scholarly journals RNA Silencing May Play a Role in but Is Not the Only Determinant of the Multiplicity of Infection

2015 ◽  
Vol 90 (1) ◽  
pp. 553-561 ◽  
Author(s):  
Livia Donaire ◽  
József Burgyán ◽  
Fernando García-Arenal
Keyword(s):  
Gene Silencing ◽  
Viral Proteins ◽  
Virus Evolution ◽  
Multiplicity Of Infection ◽  
Silencing Suppressor ◽  
Content Type ◽  
Viral Genomes ◽  
A Cell ◽  
Virus Fitness

ABSTRACTThe multiplicity of infection (MOI), i.e., the number of viral genomes that infect a cell, is an important parameter in virus evolution, which for each virus and environment may have an optimum value that maximizes virus fitness. Thus, the MOI might be controlled by virus functions, an underexplored hypothesis in eukaryote-infecting viruses. To analyze if the MOI is controlled by virus functions, we estimated the MOI in plants coinfected by two genetic variants ofTomato bushy stunt virus(TBSV); by TBSV and a TBSV-derived defective interfering RNA (DI-RNA); or by TBSV and a second tombusvirus,Cymbidium ringspot virus(CymRSV). The MOI was significantly larger in TBSV-CymRSV coinfections (∼4.0) than in TBSV-TBSV or TBSV–DI-RNA coinfections (∼1.7 to 2.2). Coinfections by CymRSV or TBSV with chimeras in which an open reading frame (ORF) of one virus species was replaced by that of the other identified a role of viral proteins in determining the MOI, which ranged from 1.6 to 3.9 depending on the coinfecting genotypes. However, no virus-encoded protein or genomic region was the sole MOI determinant. Coinfections by CymRSV and TBSV mutants in which the expression of the gene-silencing suppressor protein p19 was abolished also showed a possible role of gene silencing in MOI determination. Taken together, these results demonstrate that the MOI is a quantitative trait showing continuous variation and that as such it has a complex determination involving different virus-encoded functions.IMPORTANCEThe number of viral genomes infecting a cell, or the multiplicity of infection (MOI), is an important parameter in virus evolution affecting recombination rates, selection intensity on viral genes, evolution of multipartite genomes, or hyperparasitism by satellites or defective interfering particles. For each virus and environment, the MOI may have an optimum value that maximizes virus fitness, but little is known about MOI control in eukaryote-infecting viruses. We show here that in plants coinfected by two genotypes ofTomato bushy stunt virus(TBSV), the MOI was lower than in plants coinfected by TBSV andCymbidium ringspot virus(CymRSV). Coinfections by CymRSV or TBSV with TBSV-CymRSV chimeras showed a role of viral proteins in MOI determination. Coinfections by CymRSV and TBSV mutants not expressing the gene-silencing suppressor protein also showed a role of gene silencing in MOI determination. The results demonstrate that the MOI is a quantitative trait with a complex determination involving different viral functions.

scholarly journals Coxsackieviruses Undergo Intercellular Transmission as Pools of Sibling Viral Genomes Associated to Membranes

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 120
Author(s):  
Juan-Vicente Bou ◽  
Ron Geller ◽  
Rafael Sanjuán
Keyword(s):  
Genetic Variants ◽  
Social Evolution ◽  
Viral Population ◽  
Multiplicity Of Infection ◽  
Defective Interfering Particles ◽  
Viral Genomes ◽  
Viral Particles ◽  
A Cell ◽  
Infected Cells ◽  
Population Sequence

Some viruses are released from cells as pools of membrane-associated virions. By increasing the multiplicity of infection, this type of collective dispersal could favor viral cooperation, but also the emergence of cheater-like viruses, such as defective interfering particles. To better understand this process, we examined the genetic diversity of membrane-associated coxsackievirus infectious units. We found that infected cells released large membranous structures containing 8–21 infectious particles on average, including vesicles. However, in most cases (62–93%), these structures did not promote the co-transmission of different viral genetic variants present in a cell. Furthermore, collective dispersal had no effect on viral population sequence diversity. Our results indicate that membrane-associated collective infectious units typically contain viral particles derived from the same parental genome. Hence, if cooperation occurred, it should probably involve sibling viral particles rather than different variants. As shown by social evolution theory, cooperation among siblings should be robust against cheater invasion.

scholarly journals Pleiotropic Effects of Resistance-Breaking Mutations on Particle Stability Provide Insight into Life History Evolution of a Plant RNA Virus

