bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vitro Assembly Properties of Human Immunodeficiency Virus Type 1 Gag Protein Lacking the p6 Domain
Authors: Campbell Stephen , Rein Alan 
Published: December 2019

ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) normally assembles into particles of 100 to 120 nm in diameter by budding through the plasma membrane of the cell. The Gag polyprotein is the only viral protein that is required for the formation of these particles. We have used an in vitro assembly system to examine the assembly properties of purified, recombinant HIV-1 Gag protein and of Gag missing the C-terminal p6 domain (Gag Δp6). This system was used previously to show that the CA-NC fragment of HIV-1 Gag assembled into cylindrical particles. We now report that both HIV-1 Gag and Gag Δp6 assemble into small, 25- to 30-nm-diameter spherical particles in vitro. The multimerization of Gag Δp6 into units larger than dimers and the formation of spherical particles required nucleic acid. Removal of the nucleic acid with NaCl or nucleases resulted in the disruption of the multimerized complexes. We conclude from these results that (i) N-terminal extension of HIV-1 CA-NC to include the MA domain results in the formation of spherical, rather than cylindrical, particles; (ii) nucleic acid is required for the assembly and maintenance of HIV-1 Gag Δp6 virus-like particles in vitro and possibly in vivo; (iii) a wide variety of RNAs or even short DNA oligonucleotides will support assembly; (iv) protein-protein interactions within the particle must be relatively weak; and (v) recombinant HIV-1 Gag Δp6 and nucleic acid are not sufficient for the formation of normal-sized particles.
The Reovirus Mutant
tsA279
L2 Gene Is Associated with Generation of a Spikeless Core Particle: Implications for Capsid Assembly
Authors: Hazelton Paul R. , Coombs Kevin M. 
Published: December 2019

ABSTRACT

Previous studies which used intertypic reassortants of the wild-type reovirus serotype 1 Lang and the temperature-sensitive (
ts
) serotype 3 mutant clone
tsA279
identified two
ts
lesions; one lesion, in the M2 gene segment, was associated with defective transmembrane transport of restrictively assembled virions (P. R. Hazelton and K. M. Coombs, Virology 207:46–58, 1995). In the present study we show that the second lesion, in the L2 gene segment, which encodes the λ2 protein, is associated with the accumulation of a core-like particle defective for the λ2 pentameric spike. Physicochemical, biochemical, and immunological studies showed that these structures were deficient for genomic double-stranded RNA, the core spike protein λ2, and the minor core protein μ2. Core particles with the λ2 spike structure accumulated after temperature shift-down from a restrictive to a permissive temperature in the presence of cycloheximide. These data suggest the spike-deficient, core-like particle is an assembly intermediate in reovirus morphogenesis. The existence of this naturally occurring primary core structure suggests that the core proteins λ1, λ3, and ς2 interact to initiate the process of virion capsid assembly through a dodecahedral mechanism. The next step in the proposed capsid assembly model would be the association of the minor core protein μ2, either preceding or collateral to the condensation of the λ2 pentameric spike at the apices of the primary core structure. The assembly pathway of the reovirus double capsid is further elaborated when these observations are combined with structures identified in other studies.
Coxsackievirus and Adenovirus Receptor Cytoplasmic and Transmembrane Domains Are Not Essential for Coxsackievirus and Adenovirus Infection
Authors: Wang Xianghong , Bergelson Jeffrey M. 
Published: December 2019

ABSTRACTCoxsackievirus and adenovirus receptor (CAR) from which the cytoplasmic domain had been deleted and glycosylphosphatidylinositol (GPI)-anchored CAR lacking both transmembrane and cytoplasmic domains were both capable of facilitating adenovirus 5-mediated gene delivery and infection by coxsackievirus B3. These results indicate that the CAR extracellular domain is sufficient to permit virus attachment and entry and that the presence of a GPI anchor does not prevent infection.
Foamy Virus Capsids Require the Cognate Envelope Protein for Particle Export
Authors: Pietschmann Thomas , Heinkelein Martin , Heldmann Martina , Zentgraf Hanswalter , Rethwilm Axel , Lindemann Dirk 
Published: December 2019

