bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mass Determination of Rous Sarcoma Virus Virions by Scanning Transmission Electron Microscopy
Authors: Vogt Volker M. , Simon Martha N. 
Published: December 2019

ABSTRACT

The internal structural protein of retroviruses, Gag, comprises most of the mass of the virion, and Gag itself can give rise to virus-like particles when expressed in appropriate cells. Previously the stoichiometry of Gag in virions was inferred from indirect measurements carried out 2 decades ago. We now have directly determined the masses of individual particles of the prototypic avian retrovirus, Rous sarcoma virus (RSV), by using scanning transmission electron microscopy. In this technique, the number of scattered electrons in the dark-field image integrated over an individual freeze-dried virus particle on a grid is directly proportional to its mass. The RSV virions had a mean mass of 2.5 × 10
8
Da, corresponding to about 1,500 Gag molecules per virion. The population of virions was not homogeneous, with about one-third to two-thirds of the virions deviating from the mean by more than 10% of the mass in two respective preparations. The mean masses for virions carrying genomes of 7.4 or 9.3 kb were indistinguishable, suggesting that mass variability is not due to differences in RNA incorporation.
Broad, Intense Anti-Human Immunodeficiency Virus (HIV) Ex Vivo CD8+Responses in HIV Type 1-Infected Patients: Comparison with Anti-Epstein-Barr Virus Responses and Changes during Antiretroviral Therapy
Authors: Dalod Marc , Dupuis Marion , Deschemin Jean-Christophe , Sicard Didier , Salmon Dominique , Delfraissy Jean-Francois , Venet Alain , Sinet Martine , Guillet Jean-Gerard 
Published: December 2019

ABSTRACTThe ex vivo antiviral CD8+repertoires of 34 human immunodeficiency virus (HIV)-seropositive patients with various CD4+T-cell counts and virus loads were analyzed by gamma interferon enzyme-linked immunospot assay, using peptides derived from HIV type 1 and Epstein-Barr virus (EBV). Most patients recognized many HIV peptides, with markedly high frequencies, in association with all the HLA class I molecules tested. We found no correlation between the intensity of anti-HIV CD8+responses and the CD4+counts or virus load. In contrast, the polyclonality of anti-HIV CD8+responses was positively correlated with the CD4+counts. The anti-EBV responses were significantly less intense than the anti-HIV responses and were positively correlated with the CD4+counts. Longitudinal follow-up of several patients revealed the remarkable stability of the anti-HIV and anti-EBV CD8+responses in two patients with stable CD4+counts, while both antiviral responses decreased in two patients with obvious progression toward disease. Last, highly active antiretroviral therapy induced marked decreases in the number of anti-HIV CD8+T cells, while the anti-EBV responses increased. These findings emphasize the magnitude of the ex vivo HIV-specific CD8+responses at all stages of HIV infection and suggest that the CD8+hyperlymphocytosis commonly observed in HIV infection is driven mainly by virus replication, through intense, continuous activation of HIV-specific CD8+T cells until ultimate progression toward disease. Nevertheless, highly polyclonal anti-HIV CD8+responses may be associated with a better clinical status. Our data also suggest that a decrease of anti-EBV CD8+responses may occur with depletion of CD4+T cells, but this could be restored by highly active antiretroviral treatment.
Modulation of Hepatitis C Virus NS5A Hyperphosphorylation by Nonstructural Proteins NS3, NS4A, and NS4B
Authors: Oliver Koch Jan , Bartenschlager Ralf 
Published: December 2019

