This page has only limited features, please log in for full access.
The capsid precursor P1 constitutes the N-terminal part of the enterovirus polyprotein. It is processed into VP0, VP3, and VP1 by the viral proteases, and VP0 is cleaved autocatalytically into VP4 and VP2. We observed that poliovirus VP0 is recognized by an antibody against a cellular autophagy protein, LC3A. The LC3A-like epitope overlapped the VP4/VP2 cleavage site. Individually expressed VP0-EGFP and P1 strongly colocalized with a marker of selective autophagy, p62/SQSTM1. To assess the role of capsid proteins in autophagy development we infected different cells with poliovirus or encapsidated polio replicon coding for only the replication proteins. We analyzed the processing of LC3B and p62/SQSTM1, markers of the initiation and completion of the autophagy pathway and investigated the association of the viral antigens with these autophagy proteins in infected cells. We observed cell-type-specific development of autophagy upon infection and found that only the virion signal strongly colocalized with p62/SQSTM1 early in infection. Collectively, our data suggest that activation of autophagy is not required for replication, and that capsid proteins contain determinants targeting them to p62/SQSTM1-dependent sequestration. Such a strategy may control the level of capsid proteins so that viral RNAs are not removed from the replication/translation pool prematurely.
Anna Zimina; Ekaterina G. Viktorova; Seyedehmahsa Moghimi; Jules Nchoutmboube; George A. Belov. Interaction of Poliovirus Capsid Proteins with the Cellular Autophagy Pathway. Viruses 2021, 13, 1587 .
AMA StyleAnna Zimina, Ekaterina G. Viktorova, Seyedehmahsa Moghimi, Jules Nchoutmboube, George A. Belov. Interaction of Poliovirus Capsid Proteins with the Cellular Autophagy Pathway. Viruses. 2021; 13 (8):1587.
Chicago/Turabian StyleAnna Zimina; Ekaterina G. Viktorova; Seyedehmahsa Moghimi; Jules Nchoutmboube; George A. Belov. 2021. "Interaction of Poliovirus Capsid Proteins with the Cellular Autophagy Pathway." Viruses 13, no. 8: 1587.
The capsid precursor P1 constitutes the N-terminal part of the enterovirus poly-protein. It is processed into VP0, VP3, and VP1 by the viral proteases, and VP0 is cleaved autocatalytically into VP4 and VP2. We observed that poliovirus VP0 is recognized by an antibody against a cellular autophagy protein LC3A. The LC3A-like epitope overlapped the VP4/VP2 cleavage site. Individually expressed VP0-EGFP and P1 strongly colocalized with a marker of selective autophagy p62/SQSTM1. To assess the role of capsid proteins in autophagy development we infected different cells with poliovirus or encapsidated polio replicon coding for only the replication proteins. We analyzed the processing of LC3B and p62/SQSTM1, markers of the initiation and completion of the autophagy pathway, and systematically investigated the association of the viral antigens with these au-tophagy proteins in infected cells. We observed cell-type specific development of autophagy upon infection and found that only the virion signal strongly co-localized with p62/SQSTM1 early in infection. Collectively, our data suggest that activation of autophagy is an antiviral response, and that capsid proteins con-tain determinants targeting them to p62/SQSTM1-dependent sequestration. Such a strategy may control the level of capsid proteins so that viral RNAs are not re-moved from the replication/translation pool prematurely.
Anna Zimina; Ekaterina G. Viktorova; SeyedeMahsa Moghimi; Jules Nchoutmboube; George A. Belov. Interaction of Poliovirus Capsid Proteins with the Cellular Autophagy Pathway. 2021, 1 .
AMA StyleAnna Zimina, Ekaterina G. Viktorova, SeyedeMahsa Moghimi, Jules Nchoutmboube, George A. Belov. Interaction of Poliovirus Capsid Proteins with the Cellular Autophagy Pathway. . 2021; ():1.
Chicago/Turabian StyleAnna Zimina; Ekaterina G. Viktorova; SeyedeMahsa Moghimi; Jules Nchoutmboube; George A. Belov. 2021. "Interaction of Poliovirus Capsid Proteins with the Cellular Autophagy Pathway." , no. : 1.