ABSTRACT

Unlike other subclasses of the
Retroviridae
the
Spumavirinae
, its prototype member being the so-called human foamy virus (HFV), require the expression of the envelope (Env) glycoprotein for viral particle egress. Both the murine leukemia virus (MuLV) Env and the vesicular stomatitis virus G protein, which efficiently pseudotype other retrovirus capsids, were not able to support export of HFV particles. Analysis of deletion and point mutants of the HFV Env protein revealed that the HFV Env cytoplasmic domain (CyD) is dispensable for HFV particle envelopment, release, and infectivity, whereas deletion of the membrane-spanning-domain (MSD) led to an accumulation of naked capsids in the cytoplasm. Neither alternative membrane association of HFV Env deletion mutants lacking the MSD and CyD via phosphoglycolipid anchor nor domain swapping mutants, with the MSD or CyD of MuLV Env and VSV-G exchanged against the corresponding HFV domains, could restore particle envelopment and the release defect of pseudotypes. However, replacement of the HFV MSD with that of MuLV led to budding of HFV capsids at the intracellular membranes. These virions were of apparently wild-type morphology but were not naturally released into the supernatant and they were noninfectious.
Bacterial Lipopolysaccharide Inhibits Dengue Virus Infection of Primary Human Monocytes/Macrophages by Blockade of Virus Entry via a CD14-Dependent Mechanism
Authors: Chen Yun-Chi , Wang Sheng-Yuan , King Chwan-Chuen 
Published: December 2019

ABSTRACTMonocytes/macrophages (MO/Mφ) are the major target cells for both dengue virus (DV) and bacterial lipopolysaccharide (LPS), and the aim of this study was to define their interactions. We had found that LPS markedly suppressed DV infection of primary human MO/Mφ when it was added to cultures prior to or together with, but not after, viral adsorption. The inhibitory effect of LPS was direct and specific and was not mediated by LPS-induced secretion of cytokines and chemokines such as tumor necrosis factor alpha, interleukin-1β (IL-1β), IL-6, IL-8, IL-12, alpha interferon, MIP-1α, and RANTES. In fact, productive DV infection was not blocked but was just postponed by LPS, with a time lag equal to one viral replication cycle. Time course studies demonstrated that LPS was only effective in suppressing DV infection of MO/Mφ that had not been previously exposed to the virus. At various time points after viral adsorption, the level of unbound viruses that remained free in the culture supernatants of LPS-pretreated cultures was much higher than that of untreated controls. These observations suggest that the LPS-induced suppression of DV replication was at the level of virus attachment and/or entry. Blockade of the major LPS receptor, CD14, with monoclonal antibodies MY4 or MoS39 failed to inhibit DV infection but could totally abrogate the inhibitory effect of LPS. Moreover, human serum could significantly enhance the LPS-induced DV suppression in a CD14-dependent manner, indicating that the “binding” of LPS to CD14 was critical for the induction of virus inhibition. Taken together, our results suggest that LPS blocked DV entry into human MO/Mφ via its receptor CD14 and that a CD14-associated cell surface structure may be essential for the initiation of a DV infection.
Sufficient Length of a Poly(A) Tail for the Formation of a Potential Pseudoknot Is Required for Efficient Replication of Bamboo Mosaic Potexvirus RNA
Authors: Tsai Ching-Hsiu , Cheng Chi-Ping , Peng Chih-Weng , Lin Biing-Yuan , Lin Na-Sheng , Hsu Yau-Heiu 
Published: December 2019

ABSTRACT

RNAs transcribed from a full-length infectious cDNA clone of the bamboo mosaic potexvirus (strain O) genome, pBaMV-O, were infectious to
Nicotiana benthamiana
plants. Mutant genomes in which the poly(A) tail is absent or replaced by a 3′ tRNA-like structure from turnip yellow mosaic virus RNA failed to amplify detectably in
N. benthamiana
protoplasts. No amplification was detected in protoplasts inoculated with transcripts containing 4, 7, or 10 adenylate residues at the 3′ end, whereas transcript inocula with 15 adenylate residues resulted in coat protein accumulation to a level 26% of that resulting from inoculation with transcripts with 25 adenylate residues (designated as wild type). Coat protein accumulation levels of 69 and 98% relative to wild type were observed after inoculation of protoplasts with transcripts bearing poly(A) tails 18 and 22 nucleotides long, respectively. The presence of a putative 3′ pseudoknot structure including at least 13 adenylate residues of the 3′-terminal poly(A) tail was supported by enzymatic and chemical structural analysis. The functional relevance of this putative pseudoknot was tested by mutations that affected basepairing within the pseudoknot. These results support the existence of functional 3′ pseudoknot that includes part of the 3′ poly(A) tail.
Effect of the Attenuating Deletion and of Sequence Alterations Evolving In Vivo on Simian Immunodeficiency Virus C8-Nef Function
Authors: Carl Silke , Iafrate A. John , Skowronski Jacek , Stahl-Hennig Christiane , Kirchhoff Frank 
Published: December 2019