2017 ◽  
Vol 91 (18) ◽  
Author(s):  
Sayanta Bera ◽  
Manuel G. Moreno-Pérez ◽  
Sara García-Figuera ◽  
Israel Pagán ◽  
Aurora Fraile ◽  
...  
Keyword(s):  
Life History ◽  
Life History Traits ◽  
Virus Evolution ◽  
Virus Multiplication ◽  
Pepper Mild Mottle Virus ◽  
Content Type ◽  
Resistance Breaking ◽  
Trade Offs ◽  
Particle Stability ◽  
Virus Fitness

ABSTRACT In gene-for-gene host-virus interactions, virus evolution to infect and multiply in previously resistant host genotypes, i.e., resistance breaking, is a case of host range expansion, which is predicted to be associated with fitness penalties. Negative effects of resistance-breaking mutations on within-host virus multiplication have been documented for several plant viruses. However, understanding virus evolution requires analyses of potential trade-offs between different fitness components. Here we analyzed whether coat protein (CP) mutations in Pepper mild mottle virus that break L-gene resistance in pepper affect particle stability and, thus, survival in the environment. For this purpose, CP mutations determining the overcoming of L 3 and L 4 resistance alleles were introduced in biologically active cDNA clones. The kinetics of the in vitro disassembly of parental and mutant particles were compared under different conditions. Resistance-breaking mutations variously affected particle stability. Structural analyses identified the number and type of axial and side interactions of adjacent CP subunits in virions, which explained differences in particle stability and contribute to understanding of tobamovirus disassembly. Resistance-breaking mutations also affected virus multiplication and virulence in the susceptible host, as well as infectivity. The sense and magnitude of the effects of resistance-breaking mutations on particle stability, multiplication, virulence, or infectivity depended on the specific mutation rather than on the ability to overcome the different resistance alleles, and effects on different traits were not correlated. Thus, the results do not provide evidence of links or trade-offs between particle stability, i.e., survival, and other components of virus fitness or virulence. IMPORTANCE The effect of survival on virus evolution remains underexplored, despite the fact that life history trade-offs may constrain virus evolution. We approached this topic by analyzing whether breaking of L-gene resistance in pepper by Pepper mild mottle virus, determined by coat protein (CP) mutations, is associated with reduced particle stability and survival. Resistance-breaking mutations affected particle stability by altering the interactions between CP subunits. However, the sense and magnitude of these effects were unrelated to the capacity to overcome different resistance alleles. Thus, resistance breaking was not traded with survival. Resistance-breaking mutations also affected virus fitness within the infected host, virulence, and infectivity in a mutation-specific manner. Comparison of the effects of CP mutations on these various traits indicates that there are neither trade-offs nor positive links between survival and other life history traits. These results demonstrate that trade-offs between life history traits may not be a general constraint in virus evolution.

scholarly journals LysPBC2, a Novel Endolysin Harboring aBacillus cereusSpore Binding Domain

2018 ◽  
Vol 85 (5) ◽  
Author(s):  
Minsuk Kong ◽  
Hongjun Na ◽  
Nam-Chul Ha ◽  
Sangryeol Ryu
Keyword(s):  
Cell Wall ◽  
Catalytic Domain ◽  
Binding Activity ◽  
Binding Domain ◽  
Lytic Activity ◽  
Content Type ◽  
Cell Wall Binding ◽  
A Cell ◽  
Cell Wall Binding Domain

ABSTRACTTo control the spore-forming human pathogenBacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolatedB. cereusphage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against allBacillus,Listeria, andClostridiumspecies tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds toB. cereusspores but not to vegetative cells ofB. cereus. Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells ofB. cereuscompared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure.IMPORTANCEBacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of aBacillus cereusphage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controllingB. cereus.

scholarly journals Tulane Virus Recognizes the A Type 3 and B Histo-Blood Group Antigens

2014 ◽  
Vol 89 (2) ◽  
pp. 1419-1427 ◽  
Author(s):  
Dongsheng Zhang ◽  
Pengwei Huang ◽  
Lu Zou ◽  
Todd L. Lowary ◽  
Ming Tan ◽  
...  
Keyword(s):  
Cell Culture ◽  
Blood Group ◽  
Culture Method ◽  
Blood Group Antigens ◽  
Content Type ◽  
Tulane Virus ◽  
A Cell ◽  
Type 3