Enteroviruses include many known and emerging pathogens, such as poliovirus, enteroviruses 71 and D68, and others. However, licensed vaccines are available only against poliovirus and enterovirus 71, and specific anti-enterovirus therapeutics are lacking. Enterovirus infection induces the massive remodeling of intracellular membranes and the development of specialized domains harboring viral replication complexes, replication organelles. Here, we investigated the roles of small Arf GTPases during enterovirus infection. Arfs control distinct steps in intracellular membrane traffic, and one of the Arf-activating proteins, GBF1, is a cellular factor required for enterovirus replication. We found that all Arfs expressed in human cells, including Arf6, normally associated with the plasma membrane, are recruited to the replication organelles and that Arf1 appears to be the most important Arf for enterovirus replication. These results document the rewiring of the cellular membrane pathways in infected cells and may provide new ways of controlling enterovirus infections.
Seyedehmahsa Moghimi; Ekaterina Viktorova; Anna Zimina; Tomasz Szul; Elizabeth Sztul; George A. Belov. Enterovirus Infection Induces Massive Recruitment of All Isoforms of Small Cellular Arf GTPases to the Replication Organelles. Journal of Virology 2020, 95, 1 .
AMA StyleSeyedehmahsa Moghimi, Ekaterina Viktorova, Anna Zimina, Tomasz Szul, Elizabeth Sztul, George A. Belov. Enterovirus Infection Induces Massive Recruitment of All Isoforms of Small Cellular Arf GTPases to the Replication Organelles. Journal of Virology. 2020; 95 (2):1.
Chicago/Turabian StyleSeyedehmahsa Moghimi; Ekaterina Viktorova; Anna Zimina; Tomasz Szul; Elizabeth Sztul; George A. Belov. 2020. "Enterovirus Infection Induces Massive Recruitment of All Isoforms of Small Cellular Arf GTPases to the Replication Organelles." Journal of Virology 95, no. 2: 1.
Positive-strand RNA viruses universally remodel host intracellular membranes to form membrane-bound viral replication complexes, where viral offspring RNAs are synthesized. In the majority of cases, viral replication proteins are targeted to and play critical roles in the modulation of the designated organelle membranes. Many viral replication proteins do not have transmembrane domains, but contain single or multiple amphipathic alpha-helices. It has been conventionally recognized that these helices serve as an anchor for viral replication protein to be associated with membranes. We report here that a peptide representing the amphipathic α-helix at the N-terminus of the poliovirus 2C protein not only binds to liposomes, but also remodels spherical liposomes into tubules. The membrane remodeling ability of this amphipathic alpha-helix is similar to that recognized in other amphipathic alpha-helices from cellular proteins involved in membrane remodeling, such as BAR domain proteins. Mutations affecting the hydrophobic face of the amphipathic alpha-helix severely compromised membrane remodeling of vesicles with physiologically relevant phospholipid composition. These mutations also affected the ability of poliovirus to form plaques indicative of reduced viral replication, further underscoring the importance of membrane remodeling by the amphipathic alpha-helix in possible relation to the formation of viral replication complexes.
Jobin Varkey; Jiantao Zhang; Junghyun Kim; Gincy George; Guijuan He; George Belov; Ralf Langen; Xiaofeng Wang. An Amphipathic Alpha-Helix Domain from Poliovirus 2C Protein Tubulate Lipid Vesicles. Viruses 2020, 12, 1466 .
AMA StyleJobin Varkey, Jiantao Zhang, Junghyun Kim, Gincy George, Guijuan He, George Belov, Ralf Langen, Xiaofeng Wang. An Amphipathic Alpha-Helix Domain from Poliovirus 2C Protein Tubulate Lipid Vesicles. Viruses. 2020; 12 (12):1466.
Chicago/Turabian StyleJobin Varkey; Jiantao Zhang; Junghyun Kim; Gincy George; Guijuan He; George Belov; Ralf Langen; Xiaofeng Wang. 2020. "An Amphipathic Alpha-Helix Domain from Poliovirus 2C Protein Tubulate Lipid Vesicles." Viruses 12, no. 12: 1466.
