bluuebunny/crossref_metadata_2025 huggingface dataset:
Rescue of Newcastle Disease Virus from Cloned cDNA: Evidence that Cleavability of the Fusion Protein Is a Major Determinant for Virulence
Authors: Peeters Ben P. H. , de Leeuw Olav S. , Koch Guus , Gielkens Arno L. J.
Published: December 2019

A full-length cDNA clone of Newcastle disease virus (NDV) vaccine strain LaSota was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Transfection of this plasmid into cells that were infected with a recombinant fowlpoxvirus that expressed T7 RNA polymerase, resulted in the synthesis of antigenomic NDV RNA. This RNA was replicated and transcribed by the viral NP, P, and L proteins, which were expressed from cotransfected plasmids. After inoculation of the transfection supernatant into embryonated specific-pathogen-free eggs, infectious virus derived from the cloned cDNA was recovered. By introducing three nucleotide changes in the cDNA, we generated a genetically tagged derivative of the LaSota strain in which the amino acid sequence of the protease cleavage site (GGRQGR↓L) of the fusion protein F0 was changed to the consensus cleavage site of virulent NDV strains (GRRQRR↓F). Pathogenicity tests in day-old chickens showed that the strain derived from the unmodified cDNA was completely nonvirulent (intracerebral pathogenicity index [ICPI] = 0.00). However, the strain derived from the cDNA in which the protease cleavage site was modified showed a dramatic increase in virulence (ICPI = 1.28 out of a possible maximum of 2.0). Pulse-chase labeling of cells infected with the different strains followed by radioimmunoprecipitation of the F protein showed that the efficiency of cleavage of the F0 protein was greatly enhanced by the amino acid replacements. These results demonstrate that genetically modified NDV can be recovered from cloned cDNA and confirm the supposition that cleavage of the F0 protein is a key determinant in virulence of NDV.
Dynamics of Nontypical Apoptotic Morphological Changes Visualized by Green Fluorescent Protein in Living Cells with Infectious Pancreatic Necrosis Virus Infection
Authors: Hong Jiann-Ruey , Lin Tai-Lang , Yang Jer-Yen , Hsu Ya-Li , Wu Jen-Leih
Published: December 2019

Morphologically, apoptotic cells are characterized by highly condensed membrane blebbing and formation of apoptotic bodies. Recently, we reported that apoptosis precedes necrosis in a fish cell line infected with infectious pancreatic necrosis virus (IPNV). In the present study, we tested the possibility that nontypical apoptosis is a component of IPNV-induced fish cell death. A variant type of green fluorescent protein (EGFP) was expressed in a fish cell line such that EGFP served as a protein marker for visualizing dynamic apoptotic cell morphological changes and for tracing membrane integrity changes during IPNV infection. Direct morphological changes were visualized by fluorescence microscopy by EGFP in living cells infected with IPNV. The nontypical apoptotic morphological change stage occurred during the pre-late stage (6 to 7 h postinfection). Nontypical apoptotic features, including highly condensed membrane blebbing, occurred during the middle apoptotic stage. At the pre-late apoptotic stage, membrane vesicles quickly formed, blebbed, and were finally pinched off from the cell membrane. At the same time, at this pre-late apoptotic stage, apoptotic cells formed unique small holes in their membranes that ranged from 0.39 to 0.78 μm according to examination by scanning electron microscopy and immunoelectron microscopy. Quantitation of the intra- and extracellular release of EGFP by CHSE-214-EGFP cells after IPNV infection was done by Western blotting and fluorometry. Membrane integrity was quickly lost during the late apoptotic stage (after 8 h postinfection), and morphological change and membrane integrity loss could be prevented and blocked by treatment with apoptosis inhibitors such as cycloheximide, genistein, and EDTA before IPNV infection. Together, these findings show the apoptotic features at the onset of pathology in host cells (early and middle apoptotic stages), followed secondarily by nontypical apoptosis (pre-late apoptotic stage) and then by postapoptotic necrosis (late apoptotic stage), of a fish cell line. Our results demonstrate that nontypical apoptosis is a component of IPNV-induced fish cell death.
Microglial Activation Varies in Different Models of Creutzfeldt-Jakob Disease
Authors: Baker Christopher A. , Lu Zhi Yun , Zaitsev Igor , Manuelidis Laura
Published: December 2019

Progressive changes in host mRNA expression can illuminate crucial pathogenetic pathways in infectious disease. We examined general and specific approaches to mRNA expression in three rodent models of Creutzfeldt-Jakob disease (CJD). Each of these models displays distinctive neuropathology. Although mRNAs for the chemokine receptor CCR5, the lysosomal protease cathepsin S, and the pleiotropic cytokine transforming growth factor β1 (TGF-β1) were progressively upregulated in rodent CJD, the temporal patterns and peak magnitudes of each of these transcripts varied substantially among models. Cathepsin S and TGF-β1 were elevated more than 15-fold in mice and rats infected with two different CJD strains, but not in CJD-infected hamsters. In rats, an early activation of microglial transcripts preceded obvious deposits of prion protein (PrP) amyloid. However, in each of the three CJD models, the upregulation of CCR5, cathepsin S, and TGF-β1 was variable with respect to the onset of PrP pathology. These results show glial cell involvement varies as a consequence of the agent strain and species infected. Although neurons are generally assumed to be the primary sites for agent replication and abnormal PrP formation, microglia may be targeted by some agent strains. In such instances, microglia can both process PrP to become amyloid and can enhance neuronal destruction. Because microglia can participate in agent clearance, they may also act as chronic reservoirs of infectivity. Finally, the results here strongly suggest that TGF-β1 can be an essential signal for amyloid deposition.
RGD Inclusion in the Hexon Monomer Provides Adenovirus Type 5-Based Vectors with a Fiber Knob-Independent Pathway for Infection
Authors: Vigne Emmanuelle , Mahfouz Irene , Dedieu Jean-Francois , Brie Anne , Perricaudet Michel , Yeh Patrice
Published: December 2019

