scholarly journals Temporal compartmentalization of viral infection in bacterial cells

2021 ◽  
Vol 118 (28) ◽  
pp. e2018297118
Author(s):  
Audrey Labarde ◽  
Lina Jakutyte ◽  
Cyrille Billaudeau ◽  
Beatrix Fauler ◽  
Maria López-Sanz ◽  
...  
Keyword(s):  
Genome Replication ◽  
Bacterial Cells ◽  
Biosynthetic Activity ◽  
Genome Packaging ◽  
Viral Dna ◽  
Particle Assembly ◽  
Protein Cage ◽  
Host Cytoplasm ◽  
Total Dna ◽  
Viral Particle Assembly

Virus infection causes major rearrangements in the subcellular architecture of eukaryotes, but its impact in prokaryotic cells was much less characterized. Here, we show that infection of the bacterium Bacillus subtilis by bacteriophage SPP1 leads to a hijacking of host replication proteins to assemble hybrid viral–bacterial replisomes for SPP1 genome replication. Their biosynthetic activity doubles the cell total DNA content within 15 min. Replisomes operate at several independent locations within a single viral DNA focus positioned asymmetrically in the cell. This large nucleoprotein complex is a self-contained compartment whose boundaries are delimited neither by a membrane nor by a protein cage. Later during infection, SPP1 procapsids localize at the periphery of the viral DNA compartment for genome packaging. The resulting DNA-filled capsids do not remain associated to the DNA transactions compartment. They bind to phage tails to build infectious particles that are stored in warehouse compartments spatially independent from the viral DNA. Free SPP1 structural proteins are recruited to the dynamic phage-induced compartments following an order that recapitulates the viral particle assembly pathway. These findings show that bacteriophages restructure the crowded host cytoplasm to confine at different cellular locations the sequential processes that are essential for their multiplication.

Are phytoplankton population density maxima predictable through analysis of host and viral genomic DNA content?

2006 ◽  
Vol 86 (3) ◽  
pp. 491-498 ◽  
Author(s):  
Chris M. Brown ◽  
Janice E. Lawrence ◽  
Douglas A. Campbell
Keyword(s):  
Population Density ◽  
Dna Content ◽  
Viral Genome ◽  
Particle Assembly ◽  
Phytoplankton Population ◽  
Strong Linear Correlation ◽  
Viral Progeny ◽  
A Cell ◽  
Viral Proliferation ◽  
Viral Particle Assembly

Phytoplankton:virus interactions are important factors in aquatic nutrient cycling and community succession. The number of viral progeny resulting from an infection of a cell critically influences the propagation of infection and concomitantly the dynamics of phytoplankton populations. Host nucleotide content may be the resource limiting viral particle assembly. We present evidence for a strong linear correlation between measured viral burst sizes and viral burst sizes predicted from the host DNA content divided by the viral genome size, across a diversity of phytoplankton:viral pairs. An analysis of genome sizes therefore supports predictions of taxon-specific phytoplankton population density thresholds beyond which viral proliferation can trim populations or terminate phytoplankton blooms. We present corollaries showing that host:virus interactions may place evolutionary pressure towards genome reduction of both phytoplankton hosts and their viruses.

scholarly journals Murine Cytomegalovirus Capsid Assembly Is Dependent on US22 Family Gene M140 in Infected Macrophages

2009 ◽  
Vol 83 (15) ◽  
pp. 7449-7456 ◽  
Author(s):  
Laura K. Hanson ◽  
Jacquelyn S. Slater ◽  
Victoria J. Cavanaugh ◽  
William W. Newcomb ◽  
Lisa L. Bolin ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Early Gene ◽  
Murine Cytomegalovirus ◽  
Genome Replication ◽  
Microtubule Organizing Center ◽  
Viral Dna ◽  
Dnase Treatment ◽  
Organizing Center ◽  
Significant Impairment ◽  
The Stability

ABSTRACT Macrophages are an important target cell for infection with cytomegalovirus (CMV). A number of viral genes that either are expressed specifically in this cell type or function to optimize CMV replication in this host cell have now been identified. Among these is the murine CMV (MCMV) US22 gene family member M140, a nonessential early gene whose deletion (RVΔ140) leads to significant impairment in virus replication in differentiated macrophages. We have now determined that the defect in replication is at the stage of viral DNA encapsidation. Although the rate of RVΔ140 genome replication and extent of DNA cleavage were comparable to those for revertant virus, deletion of M140 resulted in a significant reduction in the number of viral capsids in the nucleus, and the viral DNA remained sensitive to DNase treatment. These data are indicative of incomplete virion assembly. Steady-state levels of both the major capsid protein (M86) and tegument protein M25 were reduced in the absence of the M140 protein (pM140). This effect may be related to the localization of pM140 to an aggresome-like, microtubule organizing center-associated structure that is known to target misfolded and overexpressed proteins for degradation. It appears, therefore, that pM140 indirectly influences MCMV capsid formation in differentiated macrophages by regulating the stability of viral structural proteins.

