scholarly journals Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

2015 ◽  
Vol 89 (8) ◽  
pp. 4372-4386 ◽  
Author(s):  
Ekaterina G. Viktorova ◽  
Jules Nchoutmboube ◽  
Lauren A. Ford-Siltz ◽  
George A. Belov
Keyword(s):  
Viral Replication ◽  
Small Rna ◽  
Rna Viruses ◽  
Brefeldin A ◽  
Vero Cells ◽  
Cellular Protein ◽  
Rational Approach ◽  
Cell Type ◽  
Cellular Factors ◽  
Resistant Mutants

ABSTRACTIt is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses.IMPORTANCECompared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can easily generate mutants resistant to practically any compounds targeting viral proteins. An alternative approach is to target stable cellular factors recruited for the virus-specific functions. In the present study, we analyzed the factors permitting and restricting the establishment of the resistance of poliovirus, a small (+)RNA virus, to brefeldin A (BFA), a drug targeting a cellular component of the viral replication complex. We found that the emergence and replication potential of resistant mutants is cell type dependent and that BFA resistance reduces virus fitness. Our data provide a rational approach to the development of antiviral therapeutics targeting host factors.

scholarly journals A Redundant Mechanism of Recruitment Underlies the Remarkable Plasticity of the Requirement of Poliovirus Replication for the Cellular ArfGEF GBF1

2019 ◽  
Vol 93 (21) ◽  
Author(s):  
Ekaterina G. Viktorova ◽  
Samuel Gabaglio ◽  
Justyna M. Meissner ◽  
Eunjoo Lee ◽  
Seyedehmahsa Moghimi ◽  
...  
Keyword(s):  
Viral Replication ◽  
Secretory Pathway ◽  
Rna Viruses ◽  
Nonstructural Protein ◽  
Direct Interaction ◽  
Cellular Protein ◽  
Small Gtpases ◽  
Replication Process ◽  
Redundant Mechanism ◽  
Positive Strand Rna

ABSTRACT The replication of many positive-strand RNA viruses [(+)RNA viruses] depends on the cellular protein GBF1, but its role in the replication process is not clear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and related viruses has been shown to directly interact with GBF1, likely mediating its recruitment to the replication complexes. Surprisingly, viral mutants with a severely reduced level of 3A-GBF1 interaction demonstrate minimal replication defects in cell culture. Here, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrate that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results show that the Arf-activating property, but not the primary structure of the Sec7 domain, is indispensable for viral replication. They also suggest a redundant mechanism of recruitment of GBF1 to the replication sites, which is dependent not only on direct interaction of the protein with the viral protein 3A but also on determinants located in the noncatalytic C-terminal domains of GBF1. Such a double-targeting mechanism explains the previous observations of the remarkable tolerance of different levels of GBF1-3A interaction by the virus and likely constitutes an important element of the resilience of viral replication. IMPORTANCE Enteroviruses are a vast group of viruses associated with diverse human diseases, but only two of them could be controlled with vaccines, and effective antiviral therapeutics are lacking. Here, we investigated in detail the contribution of a cellular protein, GBF1, in the replication of poliovirus, a representative enterovirus. GBF1 supports the functioning of cellular membrane metabolism and is recruited to viral replication complexes upon infection. Our results demonstrate that the virus requires a limited subset of the normal GBF1 functions and reveal the elements of GBF1 essential to support viral replication under different conditions. Since diverse viruses often rely on the same cellular proteins for replication, understanding the mechanisms by which these proteins support infection is essential for the development of broad-spectrum antiviral therapeutics.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus ori-Lyt-Dependent DNA Replication: Involvement of Host Cellular Factors

2008 ◽  
Vol 82 (6) ◽  
pp. 2867-2882 ◽  
Author(s):  
Yan Wang ◽  
Hong Li ◽  
Qiyi Tang ◽  
Gerd G. Maul ◽  
Yan Yuan
Keyword(s):  
Dna Replication ◽  
Viral Replication ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Cellular Proteins ◽  
Cellular Factors ◽  
Virally Encoded ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Lytic Dna Replication ◽  
Parp 1