ABSTRACTTulane virus (TV), the prototype of theRecovirusgenus in the calicivirus family, was isolated from the stools of rhesus monkeys and can be cultivatedin vitroin monkey kidney cells. TV is genetically closely related to the genusNorovirusand recognizes the histo-blood group antigens (HBGAs), similarly to human noroviruses (NoVs), making it a valuable surrogate for human NoVs. However, the precise structures of HBGAs recognized by TV remain elusive. In this study, we performed binding and blocking experiments on TV with extended HBGA types and showed that, while TV binds all four types (types 1 to 4) of the B antigens, it recognizes only the A type 3 antigen among four types of A antigens tested. The requirements for HBGAs in TV replication were demonstrated by blocking of TV replication in cell culture using the A type 3/4 and B saliva samples. Similar results were also observed in oligosaccharide-based blocking assays. Importantly, the previously reported, unexplained increase in TV replication by oligosaccharide in cell-based blocking assays has been clarified, which will facilitate the application of TV as a surrogate for human NoVs.IMPORTANCEOur understanding of the role of HBGAs in NoV infection has been significantly advanced in the past decade, but direct evidence for HBGAs as receptors for human NoVs remains lacking due to a lack of a cell culture method. TV recognizes HBGAs and can replicatein vitro, providing a valuable surrogate for human NoVs. However, TV binds to some but not all saliva samples from A-positive individuals, and an unexplained observation of synthetic oligosaccharide blocking of TV binding has been reported. These issues have been resolved in this study.

scholarly journals Detoxification of 7-Dehydrocholesterol Fatal to Helicobacter pylori Is a Novel Role of Cholesterol Glucosylation

2012 ◽  
Vol 195 (2) ◽  
pp. 359-367 ◽  
Author(s):  
Hirofumi Shimomura ◽  
Kouichi Hosoda ◽  
David J. McGee ◽  
Shunji Hayashi ◽  
Kenji Yokota ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Cell Membrane ◽  
Membrane Lipid ◽  
Host Immune System ◽  
Content Type ◽  
Mucosal Tissues ◽  
Gene Mutant ◽  
A Cell ◽  
H Pylori

ABSTRACTThe glucosylation of free cholesterol (FC) byHelicobacter pyloricells has various biological significances for the survival of this bacterium.H. pyloricells with glucosylated FC are capable of evading host immune systems, such as phagocytosis by macrophages and activation of antigen-specific T cells, and surviving in the gastric mucosal tissues for long periods. An additional role of cholesterol glucosylation in the survival ofH. pyloriwhich is distinct from the role of escaping the host immune system, however, has yet to be identified. This study demonstrated that 7-dehydrocholesterol (7dFC), an FC precursor, is a toxic compound fatal toH. pyloricells, but the cell membrane ofH. pyloriis capable of absorbing this toxic sterol via glucosylation. In contrast to the case with 7dFC, no toxicity toH. pyloricells was detected from the glucosylated 7dFC. In addition,cgtgene mutantH. pyloricells that cannot glucosylate cholesterols had higher susceptibility to the toxic action of 7dFC than wild-typeH. pyloricells. These results indicate that thecgtgene product ofH. pyloriserves to detoxify the sterol fatal to this bacterium and to permit this toxic sterol as a cell membrane lipid component. In summary, this study defined a novel role of cholesterol glucosylation inH. pylori.

scholarly journals The Viral Polymerase Complex Mediates the Interaction of vRNPs with Recycling Endosomes During SeV Assembly

2020 ◽  
Author(s):  
Emmanuelle Genoyer ◽  
Katarzyna Kulej ◽  
Chuan Tien Hung ◽  
Patricia A. Thibault ◽  
Kristopher Azarm ◽  
...  
Keyword(s):  
Sendai Virus ◽  
Initial Step ◽  
Viral Proteins ◽  
Viral Polymerase ◽  
Recycling Endosomes ◽  
Viral Genomes ◽  
Parainfluenza Viruses ◽  
Infected Cells ◽  
Human Parainfluenza Viruses