ABSTRACT

NS5A of the hepatitis C virus (HCV) is a highly phosphorylated protein involved in resistance against interferon and required most likely for replication of the viral genome. Phosphorylation of this protein is mediated by a cellular kinase(s) generating multiple proteins with different electrophoretic mobilities. In the case of the genotype 1b isolate HCV-J, in addition to the basal phosphorylated NS5A (designated pp56), a hyperphosphorylated form (pp58) was found on coexpression of NS4A (T. Kaneko, Y. Tanji, S. Satoh, M. Hijikata, S. Asabe, K. Kimura, and K. Shimotohno, Biochem. Biophys. Res. Commun. 205:320–326, 1994). Using a comparative analysis of two full-length genomes of genotype 1b, competent or defective for NS5A hyperphosphorylation, we investigated the requirements for this NS5A modification. We found that hyperphosphorylation occurs when NS5A is expressed as part of a continuous NS3-5A polyprotein but not when it is expressed on its own or
trans
complemented with one or several other viral proteins. Results obtained with chimeras of both genomes show that single amino acid substitutions within NS3 that do not affect polyprotein cleavage can enhance or reduce NS5A hyperphosphorylation. Furthermore, mutations in the central or carboxy-terminal NS4A domain as well as small deletions in NS4B can also reduce or block hyperphosphorylation without affecting polyprotein processing. These requirements most likely reflect the formation of a highly ordered NS3-5A multisubunit complex responsible for the differential phosphorylation of NS5A and probably also for modulation of its biological activities.
Studies of the Genomic RNA of Leukosis Viruses: Implications for RNA Dimerization
Authors: Ortiz-Conde Betty A. , Hughes Stephen H. 
Published: December 2019

ABSTRACT

Retroviral particles contain two positive-strand genomic RNAs linked together by noncovalent bonds that can be dissociated under mild conditions. We studied genomic RNAs of wild-type and mutant avian leukosis viruses (ALVs) in an attempt to (i) better understand the site(s) of RNA dimerization, (ii) examine whether the primer binding site (PBS) and tRNA primer are involved in dimerization, and (iii) determine the structure of genomic RNA in protease-deficient (PR
−
) mutants. We showed that extensively nicked wild-type ALV genomic RNAs melt cooperatively. This implies a complex secondary and/or tertiary structure for these RNAs that extends well beyond the 5′ dimerization site. To investigate the role of the PBS-tRNA complex in dimerization, we analyzed genomic RNAs from mutant viruses in which the tRNA
Trp
PBS had been replaced with sequences homologous to the 3′ end of six other chicken tRNAs. We found the genomic RNAs of these viruses are dimers that dissociate at the same temperature as wild-type viral RNA, which suggests that the identity of the PBS and the tRNA primer do not affect dimer stability. We studied two ALV PR
−
mutants: one containing a large (>1.9-kb) inversion spanning the 3′ end of
gag
and much of
pol
, rendering it deficient in PR, reverse transcriptase, and integrase, and another with a point mutation in PR. In both of these mutant viruses, the genomic RNA appears to be either primarily or exclusively monomeric. These data suggest that ALV can package its RNA as monomers that subsequently dimerize.
Picornavirus Receptor Down-Regulation by Plasminogen Activator Inhibitor Type 2
Authors: Shafren D. R. , Gardner J. , Mann V. H. , Antalis T. M. , Suhrbier A. 
Published: December 2019

ABSTRACTTherapeutic interference with virus-cell surface receptor interactions represents a viable antiviral strategy. Here we demonstrate that cytoplasmic expression of the serine protease inhibitor (serpin), plasminogen activator inhibitor type 2 (PAI-2), affords a high level of protection from lytic infection by multiple human picornaviruses. The antiviral action of PAI-2 was mediated primarily through transcriptional down-regulation of the following virus receptors: intercellular adhesion molecule 1 (ICAM-1, a cellular receptor for the major group of rhinoviruses), decay-accelerating factor (a cellular receptor for echoviruses and coxsackieviruses), and to a lesser extent the coxsackie-adenovirus receptor protein (a cellular receptor for group B coxsackieviruses and group C adenoviruses). Expression of related cell surface receptors, including membrane cofactor protein and the poliovirus receptor, remained unaffected. These findings suggest that PAI-2 and/or related serpins may form the basis of novel antiviral strategies against picornavirus infections and also therapeutic interventions against ICAM-1-mediated respiratory inflammation.
Sequence Requirements for the Assembly of Simian Virus 40 T Antigen and the T-Antigen Origin Binding Domain on the Viral Core Origin of Replication
Authors: Kim Henry Y. , Barbaro Brett A. , Joo Woo S. , Prack Andrea E. , Sreekumar K. R. , Bullock Peter A. 
Published: December 2019