The group of enteroviruses contains many important pathogens for humans, including poliovirus, coxsackievirus, rhinovirus, as well as newly emerging global health threats such as EV-A71 and EV-D68. Here, we describe an unbiased, system-wide and time-resolved analysis of the proteome and phosphoproteome of human cells infected with coxsackievirus B3. Of the ~3,200 proteins quantified throughout the time course, a large amount (~25%) shows a significant change, with the majority being downregulated. We find ~85% of the detected phosphosites to be significantly regulated, implying that most changes occur at the post-translational level. Kinase-motif analysis reveals temporal activation patterns of certain protein kinases, with several CDKs/MAPKs immediately active upon the infection, and basophilic kinases, ATM, and ATR engaging later. Through bioinformatics analysis and dedicated experiments, we identify mTORC1 signalling as a major regulation network during enterovirus infection. We demonstrate that inhibition of mTORC1 activates TFEB, which increases expression of lysosomal and autophagosomal genes, and that TFEB activation facilitates the release of virions in extracellular vesicles via secretory autophagy. Our study provides a rich framework for a system-level understanding of enterovirus-induced perturbations at the protein and signalling pathway levels, forming a base for the development of pharmacological inhibitors to treat enterovirus infections.
Piero Giansanti; Jeroen R. P. M. Strating; Kyra A. Y. Defourny; Ieva Cesonyte; Alexia M. S. Bottino; Harm Post; Ekaterina G. Viktorova; Vien Quang Tri Ho; Martijn A. Langereis; George A. Belov; Esther N. M. Nolte-‘T Hoen; Albert J. R. Heck; Frank J. M. Van Kuppeveld. Dynamic remodelling of the human host cell proteome and phosphoproteome upon enterovirus infection. Nature Communications 2020, 11, 1 -12.
AMA StylePiero Giansanti, Jeroen R. P. M. Strating, Kyra A. Y. Defourny, Ieva Cesonyte, Alexia M. S. Bottino, Harm Post, Ekaterina G. Viktorova, Vien Quang Tri Ho, Martijn A. Langereis, George A. Belov, Esther N. M. Nolte-‘T Hoen, Albert J. R. Heck, Frank J. M. Van Kuppeveld. Dynamic remodelling of the human host cell proteome and phosphoproteome upon enterovirus infection. Nature Communications. 2020; 11 (1):1-12.
Chicago/Turabian StylePiero Giansanti; Jeroen R. P. M. Strating; Kyra A. Y. Defourny; Ieva Cesonyte; Alexia M. S. Bottino; Harm Post; Ekaterina G. Viktorova; Vien Quang Tri Ho; Martijn A. Langereis; George A. Belov; Esther N. M. Nolte-‘T Hoen; Albert J. R. Heck; Frank J. M. Van Kuppeveld. 2020. "Dynamic remodelling of the human host cell proteome and phosphoproteome upon enterovirus infection." Nature Communications 11, no. 1: 1-12.
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.
Ekaterina G. Viktorova; Samuel Gabaglio; Justyna M. Meissner; Eunjoo Lee; Seyedehmahsa Moghimi; Elizabeth Sztul; George A. Belov. A Redundant Mechanism of Recruitment Underlies the Remarkable Plasticity of the Requirement of Poliovirus Replication for the Cellular ArfGEF GBF1. Journal of Virology 2019, 93, 1 .
AMA StyleEkaterina G. Viktorova, Samuel Gabaglio, Justyna M. Meissner, Eunjoo Lee, Seyedehmahsa Moghimi, Elizabeth Sztul, George A. Belov. A Redundant Mechanism of Recruitment Underlies the Remarkable Plasticity of the Requirement of Poliovirus Replication for the Cellular ArfGEF GBF1. Journal of Virology. 2019; 93 (21):1.
Chicago/Turabian StyleEkaterina G. Viktorova; Samuel Gabaglio; Justyna M. Meissner; Eunjoo Lee; Seyedehmahsa Moghimi; Elizabeth Sztul; George A. Belov. 2019. "A Redundant Mechanism of Recruitment Underlies the Remarkable Plasticity of the Requirement of Poliovirus Replication for the Cellular ArfGEF GBF1." Journal of Virology 93, no. 21: 1.
Replication complexes of (+)RNA viruses of eukaryotes are associated with specialized membranous domains, termed replication organelles. How these structures develop is poorly understood. In a recent Cell paper, Laufman et al. (2019) reveal that enteroviruses recruit lipid droplets to support lipid synthesis required for the structural development of replication organelles.
George A. Belov; Frank J.M. van Kuppeveld. Lipid Droplets Grease Enterovirus Replication. Cell Host & Microbe 2019, 26, 149 -151.
AMA StyleGeorge A. Belov, Frank J.M. van Kuppeveld. Lipid Droplets Grease Enterovirus Replication. Cell Host & Microbe. 2019; 26 (2):149-151.