ABSTRACTThe simian immunodeficiency virus macC8 (SIVmacC8) variant has been used in a European Community Concerted Action project to study the efficacy and safety of live attenuated SIV vaccines in a large number of macaques. The attenuating deletion in the SIVmacC8nef-long terminal repeat region encompasses only 12 bp and is “repaired” in a subset of infected animals. It is unknown whether C8-Nef retains some activity. Since it seems important to use only well-characterized deletion mutants in live attenuated vaccine studies, we analyzed the relevance of the deletion, and the duplications and point mutations selected in infected macaques for Nef function in vitro. The deletion, affecting amino acids 143 to 146 (DMYL), resulted in a dramatic decrease in Nef stability and function. The initial 12-bp duplication resulted in efficient Nef expression and an intermediate phenotype in infectivity assays, but it did not significantly restore the ability of Nef to stimulate viral replication and to downmodulate CD4 and class I major histocompatibility complex cell surface expression. The additional substitutions however, which subsequently evolved in vivo, gradually restored these Nef functions. It was noteworthy that coinfection experiments in the T-lymphoid 221 cell line revealed that even SIVmacnefvariants carrying the original 12-bp deletion readily outgrew an otherwise isogenic virus containing a 182-bp deletion in thenefgene. Thus, although C8-Nef is unstable and severely impaired in in vitro assays, it maintains some residual activity to stimulate viral replication.
A Single Amino Acid in the Adenovirus Type 37 Fiber Confers Binding to Human Conjunctival Cells
Authors: Huang Shuang , Reddy Vijay , Dasgupta Nabarun , Nemerow Glen R. 
Published: December 2019

ABSTRACT

A 46-kDa receptor, coxsackievirus-adenovirus (Ad) receptor (CAR), mediates cell attachment of a number of different Ad serotypes; however, not all Ad serotypes utilize this receptor for infection. Moreover, the precise amino acid sequences in the Ad fiber protein that mediate cell attachment have yet to be identified. We investigated the interaction of subgroup D Ads with human ocular cells. Ad serotype 37 (Ad37), a virus associated with epidemic keratoconjunctivitis, but not a closely related virus serotype, Ad19p, exhibited preferential binding to and infection of human conjunctival cells. A single amino acid substitution in the Ad19p fiber distal domain (knob), Glu
240
to Lys, conferred binding to conjunctival cells, while the reverse substitution in the Ad37 fiber abrogated cell binding. These findings provide new information on the fiber sequences that regulate Ad host cell tropism.
Effects of Human Cytomegalovirus Major Immediate-Early Proteins in Controlling the Cell Cycle and Inhibiting Apoptosis: Studies with
ts
13 Cells
Authors: Lukac David M. , Alwine James C. 
Published: December 2019

ABSTRACT

The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) encodes several MIE proteins (MIEPs) produced as a result of alternative splicing and polyadenylation of the primary transcript. Previously we demonstrated that the HCMV MIEPs expressed from the entire MIE gene could rescue the temperature-sensitive (
ts
) transcriptional defect in the
ts
13 cell line. This defect is caused by a
ts
mutation in TAF
II
250, the 250-kDa TATA binding protein-associated factor (TAF). These and other data suggested that the MIEPs perform a TAF-like function in complex with the basal transcription factor TFIID. In addition to the transcriptional defect, the
ts
mutation in
ts
13 cells results in a defect in cell cycle progression which ultimately leads to apoptosis. Since all of these defects can be rescued by wild-type TAF
II
250, we asked whether the MIEPs could rescue the cell cycle defect and/or affect the progression to apoptosis. We have found that the MIEPs, expressed from the entire MIE gene, do not rescue the cell cycle block in
ts
13 cells grown at the nonpermissive temperature. However, despite the maintenance of the cell cycle block, the
ts
13 cells which express the MIEPs are resistant to apoptosis. MIEP mutants, which have previously been shown to be defective in rescuing the
ts
transcriptional defect, maintained the ability to inhibit apoptosis. Hence, the MIEP functions which affect transcription appear to be separable from the functions which inhibit apoptosis. We discuss these data in the light of the HCMV life cycle and the possibility that the MIEPs promote cellular transformation by a “hit-and-run” mechanism.
Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes Delivered by Retroviral Vectors
Authors: Zufferey Romain , Donello John E. , Trono Didier , Hope Thomas J. 
Published: December 2019