scholarly journals Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Qiang Ding ◽  
Ila Nimgaonkar ◽  
Nicholas F. Archer ◽  
Yaron Bram ◽  
Brigitte Heller ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Regulatory Element ◽  
Hepatitis E ◽  
Regulatory Elements ◽  
Genome Replication ◽  
Genomic Rna ◽  
Chronic Infections ◽  
Particle Assembly ◽  
Subgenomic Rna ◽  
General Importance

ABSTRACT Approximately 20 million hepatitis E virus (HEV) infections occur annually in both developing and industrialized countries. Most infections are self-limiting, but they can lead to chronic infections and cirrhosis in immunocompromised patients, and death in pregnant women. The mechanisms of HEV replication remain incompletely understood due to scarcity of adequate experimental platforms. HEV undergoes asymmetric genome replication, but it produces an additional subgenomic (SG) RNA encoding the viral capsid and a viroporin in partially overlapping open reading frames. Using a novel transcomplementation system, we mapped the intragenomic subgenomic promoter regulating SG RNA synthesis. This cis -acting element is highly conserved across all eight HEV genotypes, and when the element is mutated, it abrogates particle assembly and release. Our work defines previously unappreciated viral regulatory elements and provides the first in-depth view of the intracellular genome dynamics of this emerging human pathogen. IMPORTANCE HEV is an emerging pathogen causing severe liver disease. The genetic information of HEV is encoded in RNA. The genomic RNA is initially copied into a complementary, antigenomic RNA that is a template for synthesis of more genomic RNA and for so-called subgenomic RNA. In this study, we identified the precise region within the HEV genome at which the synthesis of the subgenomic RNA is initiated. The nucleotides within this region are conserved across genetically distinct variants of HEV, highlighting the general importance of this segment for the virus. To identify this regulatory element, we developed a new experimental system that is a powerful tool with broad utility to mechanistically dissect many other poorly understood functional elements of HEV.

scholarly journals Function, Architecture, and Biogenesis of Reovirus Replication Neoorganelles

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 288 ◽  
Author(s):  
Raquel Tenorio ◽  
Isabel Fernández de Castro ◽  
Jonathan J. Knowlton ◽  
Paula F. Zamora ◽  
Danica M. Sutherland ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Host Cells ◽  
Genome Replication ◽  
Nonstructural Proteins ◽  
Particle Assembly ◽  
The Matrix ◽  
Viral Genome Replication ◽  
Pathogenic Viruses ◽  
Infected Cells ◽  
Reovirus Replication

Most viruses that replicate in the cytoplasm of host cells form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Reoviruses are common pathogens of mammals that have been linked to celiac disease and show promise for oncolytic applications. These viruses form nonenveloped, double-shelled virions that contain ten segments of double-stranded RNA. Replication organelles in reovirus-infected cells are nucleated by viral nonstructural proteins µNS and σNS. Both proteins partition the endoplasmic reticulum to form the matrix of these structures. The resultant membranous webs likely serve to anchor viral RNA–protein complexes for the replication of the reovirus genome and the assembly of progeny virions. Ongoing studies of reovirus replication organelles will advance our knowledge about the strategies used by viruses to commandeer host biosynthetic pathways and may expose new targets for therapeutic intervention against diverse families of pathogenic viruses.

scholarly journals A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 928 ◽  
Author(s):  
Eunji Jo ◽  
Jaewon Yang ◽  
Alexander Koenig ◽  
Seung Yoon ◽  
Marc Windisch
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Preparation Method ◽  
Cost Effective ◽  
Viral Dna ◽  
Dna Preparation ◽  
Hbv Replication ◽  
Viral Particles ◽  
B Virus ◽  
Total Dna