Chicago/Turabian StyleGeorge A. Belov; Frank J.M. van Kuppeveld. 2019. "Lipid Droplets Grease Enterovirus Replication." Cell Host & Microbe 26, no. 2: 149-151.
Membrane association is a hallmark of the genome replication of positive-strand RNA viruses [(+)RNA viruses]. All well-studied (+)RNA viruses remodel host membranes and lipid metabolism through orchestrated virus-host interactions to create a suitable microenvironment to survive and thrive in host cells. Recent research has shown that host lipids, as major components of cellular membranes, play key roles in the replication of multiple (+)RNA viruses. This review focuses on how (+)RNA viruses manipulate host lipid synthesis and metabolism to facilitate their genomic RNA replication, and how interference with the cellular lipid metabolism affects viral replication.
Zhenlu Zhang; Guijuan He; Natalie A. Filipowicz; Glenn Randall; George A. Belov; Benjamin G. Kopek; Xiaofeng Wang. Host Lipids in Positive-Strand RNA Virus Genome Replication. Frontiers in Microbiology 2019, 10, 286 .
AMA StyleZhenlu Zhang, Guijuan He, Natalie A. Filipowicz, Glenn Randall, George A. Belov, Benjamin G. Kopek, Xiaofeng Wang. Host Lipids in Positive-Strand RNA Virus Genome Replication. Frontiers in Microbiology. 2019; 10 ():286.
Chicago/Turabian StyleZhenlu Zhang; Guijuan He; Natalie A. Filipowicz; Glenn Randall; George A. Belov; Benjamin G. Kopek; Xiaofeng Wang. 2019. "Host Lipids in Positive-Strand RNA Virus Genome Replication." Frontiers in Microbiology 10, no. : 286.
The high human cost of Zika virus infections and the rapid establishment of virus circulation in novel areas, including the United States, present an urgent need for countermeasures against this emerging threat. The development of an effective vaccine against Zika virus may be problematic because of the cross reactivity of the antibodies with other flaviviruses leading to antibody-dependent enhancement of infection. Moreover, rapidly replicating positive strand RNA viruses, including Zika virus, generate large spectrum of mutant genomes (quasi species) every replication round, allowing rapid selection of variants resistant to drugs targeting virus-specific proteins. On the other hand, viruses are ultimate cellular parasites and rely on the host metabolism for every step of their life cycle, thus presenting an opportunity to manipulate host processes as an alternative approach to suppress virus replication and spread. Zika and other flaviviruses critically depend on the cellular secretory pathway, which transfers proteins and membranes from the ER through the Golgi to the plasma membrane, for virion assembly, maturation and release. In this review, we summarize the current knowledge of interactions of Zika and similar arthropod-borne flaviviruses with the cellular secretory machinery with a special emphasis on virus-specific changes of the secretory pathway. Identification of the regulatory networks and effector proteins required to accommodate the trafficking of virions, which represent a highly unusual cargo for the secretory pathway, may open an attractive and virtually untapped reservoir of alternative targets for the development of superior anti-viral drugs.
Garrett Sager; Samuel Gabaglio; Elizabeth Sztul; George A. Belov. Role of Host Cell Secretory Machinery in Zika Virus Life Cycle. Viruses 2018, 10, 559 .
AMA StyleGarrett Sager, Samuel Gabaglio, Elizabeth Sztul, George A. Belov. Role of Host Cell Secretory Machinery in Zika Virus Life Cycle. Viruses. 2018; 10 (10):559.
Chicago/Turabian StyleGarrett Sager; Samuel Gabaglio; Elizabeth Sztul; George A. Belov. 2018. "Role of Host Cell Secretory Machinery in Zika Virus Life Cycle." Viruses 10, no. 10: 559.
A new, safe, and effective vaccine against poliovirus is urgently needed not only to complete the eradication of the virus but also to be used in the future to prevent possible virus reemergence in a postpolio world. Currently, new formulations of the oral vaccine, as well as improvements to the inactivated vaccine, are being explored. In this study, we designed a viral vector with mucosal tropism that expresses poliovirus capsid proteins. Thus, poliovirus VLPs are produced in vivo , in the cells of a vaccine recipient, and are presented to the immune system in the context of vector virus replication, stimulating the development of systemic and mucosal immune responses. Such an approach allows the development of an affordable and safe vaccine that does not rely on the full-length poliovirus genome at any stage.