ABSTRACT Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors, including virally encoded origin-binding protein, DNA replication enzymes, and auxiliary factors. Two lytic DNA replication origins (ori-Lyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified, and two virally encoded proteins, namely, RTA and K8, have been shown to bind to the origins. In this study, we sought to identify cellular factors that associate with ori-Lyt by using DNA affinity purification and mass spectrometry. This approach led to identification of several cellular proteins that bind to KSHV ori-Lyt. They include topoisomerases (Topo) I and II, MSH2/6, RecQL, poly(ADP-ribose) polymerase I (PARP-1), DNA-PK, Ku86/70 autoantigens, and scaffold attachment factor A (SAF-A). RecQL appears to associate with prereplication complexes and be recruited to ori-Lyt through RTA and K8. Topoisomerases, MSH2, PARP-1, DNA-PK, and Ku86 were not detected in prereplication complexes but were present in replication initiation complexes on ori-Lyt. All these cellular proteins accumulate in viral replication compartments in the nucleus, indicating that these proteins may have a role in viral replication. Topo I and II appear to be essential for viral DNA replication as inhibition of their activities with specific inhibitors (camptothecin and ellipticine) blocked ori-Lyt-dependent DNA replication. Furthermore, inhibition of PARP-1 with chemical inhibitors (3-aminobenzamide and niacinamide) resulted in decreased ori-Lyt-dependent DNA replication, whereas hydroxyurea, which raises PARP-1 activity, caused an increase in the DNA replication, suggesting a positive role for PARP-1 in KSHV lytic DNA replication.

scholarly journals Povidone-Iodine Attenuates Viral Replication in Ocular Cells: Implications for Ocular Transmission of RNA Viruses

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 753
Author(s):  
Sneha Singh ◽  
Onkar B. Sawant ◽  
Shahzad I. Mian ◽  
Ashok Kumar
Keyword(s):  
Epithelial Cells ◽  
Viral Replication ◽  
Povidone Iodine ◽  
Rna Viruses ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Host Cells ◽  
Eye Banking ◽  
Antiviral Effects ◽  
Infected Cells

Several RNA viruses, including SARS-CoV-2, can infect or use the eye as an entry portal to cause ocular or systemic diseases. Povidone-Iodine (PVP-I) is routinely used during ocular surgeries and eye banking as a cost-effective disinfectant due to its broad-spectrum antimicrobial activity, including against viruses. However, whether PVP-I can exert antiviral activities in virus-infected cells remains elusive. In this study, using Zika (ZIKV) and Chikungunya (CHIKV) virus infection of human corneal and retinal pigment epithelial cells, we report antiviral mechanisms of PVP-I. Our data showed that PVP-I, even at the lowest concentration (0.01%), drastically reduced viral replication in corneal and retinal cells without causing cellular toxicity. Antiviral effects of PVP-I against ZIKV and CHIKV were mediated by direct viral inactivation, thus attenuating the ability of the virus to infect host cells. Moreover, one-minute PVP-I exposure of infected ocular cells drastically reduced viral replication and the production of infectious progeny virions. Furthermore, viral-induced (CHIKV) expression of inflammatory genes (TNF-α, IL-6, IL-8, and IL1β) were markedly reduced in PVP-I treated corneal epithelial cells. Together, our results demonstrate potent antiviral effects of PVP-I against ZIKV and CHIKV infection of ocular cells. Thus, a low dose of PVP-I can be used during tissue harvesting for corneal transplants to prevent potential transmission of RNA viruses via infected cells.

scholarly journals xMD-miRNA-seq to generate near in vivo miRNA expression estimates in colon epithelial cells

2018 ◽  
Author(s):  
Avi Z. Rosenberg ◽  
Carrie Wright ◽  
Karen Fox-Talbot ◽  
Anandita Rajpurohit ◽  
Courtney Williams ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Small Rna ◽  
Mirna Expression ◽  
Low Cost ◽  
Expression Patterns ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Cell Type