ABSTRACT

The regions of the simian virus 40 (SV40) core origin that are required for stable assembly of virally encoded T antigen (T-ag) and the T-ag origin binding domain (T-ag-obd
131–260
) have been determined. Binding of the purified T-ag-obd
131–260
is mediated by interactions with the central region of the core origin, site II. In contrast, T-ag binding and hexamer assembly requires a larger region of the core origin that includes both site II and an additional fragment of DNA that may be positioned on either side of site II. These studies indicate that in the context of T-ag, the origin binding domain can engage the pentanucleotides in site II only if a second region of T-ag interacts with one of the flanking sequences. The requirements for T-ag double-hexamer assembly are complex; the nucleotide cofactor present in the reaction modulates the sequence requirements for oligomerization. Nevertheless, these experiments provide additional evidence that only a subset of the SV40 core origin is required for assembly of T-ag double hexamers.
The E7 Oncoprotein of Human Papillomavirus Type 16 Stabilizes p53 through a Mechanism Independent of p19
ARF
Authors: Seavey Scott E. , Holubar Marisa , Saucedo Leslie J. , Perry Mary Ellen 
Published: December 2019

ABSTRACT

High-risk human papillomaviruses are causally associated with cervical cancer. Two viral oncogenes, E6 and E7, are expressed in most cervical cancers, and these genes cause cancer when expressed in experimental animals. The E6 protein targets the p53 tumor suppressor for degradation, while the E7 protein inactivates the retinoblastoma susceptibility protein (pRb), in part by stimulating its degradation. In contrast, expression of E7 in the absence of E6 leads to stabilization of p53. Here we show that E7 stabilizes p53 in mouse embryo fibroblasts lacking p19
ARF
. The stable p53 is active as a transcriptional activator, as evidenced by the increased expression of the p53-responsive
mdm2
gene. Normally, MDM2 protein inhibits p53 function in an autoregulatory loop. Regulation of p53 by MDM2 is required for murine development as well as for proliferation of cultured human fibroblasts. However, E7-expressing human fibroblasts continue to divide even though E7 abrogates the ability of MDM2 and p53 to bind. Furthermore, E7-expressing cells are not more sensitive to UV light, an agent that has been reported to induce apoptosis mediated by p53. These results indicate that in addition to inhibiting the ability of MDM2 to regulate p53, E7 must block signaling steps downstream of p53 to allow cell division.
A Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase Capable of Copying the Full-Length Viral RNA
Authors: Oh Jong-Won , Ito Takayoshi , Lai Michael M. C. 
Published: December 2019

ABSTRACT

All of the previously reported recombinant RNA-dependent RNA polymerases (RdRp), the NS5B enzymes, of hepatitis C virus (HCV) could function only in a primer-dependent and template-nonspecific manner, which is different from the expected properties of the functional viral enzymes in the cells. We have now expressed a recombinant NS5B that is able to synthesize a full-length HCV genome in a template-dependent and primer-independent manner. The kinetics of RNA synthesis showed that this RdRp can initiate RNA synthesis de novo and yield a full-length RNA product of genomic size (9.5 kb), indicating that it did not use the copy-back RNA as a primer. This RdRp was also able to accept heterologous viral RNA templates, including poly(A)- and non-poly(A)-tailed RNA, in a primer-independent manner, but the products in these cases were heterogeneous. The RdRp used some homopolymeric RNA templates only in the presence of a primer. By using the 3′-end 98 nucleotides (nt) of HCV RNA, which is conserved in all genotypes of HCV, as a template, a distinct RNA product was generated. Truncation of 21 nt from the 5′ end or 45 nt from the 3′ end of the 98-nt RNA abolished almost completely its ability to serve as a template. Inclusion of the 3′-end variable sequence region and the U-rich tract upstream of the X region in the template significantly enhanced RNA synthesis. The 3′ end of minus-strand RNA of HCV genome also served as a template, and it required a minimum of 239 nt from the 3′ end. These data defined the
cis
-acting sequences for HCV RNA synthesis at the 3′ end of HCV RNA in both the plus and minus senses. This is the first recombinant HCV RdRp capable of copying the full-length HCV RNA in the primer-independent manner expected of the functional HCV RNA polymerase.
A New Group of Hepadnaviruses Naturally Infecting Orangutans (
Pongo pygmaeus
)
Authors: Warren Kristin S. , Heeney Jonathan L. , Swan Ralph A. , Heriyanto  , Verschoor Ernst J. 
Published: December 2019

