replication in 10 patients on combination antiretroviral therapy for up to 1 year, the emergence of genotypic drug resistance across different compartments was studied and correlated with plasma viral RNA levels. In addition, lymph node (LN) mononuclear cells were assayed for the presence of multiply spliced RNA. Population sequencing of HIV-1
pol
was done on plasma RNA, peripheral blood mononuclear cell (PBMC) RNA, PBMC DNA, LN RNA, LN DNA, and RNA from virus isolated from PBMCs or LNs. A special effort was made to obtain sequences from patients with undetectable plasma RNA, emphasizing the rapidly emerging lamivudine-associated M184V mutation. Furthermore, concordance of drug resistance mutations across compartments was investigated. No evidence for viral replication was found in patients with plasma HIV RNA levels of <20 copies/ml. In contrast, evolving genotypic drug resistance or the presence of multiply spliced RNA provided evidence for low-level replication in subjects with plasma HIV RNA levels between 20 and 400 copies/ml. All patients failing therapy showed multiple drug resistance mutations in different compartments, and multiply spliced RNA was present upon examination. Concordance of nucleotide sequences from different tissue compartments obtained concurrently from individual patients was high: 98% in the protease and 94% in the reverse transcriptase regions. These findings argue that HIV replication differs significantly between patients on potent antiretroviral therapy with low but detectable viral loads and those with undetectable viral loads.
The Extracellular Domain of Vaccinia Virus Protein B5R Affects Plaque Phenotype, Extracellular Enveloped Virus Release, and Intracellular Actin Tail Formation
Authors: Mathew Elizabeth , Sanderson Christopher M. , Hollinshead Michael , Smith Geoffrey L.
Published: December 2019

Vaccinia virus produces two morphologically distinct forms of infectious virus, termed intracellular mature virus (IMV) and extracellular enveloped virus (EEV). EEV is important for virus dissemination within a host and has different surface proteins which bind to cell receptors different from those used by IMV. Six genes are known to encode EEV-specific proteins. One of these, B5R, encodes a 42-kDa glycoprotein with amino acid similarity to members of the complement control protein superfamily and contains four copies of a 50- to 70-amino-acid repeat called the short consensus repeat (SCR). Deletion of B5R causes a small-plaque phenotype, a 10-fold reduction in EEV formation, and virus attenuation in vivo. In this study, we inserted mutated versions of the B5R gene lacking different combinations of the SCRs into a virus deletion mutant lacking the B5R gene. The resultant viruses each formed small plaques only slightly larger than those of the deletion mutant; however, the virus containing only SCR 1 formed plaques slightly larger than those of viruses with SCRs 1 and 2 or SCRs 1, 2, and 3. All of these viruses produced approximately 50-fold more infectious EEV than wild-type virus and formed comet-shaped plaques under liquid overlay. Despite producing more EEV, the mutant viruses were unable to induce the polymerization of actin on intracellular virus particles. The implications of these results for our understanding of EEV formation, release, and infectivity are discussed.
Measles Virus DNA Vaccination: Antibody Isotype Is Determined by the Method of Immunization and by the Nature of both the Antigen and the Coimmunized Antigen
Authors: Cardoso Alicia I. , Sixt Nathalie , Vallier Agnes , Fayolle Joel , Buckland Robin , Wild T. Fabian
Published: December 2019

Plasmids encoding the measles virus hemagglutinin (HA) and nucleoprotein (NP) proteins inoculated into the skin of BALB/c mice by the gene gun method induced both humoral and cytotoxic lymphocyte class I-restrict- ed immune responses. Although intramuscular immunization induces the immunoglobulin G2a (IgG2a) antibody isotype for both antigens, with gene gun immunization, the NP still generated mainly IgG2a and the major isotype induced by the HA was IgG1. Interestingly, gene gun coimmunization of HA and NP plasmids resulted in a dominant IgG1 HA response and the switching of antibodies generated against the NP to the IgG1 isotype.
The Glycoprotein D (US6) Homolog Is Not Essential for Oncogenicity or Horizontal Transmission of Marek’s Disease Virus
Authors: Anderson Amy S. , Parcells Mark S. , Morgan Robin W.
Published: December 2019