Hypervariable region 5 (HVR5) is a hydrophilic, serotypically nonconserved loop of the hexon monomer which extrudes from the adenovirus (Ad) capsid. We have replaced the HVR5 sequence of Ad5 with that of heterologous peptides and studied their effects on virus viability and peptide accessibility. A poliovirus model epitope was first inserted in a series of nine “isogenic” viruses that differed in their flanking spacers. Whereas virus productivity was not profoundly altered by any of these modifications, immunoprecipitation experiments under nondenaturing conditions demonstrated that epitope recognition by its cognate monoclonal antibody (C3 MAb) was strongly linker dependent and correlated perfectly with the ability of C3 MAb to inhibit transgene delivery and expression. An α
v
-specific ligand (DCRGDCF) was then inserted in a suitable linker context to investigate whether hexon-modified capsids would enhance the transduction of cells displaying limiting amounts of the virus attachment receptors. Interestingly, although hexon has never been implicated in Ad entry, the modified virus significantly increased the transduction of human vascular smooth muscle cells in vitro. Competition experiments with 293 cells saturated with recombinant knob further indicated that the hexon-modified virus could use an additional, knob-independent pathway for entry. We concluded that genetic engineering of the Ad5 hexon monomer constitutes a novel and feasible approach to equip the virus with additional targeting ligands.
Intracisternal Type A Particle-Mediated Activation of the
Notch4/int3
Gene in a Mouse Mammary Tumor: Generation of Truncated
Notch4/int3
mRNAs by Retroviral Splicing Events
Authors: Lee Jong-Seo , Haruna Tatsuya , Ishimoto Akinori , Honjo Tasuku , Yanagawa Shin-ichi
Published: December 2019

The
int3
oncogene was discovered as a frequent target in mouse mammary tumor virus-induced mammary tumors and encodes the intracellular domain of a Notch4/int3 protein. In one spontaneous mammary tumor, no. 9, that developed in a BALB/c mouse, we have found an insertion of a 1.2-kb sequence, consisting of a 5′ long terminal repeat and
gag
sequences of an intracisternal type A particle (IAP) as well as an extra copy of the
Notch4/int3
genomic sequences containing exons 23 and 24, into the intron between exons 24 and 25 of the
Notch4/int3
gene. In this tumor, unique splicing events between the IAP and the
Notch4/int3
sequences generated two types of IAP-
Notch4/int3
fusion transcripts encoding two different portions of the intracellular domain of Notch4/int3 proteins: one with a RAM domain and the other without. Interestingly, these two proteins showed different subcellular localizations in a mouse mammary epithelial cell line, HC-11.
Effects of Exonuclease Activity and Nucleotide Selectivity of the Herpes Simplex Virus DNA Polymerase on the Fidelity of DNA Replication In Vivo
Authors: Hwang Ying T. , Liu Bu-Yuan , Hong Chi-Yuan , Shillitoe Edward J. , Hwang Charles B. C.
Published: December 2019

A mutagenesis system was developed for the in vivo study of the fidelity of DNA replication mediated by wild-type herpes simplex virus type 1 (HSV-1) strain KOS and its polymerase (Pol) mutant derivatives PAA
r
5, Y7, and YD12. The pHOS1 shuttle plasmid, which contained the
SupF
mutagenesis marker gene and the HSV

ori
s

sequence, was used for analysis of the mutation frequency and the mutation spectrum. All three Pol mutants induced significant increases in the mutation frequencies of the target gene, despite the fact that PAA
r
5 was previously shown to have an antimutator phenotype by the thymidine kinase mutagenesis assay (J. D. Hall, D. M. Coen, B. L. Fisher, M. Weisslitz, S. Randall, R. E. Almy, P. Gelep, and P. A. Schaffer, Virology 132:26–37, 1984; C. B. C. Hwang and J.-H. Chen, Gene 152:191–193, 1995). Altered spectra of mutated target genes induced by these three mutants were also observed. The relative frequencies of both deletion and complex mutations found in mutants induced by exonuclease-proficient Pols were significantly higher than those induced by exonuclease-deficient Pols. On the other hand, the exonuclease-deficient Pols induced significant increases in the frequency of base substitutions, which comprised predominantly G · C-to-T · A transversions, as well as mutations at additional hot spots. These results suggest that the HSV-1 DNA Pol can incorporate purine-purine or pyrimidine-pyrimidine mispaired bases which may be preferentially proofread by its intrinsic exonuclease activity. Furthermore, the effects of the sequence context of the target gene and the assay method should also be considered carefully in any analysis of replication fidelity.
Combinatorial Screening and Intracellular Antiviral Activity of Hairpin Ribozymes Directed against Hepatitis B Virus
Authors: zu Putlitz Jasper , Yu Qiao , Burke John M. , Wands Jack R.
Published: December 2019