ABSTRACTParamyxoviruses are negative sense single-stranded RNA viruses that comprise many important human and animal pathogens, including human parainfluenza viruses. These viruses bud from the plasma membrane of infected cells after the viral ribonucleoprotein complex (vRNP) is transported from the cytoplasm to the cell membrane via Rab11a-marked recycling endosomes. The viral proteins that are critical for mediating this important initial step in viral assembly are unknown. Here we use the model paramyxovirus, murine parainfluenza virus 1, or Sendai virus (SeV), to investigate the roles of viral proteins in Rab11a-driven virion assembly. We previously reported that infection with SeV containing high levels of copy-back defective viral genomes (DVGs) generates heterogenous populations of cells. Cells enriched in full-length virus produce viral particles containing standard or defective viral genomes, while cells enriched in DVGs do not, despite high levels of defective viral genome replication. Here we take advantage of this heterogenous cell phenotype to identify proteins that mediate interaction of vRNPs with Rab11a. We examine the role of matrix protein and nucleoprotein and determine that they are not sufficient to drive interaction of vRNPs with recycling endosomes. Using a combination of mass spectrometry and comparative protein abundance and localization in DVG- and FL-high cells, we identify viral polymerase complex components L and, specifically, its cofactor C proteins as interactors with Rab11a. We find that accumulation of these proteins within the cell is the defining feature that differentiates cells that proceed to viral egress from cells which remain in replication phases.IMPORTANCEParamyxoviruses are a family of viruses that include a number of pathogens with significant burdens on human health. Particularly, human parainfluenza viruses are an important cause of pneumonia and bronchiolitis in children for which there are no vaccines or direct acting antivirals. These cytoplasmic replicating viruses bud from the plasma membrane and coopt cellular endosomal recycling pathways to traffic viral ribonucleoprotein complexes from the cytoplasm to the membrane of infected cells. The viral proteins required for viral engagement with the recycling endosome pathway are still not known. Here we use the model paramyxovirus Sendai virus, or murine parainfluenza virus 1, to investigate the role of viral proteins in this initial step of viral assembly. We find that viral polymerase components large protein L and accessory C proteins are necessary for engagement with recycling endosomes. These findings are important in identifying viral proteins as potential targets for development of antivirals.

scholarly journals Combinatorial interactions between viral proteins expand the functional landscape of the viral proteome

2021 ◽  
Author(s):  
Liping Wang ◽  
Huang Tan ◽  
Laura Medina-Puche ◽  
Mengshi Wu ◽  
Borja Garnelo Gomez ◽  
...  
Keyword(s):  
Viral Proteins ◽  
Host Cells ◽  
In Planta ◽  
Viral Genomes ◽  
Intracellular Parasites ◽  
Molecular Machinery ◽  
Combinatorial Interactions ◽  
Massive Cell ◽  
Coding Capacity

As intracellular parasites, viruses need to manipulate the molecular machinery of their host cells in order to enable their own replication and spread. This manipulation is based on the activity of virus-encoded proteins. The reduced size of viral genomes imposes restrictions in coding capacity; how the action of the limited number of viral proteins results in the massive cell reprogramming observed during the viral infection is a long-standing conundrum in virology. In this work, we explore the hypothesis that combinatorial interactions expand the multifunctionality of viral proteins, which may exert different activities individually and when in combination, physical or functional. We show that the proteins encoded by a plant-infecting DNA virus physically associate with one another in an intricate network. Our results further demonstrate that these interactions can modify the subcellular localization of the viral proteins involved, and that co-expressed interacting viral proteins can exert novel biological functions in planta that go beyond the sum of their individual functions. Based on this, we propose a model in which combinatorial physical and functional interactions between viral proteins enlarge the functional landscape of the viral proteome, which underscores the importance of studying the role of viral proteins in the context of the infection.

scholarly journals The Role of Viral Proteins in the Regulation of Exosomes Biogenesis

2021 ◽  
Vol 11 ◽  
Author(s):  
Xiaonan Jia ◽  
Yiqian Yin ◽  
Yiwen Chen ◽  
Lingxiang Mao
Keyword(s):  
Nucleic Acids ◽  
Viral Infection ◽  
Viral Proteins ◽  
Viral Genomes ◽  
Extracellular Milieu ◽  
Membrane Bound ◽  
Viral Components ◽  
External Stimuli

Exosomes are membrane-bound vesicles of endocytic origin, secreted into the extracellular milieu, in which various biological components such as proteins, nucleic acids, and lipids reside. A variety of external stimuli can regulate the formation and secretion of exosomes, including viruses. Viruses have evolved clever strategies to establish effective infections by employing exosomes to cloak their viral genomes and gain entry into uninfected cells. While most recent exosomal studies have focused on clarifying the effect of these bioactive vesicles on viral infection, the mechanisms by which the virus regulates exosomes are still unclear and deserve further attention. This article is devoted to studying how viral components regulate exosomes biogenesis, composition, and secretion.