RB1BUS6
lacgpt
, a Marek’s disease virus (MDV) mutant having a disrupted glycoprotein D (gD) homolog gene, established infection and induced tumors in chickens exposed to it by inoculation or by contact. Lymphoblastoid cell lines derived from RB1BUS6
lacgpt
-induced tumors harbored only the mutant virus. These results provide strong evidence that an intact gD homolog gene is not essential for oncogenicity or horizontal transmission of MDV.
Glucagon Treatment Interferes with an Early Step of Duck Hepatitis B Virus Infection
Authors: Hild Marc , Weber Olaf , Schaller Heinz
Published: December 2019

The effect of glucagon on the establishment of hepadnavirus infection was studied in vitro with the duck hepatitis B virus (DHBV) model. The presence of the peptide hormone throughout infection or starting up to 8 h after virus uptake resulted in a dose-dependent reduction in the levels of intra- and extracellular viral gene products and of secreted virions. Treatment with forskolin or dibutyryl-cyclic AMP, two drugs that also stimulate the cyclic AMP (cAMP) signal transduction pathway, resulted in comparable inhibition, suggesting that the inhibitor effect is related to changes in the activity of protein kinase A. In persistently infected hepatocytes, only a slight, but continuous, decrease in viral replication was observed upon prolonged drug treatment. Time course analysis, including detection of DHBV covalently closed circular (ccc) DNA templates, revealed that glucagon acts late during the establishment of infection, at a time when the virus is already internalized, but before detectable ccc DNA accumulation in the nucleus. These data suggest that nuclear import (and reimport) of DHBV DNA genomes from cytosolic capsids is subject to cAMP-mediated regulation by cellular factors responding to changes in the state of the host cell.
Mutation of the Lck-Binding Motif of Tip Enhances Lymphoid Cell Activation by Herpesvirus Saimiri
Authors: Duboise S. Monroe , Lee Heuiran , Guo Jie , Choi Joong-Kook , Czajak Susan , Simon Meredith , Desrosiers Ronald C. , Jung Jae U.
Published: December 2019

The proline-rich SH3-binding (SH3B) motif of the tyrosine kinase-interacting protein (Tip) of herpesvirus saimiri (HVS) is required for binding to the cellular Src family kinase Lck. We constructed a mutant form of HVS in which prolines in the SH3B motif of Tip were altered to alanines. This mutant form of Tip was incapable of binding to Lck. The mutant virus, HVS/Tip mSH3B, retained its ability to immortalize common marmoset lymphocytes in culture. In fact, common marmoset lymphocytes immortalized by the HVS/Tip mSH3B mutant displayed increased expression of HLA-DR lymphocyte activation marker, an altered pattern of tyrosine phosphorylation, increased expression of the tyrosine kinase Lyn, and a shift in electrophoretic mobility of Lck compared to cells immortalized by wild-type HVS. Experimental infection of common marmosets resulted in fulminant lymphoma with both HVS/Tip mSH3B and wild-type HVS. However, HVS/Tip mSH3B produced greater infiltration of affected organs by proliferating lymphoid cells compared to wild-type HVS. These results demonstrate that Tip binding to Lck is not necessary for transformation and that abrogation of Tip binding to Lck alters the characteristics of transformed cells and the severity of the pathologic lesions.
Separate DNA Elements Containing ATF/CREB and IE86 Binding Sites Differentially Regulate the Human Cytomegalovirus UL112-113 Promoter at Early and Late Times in the Infection
Authors: Rodems Steven M. , Clark Charles L. , Spector Deborah H.
Published: December 2019