A combinatorial screening method has been used to identify hairpin ribozymes that inhibit hepatitis B virus (HBV) replication in transfected human hepatocellular carcinoma (HCC) cells. A hairpin ribozyme library (5 × 10
5
variants) containing a randomized substrate-binding domain was used to identify accessible target sites within 3.3 kb of full-length in vitro-transcribed HBV pregenomic RNA. Forty potential target sites were found within the HBV pregenomic RNA, and 17 sites conserved in all four subtypes of HBV were chosen for intracellular inhibition experiments. Polymerase II and III promoter expression constructs for corresponding hairpin ribozymes were generated and cotransfected into HCC cells together with a replication-competent dimer of HBV DNA. Four ribozymes inhibited HBV replication by 80, 69, 66, and 49%, respectively, while catalytically inactive mutant forms of these ribozymes affected HBV replication by 36, 28, 0, and 0%. These findings indicate that the inhibitory effects on HBV replication were largely mediated by the catalytic activity of the ribozymes. In conclusion, we have identified catalytically active RNAs by combinatorial screening that mediate intracellular antiviral effects on HBV.
Mapping of Functional Elements in the Stem-Anchor Region of Tick-Borne Encephalitis Virus Envelope Protein E
Authors: Allison Steven L. , Stiasny Karin , Stadler Konrad , Mandl Christian W. , Heinz Franz X.
Published: December 2019

Envelope protein E of the flavivirus tick-borne encephalitis virus mediates membrane fusion, and the structure of the N-terminal 80% of this 496-amino-acid-long protein has been shown to differ significantly from that of other viral fusion proteins. The structure of the carboxy-terminal 20%, the stem-anchor region, is not known. It contains sequences that are important for membrane anchoring, interactions with prM (the precursor of membrane protein M) during virion assembly, and low-pH-induced structural changes associated with the fusion process. To identify specific functional elements in this region, a series of C-terminal deletion mutants were constructed and the properties of the resulting truncated recombinant E proteins were examined. Full-length E proteins and proteins lacking the second of two predicted transmembrane segments were secreted in a particulate form when coexpressed with prM, whereas deletion of both segments resulted in the secretion of soluble homodimeric E proteins. Sites located within a predicted α-helical region of the stem (amino acids 431 to 449) and the first membrane-spanning region (amino acids 450 to 472) were found to be important for the stability of the prM-E heterodimer but not essential for prM-mediated intracellular transport and secretion of soluble E proteins. A separate site in the stem, also corresponding to a predicted α-helix (amino acids 401 to 413), was essential for the conversion of soluble protein E dimers to a homotrimeric form upon low-pH treatment, a process resembling the transition to the fusogenic state in whole virions. This functional mapping will aid in the understanding of the molecular mechanisms of membrane fusion and virus assembly.
Identification of Multiple Protective Epitopes (Protectopes) in the Central Conserved Domain of a Prototype Human Respiratory Syncytial Virus G Protein
Authors: Plotnicky-Gilquin Hélène , Goetsch Liliane , Huss Thierry , Champion Thierry , Beck Alain , Haeuw Jean-François , Nguyen Thien Ngoc , Bonnefoy Jean-Yves , Corvaïa Nathalie , Power Ultan F.
Published: December 2019

A recombinant fusion protein (BBG2Na) comprising the central conserved domain of the respiratory syncytial virus subgroup A (RSV-A) (Long) G protein (residues 130 to 230) and an albumin binding domain of streptococcal protein G was shown previously to protect mouse upper (URT) and lower (LRT) respiratory tracts against intranasal RSV challenge (U. F. Power, H. Plotnicky-Gilquin, T. Huss, A. Robert, M. Trudel, S. Stahl, M. Uhlén, T. N. Nguyen, and H. Binz, Virology 230:155–166, 1997). Panels of monoclonal antibodies (MAbs) and synthetic peptides were generated to facilitate dissection of the structural elements of this domain implicated in protective efficacy. All MAbs recognized native RSV-A antigens, and five linear B-cell epitopes were identified; these mapped to residues 152 to 163, 165 to 172, 171 to 187 (two overlapping epitopes), and 196 to 204, thereby covering the highly conserved cysteine noose domain. Antibody passive-transfer and peptide immunization studies revealed that all epitopes were implicated in protection of the LRT, but not likely the URT, against RSV-A challenge. Pepscan analyses of anti-RSV-A and anti-BBG2Na murine polyclonal sera revealed lower-level epitope usage within the central conserved region in the former, suggesting diminished immunogenicity of the implicated epitopes in the context of the whole virus. However, Pepscan analyses of RSV-seropositive human sera revealed that all of the murine B-cell protective epitopes (protectopes) that mapped to the central conserved domain were recognized in man. Should these murine protectopes also be implicated in human LRT protection, their clustering around the highly conserved cysteine noose region will have important implications for the development of RSV vaccines.
Packaging-Competent Capsids of a Herpes Simplex Virus Temperature-Sensitive Mutant Have Properties Similar to Those of In Vitro-Assembled Procapsids
Authors: Rixon Frazer J. , McNab David
Published: December 2019