scholarly journals Analysis of Viral Membranes Formed in Cells Infected by a Vaccinia Virus L2-Deletion Mutant Suggests Their Origin from the Endoplasmic Reticulum

2012 ◽  
Vol 87 (3) ◽  
pp. 1861-1871 ◽  
Author(s):  
Liliana Maruri-Avidal ◽  
Andrea S. Weisberg ◽  
Himani Bisht ◽  
Bernard Moss
Keyword(s):  
Endoplasmic Reticulum ◽  
Deletion Mutant ◽  
Viral Proteins ◽  
Primary Role ◽  
Membrane Biogenesis ◽  
Structural Components ◽  
Viral Membrane ◽  
A Cell ◽  
Immature Virion

ABSTRACTAssembly of the poxvirus immature virion (IV) membrane is a poorly understood event that occurs within the cytoplasm. At least eight viral proteins participate in formation of the viral membrane. Of these, A14, A17, and D13 are structural components whereas A6, A11, F10, H7, and L2 participate in membrane biogenesis. L2, the object of this study, is conserved in all chordopoxviruses, expressed early in infection, and associated with the endoplasmic reticulum (ER) throughout the cell and at the edges of crescent-shaped IV precursors. Previous studies with an inducible L2 mutant revealed abortive formation of the crescent membrane. However, possible low-level L2 synthesis under nonpermissive conditions led to ambiguity in interpretation. Here, we constructed a cell line that expresses L2, which allowed the creation of an L2-deletion mutant. In noncomplementing cells, replication was aborted prior to formation of mature virions and two types of aberrant structures were recognized. One consisted of short crescents, at the surface of dense masses of viroplasm, which were labeled with antibodies to the A11, A14, A17, and D13 proteins. The other structure consisted of “empty” IV-like membranes, also labeled with antibodies to the viral proteins, which appeared to be derived from adjacent calnexin-containing ER. A subset of 25 proteins examined, exemplified by components of the entry-fusion complex, were greatly diminished in amount. The primary role of L2 may be to recruit ER and modulate its transformation to viral membranes in juxtaposition with the viroplasm, simultaneously preventing the degradation of viral proteins dependent on viral membranes for stability.

scholarly journals Essential Role of the Cytoplasmic Chemoreceptor TlpT in the De Novo Formation of Chemosensory Complexes in Rhodobacter sphaeroides

2017 ◽  
Vol 199 (19) ◽  
Author(s):  
Christopher W. Jones ◽  
Judith P. Armitage
Keyword(s):  
Rhodobacter Sphaeroides ◽  
De Novo ◽  
Adaptor Proteins ◽  
Cluster Assembly ◽  
Content Type ◽  
The Reformation ◽  
A Cell ◽  
Stochastic Assembly ◽  
Single Flagellum

ABSTRACT Bacterial chemosensory proteins form large hexagonal arrays. Several key features of chemotactic signaling depend on these large arrays, namely, cooperativity between receptors, sensitivity, integration of different signals, and adaptation. The best-studied arrays are the membrane-associated arrays found in most bacteria. Rhodobacter sphaeroides has two spatially distinct chemosensory arrays, one is transmembrane and the other is cytoplasmic. These two arrays work together to control a single flagellum. Deletion of one of the soluble chemoreceptors, TlpT, results in the loss of the formation of the cytoplasmic array. Here, we show the expression of TlpT in a tlpT deletion background results in the reformation of the cytoplasmic array. The number of arrays formed is dependent on the cell length, indicating spatial limitations on the number of arrays in a cell and stochastic assembly. Deletion of PpfA, a protein required for the positioning and segregation of the cytoplasmic array, results in slower array formation upon TlpT expression and fewer arrays, suggesting it accelerates cluster assembly. IMPORTANCE Bacterial chemosensory arrays are usually membrane associated and consist of thousands of copies of receptors, adaptor proteins, kinases, and adaptation enzymes packed into large hexagonal structures. Rhodobacter sphaeroides also has cytoplasmic arrays, which divide and segregate using a chromosome-associated ATPase, PpfA. The expression of the soluble chemoreceptor TlpT is shown to drive the formation of the arrays, accelerated by PpfA. The positioning of these de novo arrays suggests their position is the result of stochastic assembly rather than active positioning.

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