Ekaterina G. Viktorova; Sunil K. Khattar; Diana Kouiavskaia; Majid Laassri; Tatiana Zagorodnyaya; Eugenia Dragunsky; Siba Samal; Konstantin Chumakov; George A. Belov. Newcastle Disease Virus-Based Vectored Vaccine against Poliomyelitis. Journal of Virology 2018, 92, 1 .
AMA StyleEkaterina G. Viktorova, Sunil K. Khattar, Diana Kouiavskaia, Majid Laassri, Tatiana Zagorodnyaya, Eugenia Dragunsky, Siba Samal, Konstantin Chumakov, George A. Belov. Newcastle Disease Virus-Based Vectored Vaccine against Poliomyelitis. Journal of Virology. 2018; 92 (17):1.
Chicago/Turabian StyleEkaterina G. Viktorova; Sunil K. Khattar; Diana Kouiavskaia; Majid Laassri; Tatiana Zagorodnyaya; Eugenia Dragunsky; Siba Samal; Konstantin Chumakov; George A. Belov. 2018. "Newcastle Disease Virus-Based Vectored Vaccine against Poliomyelitis." Journal of Virology 92, no. 17: 1.
Rapid development of complex membranous replication structures is a hallmark of picornavirus infections. However, neither the mechanisms underlying such dramatic reorganization of the cellular membrane architecture, nor the specific role of these membranes in the viral life cycle are sufficiently understood. Here we demonstrate that the cellular enzyme CCTα, responsible for the rate-limiting step in phosphatidylcholine synthesis, translocates from the nuclei to the cytoplasm upon infection and associates with the replication membranes, resulting in the rerouting of lipid synthesis from predominantly neutral lipids to phospholipids. The bulk supply of long chain fatty acids necessary to support the activated phospholipid synthesis in infected cells is provided by the hydrolysis of neutral lipids stored in lipid droplets. Such activation of phospholipid synthesis drives the massive membrane remodeling in infected cells. We also show that complex membranous scaffold of replication organelles is not essential for viral RNA replication but is required for protection of virus propagation from the cellular anti-viral response, especially during multi-cycle replication conditions. Inhibition of infection-specific phospholipid synthesis provides a new paradigm for controlling infection not by suppressing viral replication but by making it more visible to the immune system. The cellular pathways hijacked to support viral replication may provide a promising class of targets for anti-viral therapeutics, which will be effective against broad groups of viruses relying on the same cellular pathways, and will likely be refractory to the development of resistance since cellular factors are not subject to selection. All (+)RNA viruses share the requirement for cellular membranes to assemble replication complexes. Here we investigated the mechanism underlying the massive membrane remodeling in poliovirus-infected cells. Our results demonstrate reorganization of the cellular lipid synthesizing machinery upon infection and identify lipid droplets as the organelles supporting the structural development of the replication membranes. Moreover, we show that inhibition of the infection-specific phospholipid synthesis renders virus propagation much more vulnerable to the cellular anti-viral defenses, providing a new direction for the development of anti-viral therapeutics.
Ekaterina G. Viktorova; Jules Nchoutmboube; Lauren A. Ford-Siltz; Ethan Iverson; George A. Belov. Phospholipid synthesis fueled by lipid droplets drives the structural development of poliovirus replication organelles. PLOS Pathogens 2018, 14, e1007280 .
AMA StyleEkaterina G. Viktorova, Jules Nchoutmboube, Lauren A. Ford-Siltz, Ethan Iverson, George A. Belov. Phospholipid synthesis fueled by lipid droplets drives the structural development of poliovirus replication organelles. PLOS Pathogens. 2018; 14 (8):e1007280.
Chicago/Turabian StyleEkaterina G. Viktorova; Jules Nchoutmboube; Lauren A. Ford-Siltz; Ethan Iverson; George A. Belov. 2018. "Phospholipid synthesis fueled by lipid droplets drives the structural development of poliovirus replication organelles." PLOS Pathogens 14, no. 8: e1007280.