Newcomb and coworkers (W. W. Newcomb, F. L. Homa, D. R. Thomsen, F. P. Booy, B. L. Trus, A. C. Steven, J. V. Spencer, and J. C. Brown, J. Mol. Biol. 263:432–446, 1996; W. W. Newcomb, F. L. Homa, D. R. Thomsen, Z. Ye, and J. C. Brown, J. Virol. 68:6059–6063, 1994) have recently described an in vitro herpes simplex virus (HSV) capsid assembly product which, because of certain parallels between its properties and those of bacteriophage proheads, they have designated the procapsid. As in their bacteriophage counterparts, there are marked differences between the structures of the two types of particle, and conversion from the procapsid to the capsid form requires extensive reconfiguration of the subunits. This reconfiguration occurs spontaneously upon extended in vitro incubation. One of the distinctive features of the HSV procapsids is that, unlike mature capsids, they are unstable and disassemble upon storage at 2°C. Using a mutant of HSV type 1 (
ts
1201), which has a lesion in the protease responsible for maturational cleavage of the scaffolding protein, we have demonstrated that capsids present within cells infected at nonpermissive temperatures are also cryosensitive and disappear if the cells are incubated at 0°C. This suggests that
ts
1201 capsids may resemble procapsids in structure. However,
ts
1201 capsids remain cryosensitive following extended incubation at an elevated temperature and, therefore, do not appear to undergo the spontaneous reconfiguration seen with in vitro-assembled procapsids. The lesion in
ts
1201 is reversible, and capsids formed at the nonpermissive temperature can undergo maturational cleavage and go on to form infectious virions following downshift to permissive temperatures. The sensitivity of
ts
1201 capsids to low temperatures is closely correlated with the cleavage status of the scaffolding protein, suggesting that proteolysis may act to trigger their conversion to the stable form. The experiments described here provide the firmest evidence yet that the procapsid has a biologically relevant role in the virus life cycle.
DNA Microarrays of the Complex Human Cytomegalovirus Genome: Profiling Kinetic Class with Drug Sensitivity of Viral Gene Expression
Authors: Chambers James , Angulo Ana , Amaratunga Dhammika , Guo Hongqing , Jiang Ying , Wan Jackson S. , Bittner Anton , Frueh Klaus , Jackson Michael R. , Peterson Per A. , Erlander Mark G. , Ghazal Peter
Published: December 2019

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (α), early (β), early-late (γ1), and late (γ2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5′ noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for β, γ1, and γ2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.
Recombination between Two Identical Sequences within the Same Retroviral RNA Molecule
Authors: Zhang Jiayou , Sapp Christy M.
Published: December 2019

As a consequence of being diploid viruses, members of the
Retroviridae
have a high recombination rate. To measure recombination between two identical sequences within the same RNA molecule per round of retroviral replication cycle, a murine leukemia virus based vector (JZ442 + 3′ Hyg) has been constructed. It carries a drug resistance gene,
hyg
, and a 290-bp repeat sequence of the 3′
hyg
gene inserted into the 3′ untranslated region of the green fluorescent protein gene (
gfp
). Under fluorescence microscopy, Hyg
r
cells containing the recombinant proviruses were clear, while a green color was observed in the drug-resistant cells carrying the parental proviruses. The rate of recombination was determined by the ratio of the number of clear colonies to the total number of Hyg
r
colonies (green and clear colonies). The rate of recombination was found to be 62% by this method. The intermolecular recombination rate between an infectious virus bearing two copies of the 290-bp segment and a noninfectious chimeric RNA virus containing only a single copy of this sequence was also measured.
Colocalization and Membrane Association of Murine Hepatitis Virus Gene 1 Products and De Novo-Synthesized Viral RNA in Infected Cells
Authors: Shi Stephanie T. , Schiller Jennifer J. , Kanjanahaluethai Amornrat , Baker Susan C. , Oh Jong-Won , Lai Michael M. C.
Published: December 2019

Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (
pol
) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery, which may localize to different membrane structures in different cell lines.
Receptor-Mediated Moloney Murine Leukemia Virus Entry Can Occur Independently of the Clathrin-Coated-Pit-Mediated Endocytic Pathway
Authors: Lee Sunyoung , Zhao Yi , Anderson W. French
Published: December 2019