The human cytomegalovirus (HCMV) UL112-113 promoter represents a useful model for studying temporal regulation of viral gene expression. Stimulation of this promoter by the 86-kDa immediate-early protein (IE86) is controlled by sequences between nucleotides −113 and −59, which include both an ATF/CREB and an IE86 binding site. In transient assays, the ATF/CREB site is essential, and the IE86 site, although nonessential, can enhance transcription. With recombinant viruses, we have assessed the function of these promoter elements in the context of the viral genome. Transcription from the inserted UL112-113 promoter shows the same temporal pattern as the endogenous promoter, including the switch to an upstream RNA start site late in infection. Deletion of sequences containing the IE86 site results in a decrease in the level of early transcription and elimination of late transcription. In contrast, when the ATF/CREB site is deleted, early RNA synthesis is almost completely abolished, but late transcription is comparable to that of the wild type, with repositioning of the RNA start site downstream by the number of nucleotides deleted. Replacement of sequences between −108 and −95 with the HCMV
cis
-repression signal from the major immediate-early promoter had no effect on the level of late RNAs but resulted in the repositioning of the RNA start site 39 nucleotides upstream. These results suggest that the ATF/CREB site is functional only at early times, while sequences containing the IE86 site modulate the level of early RNAs and may be required for activating late transcription in a distance-dependent manner.
Herpes Simplex Virus DNA Packaging without Measurable DNA Synthesis
Authors: Church Geoffrey A. , Dasgupta Anindya , Wilson Duncan W.
Published: December 2019

Herpes simplex virus (HSV) type 1 DNA synthesis and packaging occur within the nuclei of infected cells; however, the extent to which the two processes are coupled remains unclear. Correct packaging is thought to be dependent upon DNA debranching or other repair processes, and such events commonly involve new DNA synthesis. Furthermore, the HSV UL15 gene product, essential for packaging, nevertheless localizes to sites of active DNA replication and may link the two events. It has previously been difficult to determine whether packaging requires concomitant DNA synthesis due to the complexity of these processes and of the viral life cycle; however, we have recently described a model system which simplifies the study of HSV assembly. Cells infected with HSV strain
ts
Prot.A accumulate unpackaged capsids at the nonpermissive temperature of 39°C. Following release of the temperature block, these capsids proceed to package viral DNA in a single, synchronous wave. Here we report that, when DNA replication was inhibited prior to release of the temperature block, DNA packaging and later events in viral assembly nevertheless occurred at near-normal levels. We conclude that, under our conditions, HSV DNA packaging does not require detectable levels of DNA synthesis.
Genotype-Specific Complementation of Hepatitis Delta Virus RNA Replication by Hepatitis Delta Antigen
Authors: Casey John L. , Gerin John L.
Published: December 2019

Characterizations of genetic variations among hepatitis delta virus (HDV) isolates have focused principally on phylogenetic analysis of sequences, which vary by 30 to 40% among three genotypes and about 10 to 15% among isolates of the same genotype. The significance of the sequence differences has been unclear but could be responsible for pathogenic variations associated with the different genotypes. Studies of the mechanisms of HDV replication have been limited to cDNA clones from HDV genotype I, which is the most common. To perform a comparative analysis of HDV RNA replication in genotypes I and III, we have obtained a full-length cDNA clone from an HDV genotype III isolate. In transfected Huh-7 cells, the functional roles of the two forms of the viral protein, hepatitis delta antigen (HDAg), in HDV RNA replication are similar for both genotypes I and III; the short form is required for RNA replication, while the long form inhibits replication. For both genotypes, HDAg was able to support replication of RNAs of the same genotype that were mutated so as to be defective for HDAg production. Surprisingly, however, neither genotype I nor genotype III HDAg was able to support replication of such mutated RNAs of the other genotype. The inability of genotype III HDAg to support replication of genotype I RNA could have been due to a weak interaction between the RNA and HDAg. The clear genotype-specific activity of HDAg in supporting HDV RNA replication confirms the original categorization of HDV sequences in three genotypes and further suggests that these should be referred to as types (i.e., HDV-I and HDV-III) rather than genotypes.
Specific Interactions between Retrovirus Env and Gag Proteins in Rat Neurons
Authors: Weclewicz Katarzyna , Ekström Maria , Kristensson Krister , Garoff Henrik
Published: December 2019

In this work we have studied the intracellular localization properties of the Gag and Env proteins of Moloney murine leukemia virus (MLV) and human immunodeficiency virus (HIV) in dorsal root ganglion (DRG) neurons of rat. These neurons form thick bundles of axons, which facilitates protein localization studies by immunofluorescence analyses. When such neuron cultures were infected with recombinant Semliki Forest virus particles carrying the
gag
genes of either retrovirus, the expressed Gag proteins were localized to both the somatic and the axonal regions of the DRG neurons. In contrast, the Env proteins were confined only to the somatic region. When the Gag and Env proteins were coexpressed, the Gag proteins were also excluded from the axons. This effect of the Env proteins was shown to be dependent on the concentration of the Gag proteins in the neuron and also to be specific for homologous pairs of retrovirus proteins. Therefore, the results suggest that there are specific interactions between the Env and the Gag proteins of MLV and HIV in the DRG neurons.
Increased Induction of Apoptosis by a Sendai Virus Mutant Is Associated with Attenuation of Mouse Pathogenicity
Authors: Itoh Masae , Hotta Hak , Homma Morio
Published: December 2019