AbstractAccurate, RNA-seq based, microRNA (miRNA) expression estimates from primary cells have recently been described. However, this in vitro data is mainly obtained from cell culture, which is known to alter cell maturity/differentiation status, significantly changing miRNA levels. What is needed is a robust method to obtain in vivo miRNA expression values directly from cells. We introduce expression microdissection miRNA small RNA sequencing (xMD-miRNA-seq), a method to isolate cells directly from formalin fixed paraffin-embedded (FFPE) tissues. xMD-miRNA-seq is a low-cost, high-throughput, immunohistochemistry-based method to capture any cell type of interest. As a proof-of-concept, we isolated colon epithelial cells from two specimens and performed low-input small RNA-seq. We generated up to 600,000 miRNA reads from the samples. Isolated epithelial cells, had abundant epithelial-enriched miRNA expression (miR-192; miR-194; miR-200b; miR-200c; miR-215; miR-375) and overall similar miRNA expression patterns to other epithelial cell populations (colonic enteroids and flow-isolated colon epithelium). xMD-derived epithelial cells were generally not contaminated by other adjacent cells of the colon as noted by t-SNE analysis. xMD-miRNA-seq allows for simple, economical, and efficient identification of cell-specific miRNA expression estimates. Further development will enhance rapid identification of cell-specific miRNA expression estimates in health and disease for nearly any cell type using archival FFPE material.

scholarly journals Noise-induced bistability in the quasineutral coexistence of viral RNA under different replication modes

2018 ◽  
Author(s):  
Josep Sardanyés ◽  
Andreu Arderiu ◽  
Santiago F. Elena ◽  
Tomás Alarcón
Keyword(s):  
Viral Replication ◽  
Evolutionary Dynamics ◽  
Rna Viruses ◽  
Initial Conditions ◽  
Rna Replication ◽  
Viral Rna ◽  
Stochastic Simulations ◽  
Strong Impact ◽  
Deterministic Models ◽  
The Impact

Evolutionary and dynamical investigations on real viral populations indicate that RNA replication can range between two extremes given by so-called stamping machine replication (SMR) and geometric replication (GR). The impact of asymmetries in replication for single-stranded, (+) sense RNA viruses has been up to now studied with deterministic models. However, viral replication should be better described by including stochasticity, since the cell infection process is typically initiated with a very small number of RNA macromolecules, and thus largely influenced by intrinsic noise. Under appropriate conditions, deterministic theoretical descriptions of viral RNA replication predict a quasineutral coexistence scenario, with a line of fixed points involving different strands’ equilibrium ratios depending on the initial conditions. Recent research on the quasineutral coexistence in two competing populations reveals that stochastic fluctuations fundamentally alters the mean-field scenario, and one of the two species outcompetes the other one. In this manuscript we study this phenomenon for RNA viral replication modes by means of stochastic simulations and a diffusion approximation. Our results reveal that noise has a strong impact on the amplification of viral RNA, also causing the emergence of noise-induced bistability. We provide analytical criteria for the dominance of (+) sense strands depending on the initial populations on the line of equilibria, which are in agreement with direct stochastic simulation results. The biological implications of this noise-driven mechanism are discussed within the framework of the evolutionary dynamics of RNA viruses with different modes of replication.

The high activity of fluorohydroxybenzylbenzimidazoles against some small RNA-viruses

1971 ◽  
Vol 27 (9) ◽  
pp. 1025-1027 ◽  
Author(s):  
D. G. O'Sullivan ◽  
Carolyn M. Ludlow ◽  
A. K. Wallis
Keyword(s):  
High Activity ◽  
Small Rna ◽  
Rna Viruses

scholarly journals La antigen recognizes and binds to the 3'-oligouridylate tail of a small RNA.