To investigate receptor-mediated Moloney murine leukemia virus (MoMuLV) entry, the green fluorescent protein (GFP)-tagged ecotropic receptor designated murine cationic amino acid transporter (MCAT-1) (MCAT-1-GFP) was constructed and expressed in 293 cells (293/MCAT-1-GFP). 293/MCAT-1-GFP cells displayed green fluorescence primarily at the cell membrane and supported wild-type levels of MoMuLV vector binding and transduction. Using immunofluorescence labeling and confocal microscopy, it was demonstrated that the surface envelope protein (SU) gp70 of MoMuLV virions began to appear inside cells 5 min after virus binding and was colocalized with MCAT-1-GFP. However, clathrin was not colocalized with MCAT-1-GFP, suggesting that MoMuLV entry, mediated by MCAT-1, does not involve clathrin. Double immunofluorescence labeling of SU and clathrin in 293 cells expressing untagged receptor (293/MCAT-1) gave the same results, i.e., SU and clathrin did not colocalize. In addition, we examined the transduction ability of MoMuLV vector on HeLa cells overexpressing the dominant-negative GTPase mutant of dynamin (K44A). HeLa cells overexpressing mutant dynamin have a severe block in endocytosis by the clathrin-coated-pit pathway. No significant titer difference was observed when MoMuLV vector was tranduced into HeLa cells overexpressing either wild-type or mutant dynamin, while the transduction ability of vesicular stomatitis virus glycoprotein pseudotyped vector into HeLa cells overexpressing mutant dynamin was decreased significantly. Taken together, these data suggest that MoMuLV entry does not occur through the clathrin-coated-pit-mediated endocytic pathway.
Ebola Virus Can Be Effectively Neutralized by Antibody Produced in Natural Human Infection
Authors: Maruyama Toshiaki , Rodriguez Luis L. , Jahrling Peter B. , Sanchez Anthony , Khan Ali S. , Nichol Stuart T. , Peters C. J. , Parren Paul W. H. I. , Burton Dennis R.
Published: December 2019

The activity of antibodies against filoviruses is poorly understood but has important consequences for vaccine design and passive prophylaxis. To investigate this activity, a panel of recombinant human monoclonal antibodies to Ebola virus antigens was isolated from phage display libraries constructed from RNA from donors who recovered from infection in the 1995 Ebola virus outbreak in Kikwit, Democratic Republic of Congo. Antibodies reactive with nucleoprotein (NP), envelope glycoprotein (GP), and secreted envelope glycoprotein (sGP) were characterized by immunofluorescence and radioimmunoprecipitation assays. Four antibodies reacting strongly with sGP and weakly with GP and two antibodies reacting with NP were not neutralizing. An antibody specific for GP neutralized Ebola virus to 50% at 0.4 μg/ml as the recombinant Fab fragment and to 50% at 0.3 μg/ml (90% at 2.6 μg/ml) as the corresponding whole immunoglobulin G1 molecule. The studies indicate that neutralizing antibodies are produced in infection by Ebola virus although probably at a relatively low frequency. The neutralizing antibody may be useful in vaccine design and as a prophylactic agent against Ebola virus infection.
Identification of Phosphorylation Sites within the Herpes Simplex Virus Tegument Protein VP22
Authors: Elliott Gillian , O’Reilly Dawn , O’Hare Peter
Published: December 2019

The herpes simplex virus protein VP22 is a major phosphoprotein of infected cells. In this study, we identify two serine phosphorylation sites within VP22 and show that the N-terminal site is a substrate for casein kinase II, while the extreme C-terminal site is a substrate for another, as yet unidentified, cellular kinase. Furthermore, we show that a mutant of VP22 which has both sites altered is unable to incorporate phosphate in vivo, confirming that there are no other phosphorylation sites within VP22.
Effect of Inserting Paramyxovirus Simian Virus 5 Gene Junctions at the HN/L Gene Junction: Analysis of Accumulation of mRNAs Transcribed from Rescued Viable Viruses
Authors: He Biao , Lamb Robert A.
Published: December 2019

Simian parainfluenza virus 5 (SV5) is a prototype of theParamyxoviridaefamily of nonsegmented negative-sense RNA viruses. The single-stranded RNA genomes of these viruses contain a series of tandemly linked genes separated by intergenic (IG) sequences flanked by gene-end (GE) and gene-start (GS) sequences. The viral RNA polymerase (vRNAP) complex is thought to enter the genome at its 3′ end, and synthesis of mRNAs is thought to occur by a stop-start mechanism in a sequential and polar manner, with transcriptional attenuation occurring primarily at the intergenic regions. As a result, multiple nonoverlapping mRNA species are generated for each single entry of the vRNAP. To investigate the functions of GE, IG, and GS sequences in transcription, we constructed plasmids containing cDNAs of the full-length SV5 genome in which the gene junction sequences (GE, IG, and GS sequences) located between the hemagglutinin-neuraminidase (HN) and the polymerase (L) genes were replaced with the counterpart sequences from other gene junctions. By using reverse genetics, we recovered viable viruses from each cDNA construct, although their growth characteristics varied. Analysis of the HN and L mRNAs by quantitative RNase protection assay indicated that the ratios of HN to L mRNAs varied over a fourfold range. The alteration of the gene junction sequences also permitted examination of the hypothesized requirement for hexamer nucleotide position of the GS sites. The recovery of infectious viruses with transcription initiation sites that occurred at nucleotide positions 1, 2, 3, 5, and 6 of the hexamer suggest that the requirement is nonstringent.
Bicyclams, Selective Antagonists of the Human Chemokine Receptor CXCR4, Potently Inhibit Feline Immunodeficiency Virus Replication
Authors: Egberink Herman F. , De Clercq Erik , Van Vliet Arno L. W. , Balzarini Jan , Bridger Gary J. , Henson Geoffrey , Horzinek Marian C. , Schols Dominique
Published: December 2019