ABSTRACT
The expression of genes delivered by retroviral vectors is often inefficient, a potential obstacle for their widespread use in human gene therapy. Here, we explored the possibility that the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE) might help resolve this problem. Insertion of the WPRE in the 3′ untranslated region of coding sequences carried by either oncoretroviral or lentiviral vectors substantially increased their levels of expression in a transgene-, promoter- and vector-independent manner. The WPRE thus increased either luciferase or green fluorescent protein production five- to eightfold, and effects of a comparable magnitude were observed with either the immediate-early cytomegalovirus or the herpesvirus thymidine kinase promoter and with both human immunodeficiency virus- and murine leukemia virus-based vectors. The WPRE exerted this influence only when placed in the sense orientation, consistent with its predicted posttranscriptional mechanism of action. These results demonstrate that the WPRE significantly improves the performance of retroviral vectors and emphasize that posttranscriptional regulation of gene expression should be taken into account in the design of gene delivery systems.
Acute Hepatitis C Virus Structural Gene Sequences as Predictors of Persistent Viremia: Hypervariable Region 1 as a Decoy
Authors: Ray Stuart C. , Wang Yu-Ming , Laeyendecker Oliver , Ticehurst John R. , Villano Stephen A. , Thomas David L. 
Published: December 2019

ABSTRACT

We hypothesized that hepatitis C virus (HCV) persistence is related to the sequence variability of putative envelope genes. This hypothesis was tested by characterizing quasispecies in specimens collected every six months from a cohort of acutely HCV-infected subjects (mean duration of specimen collection, 72 months after seroconversion). We evaluated 5 individuals who spontaneously cleared viremia and 10 individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5′ half of E2, including hypervariable region 1 (HVR1). To assess the quasispecies complexity and to detect variants for sequencing, the first PCR-positive sample was examined by using a previously described method that combines heteroduplex analysis and analysis of single-stranded conformational polymorphisms. The ratio of nonsynonymous to synonymous substitutions (

d
N
/d
S

) within each sample was evaluated as an indicator of relative selective pressure. Amino acid sequences were analyzed for signature patterns, glycosylation signals, and charge. Quasispecies complexity was higher and E1

d
N
/d
S

ratios (selective pressure) were lower in those with persistent viremia; the association with persistence was strengthened by the presence of a combination of both characteristics. In contrast, a trend toward higher HVR1

d
N
/d
S

ratios was detected among those with persistent viremia. We did not detect any such association for factors that may affect complexity such as serum HCV RNA concentration. HVR1 had a lower positive charge in subjects with persistent viremia, although no consistent motifs were detected. Our data suggest that HCV persistence is associated with a complex quasispecies and immune response to HVR1.
The Retinoblastoma Protein Alters the Phosphorylation State of Polyomavirus Large T Antigen in Murine Cell Extracts and Inhibits Polyomavirus Origin DNA Replication
Authors: Reynisdóttir Inga , Bhattacharyya Subarna , Zhang Dong , Prives Carol 
Published: December 2019