ABSTRACT
A high prevalence (42.6%) of hepatitis B virus (HBV) infection was suspected in 195 formerly captive orangutans due to a large number of serum samples which cross-reacted with human HBV antigens. It was assumed that such viral infections were contracted from humans during captivity. However, two wild orangutans were identified which were HBV surface antigen positive, indicating that HBV or related viruses may be occurring naturally in the orangutan populations. Sequence analyses of seven isolates revealed that orangutans were infected with hepadnaviruses but that these were clearly divergent from the six known human HBV genotypes and those of other nonhuman hepadnaviruses reported. Phylogenetic analyses revealed geographic clustering with Southeast Asian genotype C viruses and gibbon ape HBV. This implies a common origin of infection within this geographic region, with cross-species transmission of hepadnaviruses among hominoids.
Induction of Antiviral Antibodies by DNA Immunization Requires neither Perforin-Mediated nor CD8+-T-Cell-Mediated Lysis of Antigen-Expressing Cells
Authors: Hassett Daniel E. , Zhang Jie , Whitton J. Lindsay 
Published: December 2019

ABSTRACTDNA immunization induces antibodies to the encoded protein, which indicates that the protein must gain access to the extracellular milieu, allowing it to interact with naı̈ve B lymphocytes. It has been suggested that antigen release may be effected by cytotoxic-T-lymphocyte-mediated lysis of transfected antigen-expressing cells; this might be particularly important for the induction of responses to a noncytopathic, cytosolic protein. Here we show that the induction of antibody responses to one such DNA-encoded protein required neither perforin nor CD8+T cells. In addition, there was no skewing of the immunoglobulin G isotypes in the absence of perforin.
Rhesus Rhadinovirus Establishes a Latent Infection in B Lymphocytes In Vivo
Authors: Bergquam Eric P. , Avery Nancy , Shiigi Stanley M. , Axthelm Michael K. , Wong Scott W. 
Published: December 2019

ABSTRACT

Recent DNA sequence analysis indicates that rhesus rhadinovirus (RRV) is a member of the lymphotropic gamma-2 herpesvirus family. To determine if RRV is lymphotropic, peripheral blood mononuclear cells from naturally infected monkeys were separated by immunomagnetic bead depletion and analyzed for the presence of RRV by virus isolation and nested PCR. The recovery and consistent detection of RRV in the CD20
+
-enriched fraction clearly demonstrates that B lymphocytes are a major site of virus persistence.
Molecular Characterization of Proteolytic Processing of the Gag Proteins of Human Spumavirus
Authors: Pfrepper Klaus-Ingmar , Löchelt Martin , Rackwitz Hans-Richard , Schnölzer Martina , Heid Hans , Flügel Rolf M. 
Published: December 2019

ABSTRACT
Spumaviruses, or foamy viruses, express Gag proteins that are incompletely processed by the viral protease in cell cultures. To delineate the proteolytic cleavage sites between potential Gag subdomains, recombinant human spumaretrovirus (HSRV) Gag proteins of different lengths were expressed, purified by affinity chromatography, and subjected to HSRV protease assays. HSRV-specific proteolytic cleavage products were isolated and characterized by Western blotting. Peptides spanning potential cleavage sites, as deduced from the sizes of the proteolytic cleavage products, were chemically synthesized and assayed with HSRV protease. The cleaved peptides were then subjected to mass spectrometry. In control experiments, HSRV protease-deficient mutant proteins were used to rule out unspecific processing by nonviral proteases. The cleavage site junctions identified and the calculated sizes of the cleavage products were in agreement with those of the authentic cleavage products of the HSRV Gag proteins detectable in viral proteins from purified HSRV particles and in virus-infected cells. The biological significance of the data was confirmed by mutational analysis of the cleavage sites in a recombinant Gag protein and in the context of the infectious HSRV DNA provirus.
Cell Wall and Secreted Proteins ofCandida albicans: Identification, Function, and Expression
Authors: Chaffin W. Lajean , López-Ribot José Luis , Casanova Manuel , Gozalbo Daniel , Martínez José P. 
Published: December 2019