Bicyclams are low-molecular-weight anti-human immunodeficiency virus (HIV) agents that have been shown to act as potent and selective CXC chemokine receptor 4 (CXCR4) antagonists. Here, we demonstrate that bicyclams are potent inhibitors of feline immunodeficiency virus (FIV) replication when evaluated in Crandell feline kidney (CRFK) cells. With a series of bicyclam derivatives, 50% inhibitory concentrations (IC
50
s) against FIV were obtained in this cell system that were comparable to those obtained for HIV-1 IIIB replication in the human CD4
+
MT-4 T-cell line. The bicyclams were also able to block FIV replication in feline thymocytes, albeit at higher concentrations than in the CRFK cells. The prototype bicyclam AMD3100, 1-1′-[1,4-phenylene-bis(methylene)]-bis(1,4,8,11-tetraazacyclotetradecane), was only fourfold less active in feline thymocytes (IC
50
, 62 ng/ml) than in CRFK cells (IC
50
, 14 ng/ml). AMD2763, 1,1′-propylene-bis(1,4,8,11-tetraazacyclotetradecane), which is a less potent CXCR4 antagonist, was virtually inactive against FIV in feline thymocytes (IC
50
, >66.5 μg/ml), while it was clearly active in CRFK cells (IC
50
, 0.9 μg/ml). The CXC chemokine stromal-cell-derived factor 1α had anti-FIV activity in CRFK cells (IC
50
, 200 ng/ml) but not in feline thymocytes (IC
50
, >2.5 μg/ml). When primary FIV isolates were evaluated for their drug susceptibility in feline thymocytes, the bicyclams AMD3100 and its Zn
2+
complex, AMD3479, inhibited all six primary isolates at equal potency. The marked susceptibility of FIV to the bicyclams suggests that FIV predominantly uses feline CXCR4 for entering its target cells.
Subcellular Localization and Rolling Circle Replication of Peach Latent Mosaic Viroid: Hallmarks of Group A Viroids
Authors: Bussière F. , Lehoux J. , Thompson D. A. , Skrzeczkowski L. J. , Perreault J.-P.
Published: December 2019

We characterized the peach latent mosaic viroid (PLMVd) replication intermediates that accumulate in infected peach leaves and determined the tissue and subcellular localization of the RNA species. Using in situ hybridization, we showed that PLMVd strands of both plus and minus polarities concentrate in the cells forming the palisade parenchyma. At the cellular level, PLMVd was found to accumulate predominantly in chloroplasts. Northern blot analyses demonstrated that PLMVd replicates via a symmetric mode involving the accumulation of both circular and linear monomeric strands of both polarities. No multimeric conformer was detected, indicating that both strands self-cleave efficiently via their hammerhead sequences. Dot blot hybridizations revealed that PLMVd strands of both polarities accumulate equally but that the relative concentrations vary by more than 50-fold between peach cultivars. Taken together these results establish two hallmarks for the classification of viroids. Group A viroids (e.g., PLMVd), which possess hammerhead structures, replicate in the chloroplasts via the symmetric mode. By contrast, group B viroids, which share a conserved central region, replicate in the nucleus via an asymmetric mechanism. This is an important difference between self-cleaving and non-self-cleaving viroids, and the implications for the evolutionary origin and replication are discussed.
Use of DNA, RNA, and Chimeric Templates by a Viral RNA-Dependent RNA Polymerase: Evolutionary Implications for the Transition from the RNA to the DNA World
Authors: Siegel Robert W. , Bellon Laurent , Beigelman Leonid , Kao C. Cheng
Published: December 2019

All polynucleotide polymerases have a similar structure and mechanism of catalysis, consistent with their evolution from one progenitor polymerase. Viral RNA-dependent RNA polymerases (RdRp) are expected to have properties comparable to those from this progenitor and therefore may offer insight into the commonalities of all classes of polymerases. We examined RNA synthesis by the brome mosaic virus RdRp on DNA, RNA, and hybrid templates and found that precise initiation of RNA synthesis can take place from all of these templates. Furthermore, initiation can take place from either internal or penultimate initiation sites. Using a template competition assay, we found that the BMV RdRp interacts with DNA only three- to fourfold less well than it interacts with RNA. Moreover, a DNA molecule with a ribonucleotide at position −11 relative to the initiation nucleotide was able to interact with RdRp at levels comparable to that observed with RNA. These results suggest that relatively few conditions were needed for an ancestral RdRp to replicate DNA genomes.
Both Memory and CD45RA+/CD62L+Naive CD4+T Cells Are Infected in Human Immunodeficiency Virus Type 1-Infected Individuals
Authors: Ostrowski Mario A. , Chun Tae-Wook , Justement Shawn J. , Motola Ivette , Spinelli Michael A. , Adelsberger Joseph , Ehler Linda A. , Mizell Stephanie B. , Hallahan Claire W. , Fauci Anthony S.
Published: December 2019