ABSTRACT

The retinoblastoma tumor suppressor protein (pRb) can associate with the transforming proteins of several DNA tumor viruses, including the large T antigen encoded by polyomavirus (Py T Ag). Although pRb function is critical for regulating progression from G
1
to S phase, a role for pRb in S phase has not been demonstrated or excluded. To identify a potential effect of pRb on DNA replication, pRb protein was added to reaction mixtures containing Py T Ag, Py origin-containing DNA (Py ori-DNA), and murine FM3A cell extracts. We found that pRb strongly represses Py ori-DNA replication in vitro. Unexpectedly, however, this inhibition only partially depends on the interaction of pRb with Py T Ag, since a mutant Py T Ag (
dl
141) lacking the pRb interaction region was also significantly inhibited by pRb. This result suggests that pRb interferes with or alters one or more components of the murine cell replication extract. Furthermore, the ability of Py T Ag to be phosphorylated in such extracts is markedly reduced in the presence of pRb. Since cyclin-dependent kinase (CDK) phosphorylation of Py T Ag is required for its replication function, we hypothesize that pRb interferes with this phosphorylation event. Indeed, the S-phase CDK complex (cyclin A-CDK2), which phosphorylates both pRb and Py T Ag, alleviates inhibition caused by pRb. Moreover, hyperphosphorylated pRb is incapable of inhibiting replication of Py ori-DNA in vitro. We propose a new requirement for maintaining pRb phosphorylation in S phase, namely, to prevent deleterious effects on the cellular replication machinery.
T Cells Contribute to Disease Severity during Coxsackievirus B4 Infection
Authors: Ramsingh Arlene I. , Lee William T. , Collins Doris N. , Armstrong Lori E. 
Published: December 2019

ABSTRACTBy using a model of coxsackievirus B4-induced disease, the question of whether tissue damage is due to the virus or to immune-mediated mechanisms was addressed. Both viral replication and T-cell function were implicated in contributing to the severity of disease. Three stages (I to III) of disease, which correspond to periods of high viral titers, low viral titers, and no infectious virus, have been identified. Stage I disease is considered to be primarily the result of viral replication. Immunopathological mechanisms appear to contribute to the severity of stage II and III disease. To investigate the role of T cells in contributing to the severity of disease, viral infection in CD8 knockout (ko) mice and CD4 ko mice was analyzed. CD8 T-cell responses appear to be beneficial during early, viral disease but detrimental in later disease when viral titers are diminishing. CD4 ko mice, unlike the parental strain, survived infection. Viral replication was lower in the CD4 ko mice. Was survival due to decreased viral replication or to the lack of T-helper-cell function? To investigate further the role of T helper cells in contributing to tissue damage, viral infection in two additional ko strains (interleukin-4 [IL-4] ko and gamma interferon ko strains) was examined. A clear correlation between viral replication and the outcome of infection was not observed. The absence of IL-4, which may influence T-helper-cell subset development, was advantageous during early viral disease but deleterious in later disease. The results suggest that T-cell-mediated immunity is both beneficial and detrimental during coxsackievirus B4 infection.
Sendai Virus and Simian Virus 5 Block Activation of Interferon-Responsive Genes: Importance for Virus Pathogenesis
Authors: Didcock L. , Young D. F. , Goodbourn S. , Randall R. E. 
Published: December 2019

ABSTRACTSendai virus (SeV) is highly pathogenic for mice. In contrast, mice (including SCID mice) infected with simian virus 5 (SV5) showed no overt signs of disease. Evidence is presented that a major factor which prevented SV5 from productively infecting mice was its inability to circumvent the interferon (IFN) response in mice. Thus, in murine cells that produce and respond to IFN, SV5 protein synthesis was rapidly switched off. In marked contrast, once SeV protein synthesis began, it continued, even if the culture medium was supplemented with alpha/beta IFN (IFN-α/β). However, in human cells, IFN-α/β did not inhibit the replication of either SV5 or SeV once virus protein synthesis was established. To begin to address the molecular basis for these observations, the effects of SeV and SV5 infections on the activation of an IFN-α/β-responsive promoter and on that of the IFN-β promoter were examined in transient transfection experiments. The results demonstrated that (i) SeV, but not SV5, inhibited an IFN-α/β-responsive promoter in murine cells; (ii) both SV5 and SeV inhibited the activation of an IFN-α/β-responsive promoter in human cells; and (iii) in both human and murine cells, SeV was a strong inducer of the IFN-β promoter, whereas SV5 was a poor inducer. The ability of SeV and SV5 to inhibit the activation of IFN-responsive genes in human cells was confirmed by RNase protection experiments. The importance of these results in terms of paramyxovirus pathogenesis is discussed.