Cellular life requires the activation of the ADP-ribosylation factors (ARFs) by Golgi brefeldin A-resistant factor 1 (GBF1), a guanine nucleotide exchange factor (GEF) with a highly conserved catalytic Sec7 domain (Sec7d). In addition to the Sec7d, GBF1 contains other conserved domains whose functions remain unclear. Here, we focus on HDS2 (homology downstream of Sec7d 2) domain because the L1246R substitution within the HDS2 α-helix 5 of the zebrafish GBF1 ortholog causes vascular hemorrhaging and embryonic lethality (13). To dissect the structure/function relationships within HDS2, we generated six variants, in which the most conserved residues within α-helices 1, 2, 4, and 6 were mutated to alanines. Each HDS2 mutant was assessed in a cell-based “replacement” assay for its ability to support cellular functions normally supported by GBF1, such as maintaining Golgi homeostasis, facilitating COPI recruitment, supporting secretion, and sustaining cellular viability. We show that cells treated with the pharmacological GBF1 inhibitor brefeldin A (BFA) and expressing a BFA-resistant GBF1 variant with alanine substitutions of RDR1168 or LF1266 are compromised in Golgi homeostasis, impaired in ARF activation, unable to sustain secretion, and defective in maintaining cellular viability. To gain insight into the molecular mechanism of this dysfunction, we assessed the ability of each GBF1 mutant to target to Golgi membranes and found that mutations in RDR1168 and LF1266 significantly decrease targeting efficiency. Thus, these residues within α-helix 2 and α-helix 6 of the HDS2 domain in GBF1 are novel regulatory determinants that support GBF1 cellular function by impacting the Golgi-specific membrane association of GBF1.
Cristian A. Pocognoni; Ekaterina G. Viktorova; John Wright; Justyna M. Meissner; Garrett Sager; Eunjoo Lee; George A. Belov; Elizabeth Sztul. Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity. American Journal of Physiology-Cell Physiology 2018, 314, C675 -C689.
AMA StyleCristian A. Pocognoni, Ekaterina G. Viktorova, John Wright, Justyna M. Meissner, Garrett Sager, Eunjoo Lee, George A. Belov, Elizabeth Sztul. Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity. American Journal of Physiology-Cell Physiology. 2018; 314 (6):C675-C689.
Chicago/Turabian StyleCristian A. Pocognoni; Ekaterina G. Viktorova; John Wright; Justyna M. Meissner; Garrett Sager; Eunjoo Lee; George A. Belov; Elizabeth Sztul. 2018. "Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity." American Journal of Physiology-Cell Physiology 314, no. 6: C675-C689.
Poliovirus is a prototype member of the Enterovirus genus of the Picornaviridae family of small positive strand RNA viruses, which include important human and animal pathogens. Quantitative assessment of viral replication is very important for investigation of the virus biology and the development of anti‐viral strategies. The poliovirus genome structure allows replacement of structural genes with a reporter protein, such as a luciferase or a fluorescent protein, whose signals can be detected and quantified in vivo, thus permitting observation of replication kinetics in live cells. This paper presents protocols for poliovirus replicon RNA production, purification, packaging and transfection, as well as techniques for monitoring Renilla luciferase replication signal in living cells. © 2018 by John Wiley & Sons, Inc.
Ekaterina G. Viktorova; Sunil Khattar; Siba Samal; George A. Belov. Poliovirus Replicon RNA Generation, Transfection, Packaging, and Quantitation of Replication. Current Protocols in Microbiology 2018, 48, 15H.4.1 -15H.4.15.
AMA StyleEkaterina G. Viktorova, Sunil Khattar, Siba Samal, George A. Belov. Poliovirus Replicon RNA Generation, Transfection, Packaging, and Quantitation of Replication. Current Protocols in Microbiology. 2018; 48 (1):15H.4.1-15H.4.15.
Chicago/Turabian StyleEkaterina G. Viktorova; Sunil Khattar; Siba Samal; George A. Belov. 2018. "Poliovirus Replicon RNA Generation, Transfection, Packaging, and Quantitation of Replication." Current Protocols in Microbiology 48, no. 1: 15H.4.1-15H.4.15.
Picornavirus infection induces rapid reorganization of the cellular membrane architecture and appearance of novel membranous structures associated with the viral RNA replication and virion assembly — replication organelles. Recent studies significantly advanced our understanding of their lipid composition and cellular mechanisms involved in their development. Picornaviruses activate synthesis of both structural and signaling lipids and reroute cellular cholesterol trafficking pathways to create unique membranous domains favoring viral replication. Rapidly replicating picornaviruses rely on posttranslational activation and/or specific recruitment of cellular proteins rather than on modulation of expression of cellular genes to create favorable membrane microenvironment. At the same time picornaviruses demonstrate remarkable adaptability to changes in the lipid landscape which should be taken into account when developing novel antiviral strategies.