SUMMARYThe cell wall is essential to nearly every aspect of the biology and pathogenicity of Candida albicans. Although it was intially considered an almost inert cellular structure that protected the protoplast against osmotic offense, more recent studies have demonstrated that it is a dynamic organelle. The major components of the cell wall are glucan and chitin, which are associated with structural rigidity, and mannoproteins. The protein component, including both mannoprotein and nonmannoproteins, comprises some 40 or more moieties. Wall proteins may differ in their expression, secretion, or topological location within the wall structure. Proteins may be modified by glycosylation (primarily addition of mannose residues), phosphorylation, and ubiquitination. Among the secreted enzymes are those that are postulated to have substrates within the cell wall and those that find substrates in the extracellular environment. Cell wall proteins have been implicated in adhesion to host tissues and ligands. Fibrinogen, complement fragments, and several extracellular matrix components are among the host proteins bound by cell wall proteins. Proteins related to the hsp70 and hsp90 families of conserved stress proteins and some glycolytic enzyme proteins are also found in the cell wall, apparently as bona fide components. In addition, the expression of some proteins is associated with the morphological growth form of the fungus and may play a role in morphogenesis. Finally, surface mannoproteins are strong immunogens that trigger and modulate the host immune response during candidiasis.
Growth Kinetics of Suspended Microbial Cells: From Single-Substrate-Controlled Growth to Mixed-Substrate Kinetics
Authors: Kovárová-Kovar Karin , Egli Thomas 
Published: December 2019

SUMMARY

Growth kinetics, i.e., the relationship between specific growth rate and the concentration of a substrate, is one of the basic tools in microbiology. However, despite more than half a century of research, many fundamental questions about the validity and application of growth kinetics as observed in the laboratory to environmental growth conditions are still unanswered. For pure cultures growing with single substrates, enormous inconsistencies exist in the growth kinetic data reported. The low quality of experimental data has so far hampered the comparison and validation of the different growth models proposed, and only recently have data collected from nutrient-controlled chemostat cultures allowed us to compare different kinetic models on a statistical basis. The problems are mainly due to (i) the analytical difficulty in measuring substrates at growth-controlling concentrations and (ii) the fact that during a kinetic experiment, particularly in batch systems, microorganisms alter their kinetic properties because of adaptation to the changing environment. For example, for Escherichia coli growing with glucose, a physiological long-term adaptation results in a change in K
S
for glucose from some 5 mg liter
−1
to ca. 30 μg liter
−1
. The data suggest that a dilemma exists, namely, that either “intrinsic” K
S
(under substrate-controlled conditions in chemostat culture) or μ
max
(under substrate-excess conditions in batch culture) can be measured but both cannot be determined at the same time. The above-described conventional growth kinetics derived from single-substrate-controlled laboratory experiments have invariably been used for describing both growth and substrate utilization in ecosystems. However, in nature, microbial cells are exposed to a wide spectrum of potential substrates, many of which they utilize simultaneously (in particular carbon sources). The kinetic data available to date for growth of pure cultures in carbon-controlled continuous culture with defined mixtures of two or more carbon sources (including pollutants) clearly demonstrate that simultaneous utilization results in lowered residual steady-state concentrations of all substrates. This should result in a competitive advantage of a cell capable of mixed-substrate growth because it can grow much faster at low substrate concentrations than one would expect from single-substrate kinetics. Additionally, the relevance of the kinetic principles obtained from defined culture systems with single, mixed, or multicomponent substrates to the kinetics of pollutant degradation as it occurs in the presence of alternative carbon sources in complex environmental systems is discussed. The presented overview indicates that many of the environmentally relevant apects in growth kinetics are still waiting to be discovered, established, and exploited.