Cellular activation is critical for the propagation of human immunodeficiency virus type 1 (HIV-1) infection. It has been suggested that truly naive CD4+T cells are resistant to productive HIV-1 infection because of their constitutive resting state. Memory and naive CD4+T-cell subsets from 11 HIV-1-infected individuals were isolated ex vivo by a combination of magnetic bead depletion and fluorescence-activated cell sorting techniques with stringent criteria of combined expression of CD45RA and CD62L to identify naive CD4+T-cell subsets. In all patients HIV-1 provirus could be detected within naive CD45RA+/CD62L+CD4+T cells; in addition, replication-competent HIV-1 was isolated from these cells upon CD4+T-cell stimulation in tissue cultures. Memory CD4+T cells had a median of fourfold more replication-competent virus and a median of sixfold more provirus than naive CD4+T cells. Overall, there was a median of 16-fold more integrated provirus identified in memory CD4+T cells than in naive CD4+T cells within a given patient. Interestingly, there was a trend toward equalization of viral loads in memory and naive CD4+T-cell subsets in those patients who harbored CXCR4-using (syncytium-inducing) viruses. Within any given patient, there was no selective usage of a particular coreceptor by virus isolated from memory versus naive CD4+T cells. Our findings suggest that naive CD4+T cells may be a significant viral reservoir for HIV, particularly in those patients harboring CXCR4-using viruses.
Lymphocyte Deficiencies Increase Susceptibility to Friend Virus-Induced Erythroleukemia inFv-2Genetically Resistant Mice
Authors: Hasenkrug Kim J.
Published: December 2019

The study of genetic resistance to retroviral diseases provides insights into the mechanisms by which organisms overcome potentially lethal infections.Fv-2resistance to Friend virus-induced erythroleukemia acts through nonimmunological mechanisms to prevent early virus spread, but it does not completely block infection. The current experiments were done to determine whether Fv-2 alone could provide resistance or whether immunological mechanisms were also required to bring infection under control.Fv-2-resistant mice that were CD4+T-cell deficient were able to restrict early virus replication and spread as well as normalFv-2-resistant mice, but they could not maintain control and developed severe Friend virus-induced splenomegaly and erythroleukemia by 6 to 8 weeks postinfection. Mice deficient in CD8+T cells and, to a lesser extent, B cells were also susceptible to late Friend virus-induced disease. Thus,Fv-2resistance does not independently prevent FV-induced erythroleukemia but works in concert with the immune system by limiting early infection long enough to allow virus-specific immunity time to develop and facilitate recovery.
Reverse Transcriptase Inhibitors Can Selectively Block the Synthesis of Differently Sized Viral DNA Transcripts in Cells Acutely Infected with Human Immunodeficiency Virus Type 1
Authors: Quan Yudong , Rong Liwei , Liang Chen , Wainberg Mark A.
Published: December 2019

We have recently reported that the in vitro inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcription by inhibitors of reverse transcriptase (RT) occurred most efficiently when the expected DNA products of RT reactions were long (Quan et al., Nucleic Acids Res. 26:5692–5698, 1998). Here, we have used a quantitative PCR to analyze HIV-1 reverse transcription within acutely infected cells treated with RT inhibitors. We found that levels of minus-strand strong-stop DNA [(−)ssDNA] formed in acutely infected MT2 cells were only slightly reduced if cells were infected with viruses that had been generated in the presence of either azidothymidine or nevirapine (5 μM) and maintained in the presence of this drug throughout the viral adsorption period and thereafter. Control experiments in which virus inoculation of cells was performed at 4°C, followed directly by cell extraction, showed that less than 1% of total (−)ssDNA within acutely infected cells was attributable to its presence within adsorbed virions. In contrast, synthesis of intermediate-length reverse-transcribed DNA products decreased gradually as viral DNA strand elongation took place in the presence of either of these inhibitors. This establishes that nucleoside and nonnucleoside RT inhibitors can exert similar temporal impacts in regard to inhibition of viral DNA synthesis. Generation of full-length viral DNA, as expected, was almost completely blocked in the presence of these antiviral drugs. These results provide insight into the fact that high concentrations of drugs are often needed to yield inhibitory effects in cell-free RT assays performed with short templates, whereas relatively low drug concentrations are often strongly inhibitory in cellular systems.
Association between Virus-Specific Cytotoxic T-Lymphocyte and Helper Responses in Human Immunodeficiency Virus Type 1 Infection
Authors: Kalams Spyros A. , Buchbinder S. P. , Rosenberg E. S. , Billingsley J. M. , Colbert D. S. , Jones N. G. , Shea A. K. , Trocha A. K. , Walker B. D.
Published: December 2019

Cellular immune responses are thought to be an important antiviral host defense, but the relationship between virus-specific T-helper and cytotoxic-T-lymphocyte (CTL) responses has not been defined. To investigate a potential link between these responses, we examined functional human immunodeficiency virus type 1 (HIV-1)-specific memory CTL precursor frequencies and p24-specific proliferative responses in a cohort of infected untreated persons with a wide range of viral loads and CD4 cell counts. Levels of p24-specific proliferative responses positively correlated with levels of Gag-specific CTL precursors and negatively correlated with levels of plasma HIV-1 RNA. These data linking the levels of HIV-specific CTL with virus-specific helper cell function during chronic viral infection provide cellular immunologic parameters to guide therapeutic and prophylactic vaccine development.
Levels of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T-Lymphocyte Effector and Memory Responses Decline after Suppression of Viremia with Highly Active Antiretroviral Therapy
Authors: Kalams Spyros A. , Goulder Philip J. , Shea Amy K. , Jones Norman G. , Trocha Alicja K. , Ogg Graham S. , Walker Bruce D.
Published: December 2019