George A Belov. Dynamic lipid landscape of picornavirus replication organelles. Current Opinion in Virology 2016, 19, 1 -6.
AMA StyleGeorge A Belov. Dynamic lipid landscape of picornavirus replication organelles. Current Opinion in Virology. 2016; 19 ():1-6.
Chicago/Turabian StyleGeorge A Belov. 2016. "Dynamic lipid landscape of picornavirus replication organelles." Current Opinion in Virology 19, no. : 1-6.
Members of the large Sec7 domain-containing Arf guanine nucleotide exchange factor (GEF) family have been shown to dimerize through their NH2-terminal dimerization and cyclophilin binding (DCB) and homology upstream of Sec7 (HUS) domains. However, the importance of dimerization in GEF localization and function has not been assessed. We generated a GBF1 mutant (91/130) in which two residues required for oligomerization (K91 and E130 within the DCB domain) were replaced with A and assessed the effects of these mutations on GBF1 localization and cellular functions. We show that 91/130 is compromised in oligomerization but that it targets to the Golgi in a manner indistinguishable from wild-type GBF1 and that it rapidly exchanges between the cytosolic and membrane-bound pools. The 91/130 mutant appears active as it integrates within the functional network at the Golgi, supports Arf activation and COPI recruitment, and sustains Golgi homeostasis and cargo secretion when provided as a sole copy of functional GBF1 in cells. In addition, like wild-type GBF1, the 91/130 mutant supports poliovirus RNA replication, a process requiring GBF1 but believed to be independent of GBF1 catalytic activity. However, oligomerization appears to stabilize GBF1 in cells, and the 91/130 mutant is degraded faster than the wild-type GBF1. Our data support a model in which oligomerization is not a key regulator of GBF1 activity but impacts its function by regulating the cellular levels of GBF1.
Jay M. Bhatt; Ekaterina G. Viktorova; Theodore Busby; Paulina Wyrozumska; Laura Newman; Wyrozumska Paulina; Eunjoo Lee; John Wright; George Belov; Richard A. Kahn; Elizabeth Sztul. Oligomerization of the Sec7 domain Arf guanine nucleotide exchange factor GBF1 is dispensable for Golgi localization and function but regulates degradation. American Journal of Physiology-Cell Physiology 2016, 310, C456 -C469.
AMA StyleJay M. Bhatt, Ekaterina G. Viktorova, Theodore Busby, Paulina Wyrozumska, Laura Newman, Wyrozumska Paulina, Eunjoo Lee, John Wright, George Belov, Richard A. Kahn, Elizabeth Sztul. Oligomerization of the Sec7 domain Arf guanine nucleotide exchange factor GBF1 is dispensable for Golgi localization and function but regulates degradation. American Journal of Physiology-Cell Physiology. 2016; 310 (6):C456-C469.
Chicago/Turabian StyleJay M. Bhatt; Ekaterina G. Viktorova; Theodore Busby; Paulina Wyrozumska; Laura Newman; Wyrozumska Paulina; Eunjoo Lee; John Wright; George Belov; Richard A. Kahn; Elizabeth Sztul. 2016. "Oligomerization of the Sec7 domain Arf guanine nucleotide exchange factor GBF1 is dispensable for Golgi localization and function but regulates degradation." American Journal of Physiology-Cell Physiology 310, no. 6: C456-C469.
All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting theCHO2gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes.
Jiantao Zhang; Zhenlu Zhang; Vineela Chukkapalli; Jules Nchoutmboube; Jianhui Li; Glenn Randall; George Belov; Xiaofeng Wang. Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites. Proceedings of the National Academy of Sciences 2016, 113, E1064 -E1073.
AMA StyleJiantao Zhang, Zhenlu Zhang, Vineela Chukkapalli, Jules Nchoutmboube, Jianhui Li, Glenn Randall, George Belov, Xiaofeng Wang. Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites. Proceedings of the National Academy of Sciences. 2016; 113 (8):E1064-E1073.
Chicago/Turabian StyleJiantao Zhang; Zhenlu Zhang; Vineela Chukkapalli; Jules Nchoutmboube; Jianhui Li; Glenn Randall; George Belov; Xiaofeng Wang. 2016. "Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites." Proceedings of the National Academy of Sciences 113, no. 8: E1064-E1073.