An avirulent mutant of Sendai virus, Ohita-MVC11 (MVC11), was generated from a highly virulent field strain, Ohita-M1 (M1), through successive passages in LLC-MK
2
cell cultures (M. Itoh, Y. Isegawa, H. Hotta, and M. Homma, J. Gen. Virol. 78:3207-3215, 1997). In LLC-MK
2
cells, MVC11 induced a high degree of apoptotic cell death that was demonstrated by chromatin condensation of the nucleus and DNA fragmentation, and production of MVC11 declined markedly after prolonged culture. On the other hand, M1 did not induce prominent apoptosis and maintained high virus titers. In primary mouse pulmonary epithelial cell cultures, M1 replicated rather slowly to reach maximum level of virus production at 3 days postinfection, and high levels of virus production were maintained thereafter without causing apoptosis. In contrast, MVC11, which produced 20 times more progeny virus than M1 at 1 day postinfection, induced a high degree of apoptotic cell death before the virus replication cycle was completed. Accordingly, the production of progeny virus was strongly inhibited thereafter. In the lungs of mice infected with MVC11, virus antigens and signals of DNA fragmentation detected by the in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique colocalized in bronchial epithelial cells, clearly demonstrating that infection by MVC11 triggered apoptosis in vivo as well as in vitro. These results suggest the possibility that induction of apoptosis by MVC11 plays an important role in attenuation of mouse pathogenicity by restricting progeny virus production in the lung. The C protein was shown to have the capacity to induce apoptosis, and the increased level of the C protein in MVC11-infected cells was considered to account partly, if not entirely, for the induction of apoptosis.
Human Parvovirus B19 Nonstructural (NS1) Protein Induces Apoptosis in Erythroid Lineage Cells
Authors: Moffatt Stanley , Yaegashi Nobuo , Tada Kohtaro , Tanaka Nobuyuki , Sugamura Kazuo
Published: December 2019

Infection of erythroid-lineage cells by human parvovirus B19 is characterized by a gradual cytocidal effect. Accumulating evidence now implicates the nonstructural (NS1) protein of the virus in cytotoxicity, but the mechanism underlying the NS1-induced cell death is not known. Using a stringent regulatory system, we demonstrate that NS1 cytotoxicity is closely related to apoptosis, as evidenced by cell morphology, genomic DNA fragmentation, and cell cycle analysis with the human erythroleukemia cell line K562 and the erythropoietin-dependent megakaryocytic cell line UT-7/Epo. Apoptosis was significantly inhibited by an interleukin-1β (IL-1β)-converting enzyme (ICE)/CED-3 family protease inhibitor, Ac-DEVD-CHO (CPP32; caspase 3), whereas a similar inhibitor of ICE (caspase 1), Ac-YVAD-CHO, had no effect. Furthermore, stable expression of the human Bcl-2 proto-oncogene resulted in near-total protection from cell death in response to NS1 induction. Mutations engineered into the nucleoside triphosphate-binding domain of NS1 significantly rescued cells from NS1-induced apoptosis without having any effect on NS1-induced activation of the IL-6 gene expression which is mediated by NF-κB. Furthermore, using pentoxifylline, an inhibitor of NF-κB activation, we demonstrate that the NF-κB-mediated IL-6 activation by NS1 is uncoupled from the apoptotic pathway. This functional dissection indicates a complexity underlying the biochemical function of human parvovirus NS1 in transcriptional activation and induction of apoptosis. Our findings indicate that NS1 of parvovirus B19 induces cell death by apoptosis in at least erythroid-lineage cells by a pathway that involves caspase 3, whose activation may be a key event during NS1-induced cell death.
Detection of Hepatitis G Virus Replication Sites by Using Highly Strand-Specific Tth-Based Reverse Transcriptase PCR
Authors: Laskus Tomasz , Radkowski Marek , Wang Lian-Fu , Vargas Hugo , Rakela Jorge
Published: December 2019