Therapeutic suppression of human immunodeficiency virus type 1 (HIV-1) replication may help elucidate interactions between the host cellular immune responses and HIV-1 infection. We performed a detailed longitudinal evaluation of two subjects before and after the start of highly active antiretroviral therapy (HAART). Both subjects had evidence of in vivo-activated and memory cytotoxic T-lymphocyte precursor (CTLp) activity against multiple HIV-1 gene products. After the start of therapy, both subjects had declines in the levels of in vivo-activated HIV-1-specific CTLs and had immediate increases in circulating HIV-1-specific CTL memory cells. With continued therapy, and continued suppression of viral load, levels of memory CTLps declined. HLA A*0201 peptide tetramer staining demonstrated that declining levels of in vivo-activated CTL activity were associated with a decrease in the expression of the CD38+activation marker. Transient increases in viral load during continued therapy were associated with increases in the levels of virus-specific CTLps in both individuals. The results were confirmed by measuring CTL responses to discrete optimal epitopes. These studies illustrate the dynamic equilibrium between the host immune response and levels of viral antigen burden and suggest that efforts to augment HIV-1-specific immune responses in subjects on HAART may decrease the incidence of virologic relapse.
Identification of a Spliced Gene from Kaposi’s Sarcoma-Associated Herpesvirus Encoding a Protein with Similarities to Latent Membrane Proteins 1 and 2A of Epstein-Barr Virus
Authors: Glenn Mark , Rainbow Lucille , Auradé Frédéric , Davison Andrew , Schulz Thomas F.
Published: December 2019

Kaposi’s sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) is a novel herpesvirus implicated as the causative agent of Kaposi’s sarcoma (KS), primary effusion lymphoma, and some cases of multicentric Castleman’s disease. KSHV persists in the majority of KS spindle (endothelial tumor) cells and lymphoid cells in a latent form, with only a limited set of viral genes expressed in a tissue-specific manner. Here, we report the identification of a family of alternatively-spliced transcripts of approximately 7.5 kb expressed in latently infected body cavity-based lymphoma (BCBL) cell lines which are predicted to encode membrane proteins with similarities to the LMP2A and LMP1 proteins of Epstein-Barr virus. In two highly divergent sequence variants of the right end of the KSHV genome, alternative splicing of eight exons located between KSHV ORF 75 and the terminal repeats yields transcripts appropriate for proteins with up to 12 transmembrane domains, followed by a hydrophilic C-terminal, presumably cytoplasmic, domain. This C-terminal domain contains several YxxI/L motifs reminiscent of LMP2A and a putative TRAF binding site as in LMP1. In latently (persistently) infected BCBL cells the predominant transcript utilizes all eight exons, whereas in phorbol-ester-induced cells, a shorter transcript, lacking exons 4 and 5, is also abundant. We also found evidence for an alternative use of exon 1. Transfection of an epitope-tagged cDNA construct containing all exons indicates that the encoded protein is localized on cell surface and intracellular membranes, and glutathione
S
-transferase pull-down experiments indicate that its cytoplasmic domain, like that of LMP1, interacts with TRAF1, -2, and -3. Two of 20 KS patients had antibodies to the hydrophilic C-terminal domain, suggesting that the protein is expressed in vivo.
Mutations within Four Distinct Gag Proteins Are Required To Restore Replication of Human Immunodeficiency Virus Type 1 after Deletion Mutagenesis within the Dimerization Initiation Site
Authors: Liang Chen , Rong Liwei , Quan Yudong , Laughrea Michael , Kleiman Lawrence , Wainberg Mark A.
Published: December 2019

Human immunodeficiency virus type 1 (HIV-1) genomic RNA segments at nucleotide (nt) positions +240 to +274 are thought to form a stem-loop secondary structure, termed SL1, that serves as a dimerization initiation site for viral genomic RNA. We have generated two distinct deletion mutations within this region, termed BH10-LD3 and BH10-LD4, involving nt positions +238 to +253 and +261 to +274, respectively, and have shown that each of these resulted in significant diminutions in levels of viral infectiousness. However, long-term culture of each of these viruses in MT-2 cells resulted in a restoration of infectiousness, due to a series of compensatory point mutations within four distinct proteins that are normally cleaved from the Gag precursor. In the case of BH10-LD3, these four mutations were MA1, CA1, MP2, and MNC, and they involved changes of amino acid Val-35 to Ile within the matrix protein (MA), Ile-91 to Thr within the capsid (CA), Thr-12 to Ile within p2, and Thr-24 to Ile within the nucleocapsid (NC). The order in which these mutations were acquired by the mutated BH10-LD3 was MNC > CA1 > MP2 > MA1. The results of site-directed mutagenesis studies confirmed that each of these four substitutions contributed to the increased viability of the mutated BH10-LD3 viruses and that the MNC substitution, which was acquired first, played the most important role in this regard. Three point mutations, MP2, MNC, and MA2, were also shown to be sequentially acquired by viruses that had emerged in culture from the BH10-LD4 deletion. The first two of these were identical to those described above, while the last involved a change of Val-35 to Leu. All three of these substitutions were necessary to restore the infectiousness of mutated BH10-LD4 viruses to wild-type levels, although the MP2 mutation alone, but neither of the other two substitutions, was able to confer some viability on BH10-LD4 viruses. Studies of viral RNA packaging showed that the BH10-LD4 deletion only marginally impaired encapsidation while the BH10-LD3 deletion caused a severe deficit in this regard.