All positive strand RNA viruses of eukaryotes replicate their genomes in association with membranes. These viruses actively change cellular lipid metabolism to build replication membranes enriched in specific lipids. The ubiquitous use of membranes by positive strand RNA viruses apparently holds major evolutionary advantages; however our understanding of the mechanistic role of membranes, let alone of specific lipid components of the membrane bilayer, in the viral replication cycle is minimal. The replication complexes that can be isolated from infected cells, or reconstituted in vitro from crude cell lysates, do not allow controlled manipulation of the membrane constituents thus limiting their usefulness for understanding how exactly membranes support the replication reaction. Recent work from Peter Nagy group demonstrates that replication of a model positive strand RNA virus can be reconstituted in the in vitro reaction with liposomes of chemically defined composition and reveals an exclusive role of phosphatidylethanolamine in sustaining efficient viral RNA replication. This study opens new possibilities for investigation of membrane contribution in the replication process that may ultimately lead to development of novel broad spectrum antiviral compounds targeting the membrane-dependent elements of the replication cycle conserved among diverse groups of viruses.
George A. Belov. Less Grease, Please. Phosphatidylethanolamine Is the Only Lipid Required for Replication of a (+)RNA Virus. Viruses 2015, 7, 3500 -3505.
AMA StyleGeorge A. Belov. Less Grease, Please. Phosphatidylethanolamine Is the Only Lipid Required for Replication of a (+)RNA Virus. Viruses. 2015; 7 (7):3500-3505.
Chicago/Turabian StyleGeorge A. Belov. 2015. "Less Grease, Please. Phosphatidylethanolamine Is the Only Lipid Required for Replication of a (+)RNA Virus." Viruses 7, no. 7: 3500-3505.
All (+)RNA viruses replicate on distinct membranous domains; however, how they induce and maintain their unique lipid composition is largely unknown. Two recent studies reveal that enteroviruses harness the PI4P-cholestrol exchange cycle driven by OSBP1 protein and PI4 kinase(s), and that blocking the dynamic lipid flow inhibits virus replication.
Jules Nchoutmboube; Lauren A. Ford-Siltz; George A. Belov. Enterovirus replication: go with the (counter)flow. Trends in Microbiology 2015, 23, 183 -184.
AMA StyleJules Nchoutmboube, Lauren A. Ford-Siltz, George A. Belov. Enterovirus replication: go with the (counter)flow. Trends in Microbiology. 2015; 23 (4):183-184.
Chicago/Turabian StyleJules Nchoutmboube; Lauren A. Ford-Siltz; George A. Belov. 2015. "Enterovirus replication: go with the (counter)flow." Trends in Microbiology 23, no. 4: 183-184.
It 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.
Ekaterina G. Viktorova; Jules Nchoutmboube; Lauren Siltz; George A. Belov. Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein. Journal of Virology 2015, 89, 4372 -4386.
AMA StyleEkaterina G. Viktorova, Jules Nchoutmboube, Lauren Siltz, George A. Belov. Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein. Journal of Virology. 2015; 89 (8):4372-4386.
Chicago/Turabian StyleEkaterina G. Viktorova; Jules Nchoutmboube; Lauren Siltz; George A. Belov. 2015. "Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein." Journal of Virology 89, no. 8: 4372-4386.
Picornaviruses include rapidly replicating viruses that may complete their infectious cycle within a few hours. During this short time the massive development of viral replication organelles completely transforms the cellular membrane landscape. The origin of these structures and mechanism(s) underlying their rapid expansion are still poorly understood. Recent studies revealed profound reorganization of synthesis and distribution of major structural lipids in infected cells. These data show that the lipid composition of the replication organelles is significantly different from that of preexisting cellular membranes. The apparently universal activation of specific lipid syntheses by diverse picornaviruses and at least some other (+)RNA viruses suggests that the mechanism(s) of replication organelle development may be conserved among distantly related viruses.
George A. Belov. Modulation of lipid synthesis and trafficking pathways by picornaviruses. Current Opinion in Virology 2014, 9, 19 -23.
AMA StyleGeorge A. Belov. Modulation of lipid synthesis and trafficking pathways by picornaviruses. Current Opinion in Virology. 2014; 9 ():19-23.
Chicago/Turabian StyleGeorge A. Belov. 2014. "Modulation of lipid synthesis and trafficking pathways by picornaviruses." Current Opinion in Virology 9, no. : 19-23.