The replication sites of the recently discovered hepatitis G virus (HGV) remain unknown. Using highly strand-specific Tth-based reverse transcriptase PCR, we searched for the presence of viral RNA negative strand in multiple autopsy tissues from four patients with AIDS and in peripheral blood mononuclear cells from six other human immunodeficiency virus-positive patients. Negative-strand HGV RNA was detected in three of four bone marrow samples, in two of two spleen samples, and in one of four liver tissue samples. However, the specific cellular site of replication within the positive tissues was not determined. This study does not support HGV as a primary hepatotropic virus.
Viruses and Cells with Mutations Affecting Viral Entry Are Selected during Persistent Rotavirus Infections of MA104 Cells
Authors: Mrukowicz Jacek Z. , Wetzel J. Denise , Goral Mehmet I. , Fogo Agnes B. , Wright Peter F. , Dermody Terence S.
Published: December 2019

To better understand mechanisms of persistent rotavirus infections of cultured cells, we established independent, persistently infected cultures of MA104 cells, using rotavirus strain SA11. The cultures were either passaged when the cells reached confluence or supplemented with fresh medium every 7 days. Viral titers in culture lysates varied from 10
4
to 10
7
PFU per ml during 350 days of culture maintenance. Trypan blue staining indicated that 72 to 100% of cells in the cultures were viable, and immunocytochemical staining using a monoclonal antibody directed against viral protein VP6 demonstrated that 38 to 63% of the cells contained rotavirus antigen. We tested the capacity of rotaviruses isolated from the persistently infected cultures (PI viruses) to infect cells cured of persistent infection. Although wild-type (wt) and PI viruses produced equivalent yields in parental MA104 cells, PI viruses produced greater yields than wt virus in cured cells, which indicates that viruses and cells coevolve during persistent rotavirus infections of MA104 cells. To determine whether mutations in viruses and cells selected during these persistent infections affect viral entry, we tested the effect of trypsin treatment of the viral inoculum on growth of wt and PI viruses. Trypsin pretreatment is required for postattachment penetration of rotavirus virions into cells. In contrast to the case with wt virus, PI viruses produced equivalent yields with and without trypsin pretreatment in parental MA104 cells. However, PI viruses required trypsin pretreatment for efficient growth in cured cells. These results indicate that mutant viruses and cells are selected during maintenance of persistent rotavirus infections of MA104 cells and suggest that mutations in each affect trypsin-dependent steps in rotavirus entry.
CBF, Myb, and Ets Binding Sites Are Important for Activity of the Core I Element of the Murine Retrovirus SL3-3 in T Lymphocytes
Authors: Zaiman Ari L. , Nieves Angel , Lenz Jack
Published: December 2019

Transcriptional enhancers within the long terminal repeats of murine leukemia viruses are major determinants of the pathogenic properties of these viruses. Mutations were introduced into the adjacent binding sites for three transcription factors within the enhancer of the T-cell-lymphomagenic virus SL3-3. The sites that were tested were, in 5′-to-3′ order, a binding site for core binding factor (CBF) called core II, a binding site for c-Myb, a site that binds members of the Ets family of factors, and a second CBF binding site called core I. Mutation of each site individually reduced transcriptional activity in T lymphocytes. However, mutation of the Myb and core I binding sites had larger effects than mutation of the Ets or core II site. The relative effects on transcription in T cells paralleled the effects of the same mutations on viral lymphomagenicity, consistent with the idea that the role of these sequences in viral lymphomagenicity is indeed to regulate transcription in T cells. Mutations were also introduced simultaneously into multiple sites in the SL3-3 enhancer. The inhibitory effects of these mutations indicated that the transcription factor in T cells that recognizes the core I element of SL3-3, presumably CBF, needed to synergize with one or more factors bound at the upstream sites to function. This was tested further by generating a multimer construct that contained five tandem core I elements linked to a basal long terminal repeat promoter. This construct was inactive in T cells. However, transcriptional activity was detected with a multimer construct in which the transcription factor binding sites upstream of the core were also present. These results are consistent with the hypothesis that CBF requires heterologous transcription factors bound at nearby sites to function in T cells.