Hepatitis B virus (HBV) is a para-retrovirus that reverse transcribes its pregenomic RNA into relaxed circular DNA inside viral nucleocapsids. The number of HBV genomes produced in vitro is typically quantified using commercial silica-membrane-based nucleic acid purification kits to isolate total DNA followed by HBV-specific quantitative PCR (qPCR). However, despite the convenience of commercial kits, this procedure is costly and time-consuming due to multiple centrifugation steps, which produce unnecessary waste. Here, we report a rapid, cost-effective, and environmentally friendly total DNA preparation method. The assay is based on the simple incubation of detergent and proteinase K with cells or cell-free supernatants to permeabilize cells and disrupt viral particles. After heat inactivation and subsequent centrifugation to clear the lysates, DNA samples are directly subjected to qPCR to quantify HBV genomes. As a proof of concept, the assay was developed in 12-well plates to assess intra- and extracellular HBV genome equivalents (GEqs) of stably viral-replicating cell lines (e.g., HepAD38) and HBV-infected HepG2-NTCP cells, both treated with lamivudine (LMV), an HBV replication inhibitor. Viral DNA was also prepared from the serum of patients chronically infected with HBV. To validate the assay, a representative commercial DNA isolation kit was used side-by-side to isolate intra- and extracellular HBV DNA. Both methods yielded comparable amounts of HBV GEqs with comparable LMV 50% efficient concentration (EC50) values. The assay was subsequently adapted to 96- and 384-well microtiter plates using HepAD38 cells. The EC50 values were comparable to those obtained in 12-well plates. In addition, the calculated coefficient of variation, Z’ values, and assay window demonstrated high reproducibility and quality. We devised a novel, robust, reproducible, high-throughput microtiter plate DNA preparation method suitable for quantifying HBV GEqs by qPCR analysis. This strategy enables rapid and convenient quantitative analysis of multiple viral DNA samples in parallel to investigate intracellular HBV replication and the secretion of DNA-containing viral particles.

scholarly journals The GLN Family of Murine Endogenous Retroviruses Contains an Element Competent for Infectious Viral Particle Formation

2008 ◽  
Vol 82 (9) ◽  
pp. 4413-4419 ◽  
Author(s):  
David Ribet ◽  
Francis Harper ◽  
Cécile Esnault ◽  
Gérard Pierron ◽  
Thierry Heidmann
Keyword(s):  
Viral Particle ◽  
Leukemia Virus ◽  
Endogenous Retroviruses ◽  
Particle Assembly ◽  
New Class ◽  
Mouse Mammary Tumor ◽  
The Family ◽  
Tumor Virus ◽  
Mouse Cells ◽  
Viral Particle Assembly

ABSTRACT Several families of endogenous retroviruses (ERVs) have been identified in the mouse genome, in several instances by in silico searches, but for many of them it remains to be determined whether there are elements that can still encode functional retroviral particles. Here, we identify, within the GLN family of highly reiterated ERVs, one, and only one, copy that encodes retroviral particles prone to infection of mouse cells. We show that its envelope protein confers an ecotropic host range and recognizes a receptor different from mCAT1 and mSMIT1, the two previously identified receptors for other ecotropic mouse retroviruses. Electron microscopy disclosed viral particle assembly and budding at the cell membrane, as well as release of mature particles into the extracellular space. These particles are closely related to murine leukemia virus (MLV) particles, with which they have most probably been confused in the past. This study, therefore, identifies a new class of infectious mouse ERVs belonging to the family Gammaretroviridae, with one family member still functional today. This family is in addition to the two MLV and mouse mammary tumor virus families of active mouse ERVs with an extracellular life cycle.

scholarly journals Suppression of Adenovirus Replication by Cardiotonic Steroids

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Filomena Grosso ◽  
Peter Stoilov ◽  
Clifford Lingwood ◽  
Martha Brown ◽  
Alan Cochrane
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Rna Splicing ◽  
Antiviral Effect ◽  
Early Gene ◽  
Genome Replication ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Human Adenoviruses ◽  
Adenovirus Replication

ABSTRACT The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies. IMPORTANCE Despite human adenoviruses being a common and, in some instances, life-threating pathogen in humans, there are few well-tolerated therapies. In this report, we demonstrate that two cardiotonic steroids already in use in humans, digoxin and digitoxin, are potent inhibitors of multiple adenovirus species. A synthetic derivative of the cardiotonic steroid convallotoxin was even more potent than digoxin and digitoxin when tested with HAdV-C5. These drugs alter the cascade of adenovirus gene expression, acting after initiation of early gene expression to block viral DNA replication and synthesis of viral structural proteins. These findings validate a novel approach to treating adenovirus infections through the modulation of host cell processes.

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