1984 ◽  
Vol 4 (6) ◽  
pp. 1134-1140 ◽  
Author(s):  
M B Mathews ◽  
A M Francoeur
Keyword(s):  
Rna Polymerase ◽  
Small Rna ◽  
Rna Polymerase Iii ◽  
Cellular Protein ◽  
Cross Linking ◽  
Antigen Binding ◽  
Polymerase Iii ◽  
La Antigen ◽  
Covalently Linked

The La antigen is a cellular protein which interacts with many RNA species that are products of RNA polymerase III, including the adenovirus virus-associated (VA) RNAs. We demonstrate that the efficiency of antigen binding in vitro is determined by the number of U residues at the RNA 3' terminus. Forms of VA RNAI with more than two terminal U residues are fully bound, forms with two U residues are partially bound, and forms with fewer than two U residues are not bound at all. The antigen can be covalently linked to VA RNA by UV irradiation, and the site of cross-linking is shown to contain the 3' terminus of the RNA. We conclude that the antigen recognizes the U-rich 3' tail of VA RNA, and presumably that of other polymerase III products, and that it binds at or close to this site.

La antigen recognizes and binds to the 3'-oligouridylate tail of a small RNA

1984 ◽  
Vol 4 (6) ◽  
pp. 1134-1140
Author(s):  
M B Mathews ◽  
A M Francoeur
Keyword(s):  
Rna Polymerase ◽  
Small Rna ◽  
Rna Polymerase Iii ◽  
Cellular Protein ◽  
Cross Linking ◽  
Antigen Binding ◽  
Polymerase Iii ◽  
La Antigen ◽  
Covalently Linked

The La antigen is a cellular protein which interacts with many RNA species that are products of RNA polymerase III, including the adenovirus virus-associated (VA) RNAs. We demonstrate that the efficiency of antigen binding in vitro is determined by the number of U residues at the RNA 3' terminus. Forms of VA RNAI with more than two terminal U residues are fully bound, forms with two U residues are partially bound, and forms with fewer than two U residues are not bound at all. The antigen can be covalently linked to VA RNA by UV irradiation, and the site of cross-linking is shown to contain the 3' terminus of the RNA. We conclude that the antigen recognizes the U-rich 3' tail of VA RNA, and presumably that of other polymerase III products, and that it binds at or close to this site.

Characterization of a novel 63 kDa membrane protein. Implications for the organization of the ER-to-Golgi pathway

1993 ◽  
Vol 104 (3) ◽  
pp. 671-683 ◽  
Author(s):  
A. Schweizer ◽  
M. Ericsson ◽  
T. Bachi ◽  
G. Griffiths ◽  
H.P. Hauri
Keyword(s):  
Membrane Protein ◽  
Laser Scanning ◽  
Protein Transport ◽  
Brefeldin A ◽  
Vero Cells ◽  
Subcellular Fractionation ◽  
Confocal Laser ◽  
Similar Degree ◽  
Fractionation Procedure ◽  
Intermediate Compartment

Owing to the lack of appropriate markers the structural organization of the ER-to-Golgi pathway and the dynamics of its membrane elements have been elusive. To elucidate this organization we have taken a monoclonal antibody (mAb) approach. A mAb against a novel 63 kDa membrane protein (p63) was produced that identifies a large tubular network of smooth membranes in the cytoplasm of primate cells. The distribution of p63 overlaps with the ER-Golgi intermediate compartment, defined by a previously described 53 kDa marker protein (here termed ERGIC-53), as visualized by confocal laser scanning immunofluorescence microscopy and immunoelectron microscopy. The p63 compartment mediates protein transport from the ER to Golgi apparatus, as indicated by partial colocalization of p63 and vesicular stomatitis virus G protein in Vero cells cultured at 15 degrees C. Low temperatures and brefeldin A had little effect on the cellular distribution of p63, suggesting that this novel marker is a stably anchored resident protein of these pre-Golgi membranes. p63 and ERGIC-53 were enriched to a similar degree by the same subcellular fractionation procedure. These findings demonstrate an unanticipated complexity of the ER-Golgi interface and suggest that the ER-Golgi intermediate compartment defined by ERGIC-53 may be part of a greater network of smooth membranes.

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