Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex

The replicase polyproteins of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are processed by three viral proteases to yield 12 non-structural proteins (nsps). The nsp2 and nsp3 cleavage products have previously been found to interact, a property that allows nsp2 to act as a co-factor in the processing of the downstream part of the polyprotein by the nsp4 protease. Remarkably, upon infection of Vero cells, but not of BHK-21 or RK-13 cells, EAV nsp2 is now shown to be subject to an additional, internal, cleavage. In Vero cells, approximately 50% of nsp2 (61 kDa) was cleaved into an 18 kDa N-terminal part and a 44 kDa C-terminal part, most likely by a host cell protease that is absent in BHK-21 and RK-13 cells. Although the functional consequences of this additional processing step are unknown, the experiments in Vero cells revealed that the C-terminal part of nsp2 interacts with nsp3. Most EAV nsps localize to virus-induced double-membrane structures in the perinuclear region of the infected cell, where virus RNA synthesis takes place. It is now shown that, in an expression system, the co-expression of nsp2 and nsp3 is both necessary and sufficient to induce the formation of double-membrane structures that strikingly resemble those found in infected cells. Thus, the nsp2 and nsp3 cleavage products play a crucial role in two processes that are common to positive-strand RNA viruses that replicate in mammalian cells: controlled proteolysis of replicase precursors and membrane association of the virus replication complex.

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Chapter 2a: Virology

Tick-borne encephalitis - The Book ◽

10.33442/26613980_2a-4 ◽

2021 ◽

Author(s):

Daniel Růžek ◽

Kentaro Yoshii ◽

Marshall E. Bloom ◽

Ernest A. Gould

Keyword(s):

Host Cell ◽

Secretory Pathway ◽

Virus Assembly ◽

Close Association ◽

Spherical Particles ◽

Structural Proteins ◽

Membrane Structures ◽

Vesicle Membrane ◽

Reading Frame ◽

Tbe Virus

TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae. Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). An additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalayan subtype (Himalayan and “178-79” group; TBEV-HIM). TBEV virions are small enveloped spherical particles about 50 nm in diameter. The TBEV genome consists of a single-stranded positive sense RNA molecule. The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs). The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity. The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.

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The arterivirus replicase is the only viral protein required for genome replication and subgenomic mRNA transcription

Journal of General Virology ◽

10.1099/0022-1317-81-10-2491 ◽

2000 ◽

Vol 81 (10) ◽

pp. 2491-2496 ◽

Cited By ~ 87

Author(s):

Richard Molenkamp ◽

Hans van Tol ◽

Babette C. D. Rozier ◽

Yvonne van der Meer ◽

Willy J. M. Spaan ◽

...

Keyword(s):

Rna Synthesis ◽

Point Mutations ◽

Equine Arteritis Virus ◽

Viral Rna ◽

Structural Proteins ◽

Genome Replication ◽

Mrna Transcription ◽

Replication Complex ◽

N Protein ◽

Wild Type Phenotype

Equine arteritis virus (EAV) (Arteriviridae) encodes several structural proteins. Whether any of these also function in viral RNA synthesis is unknown. For the related mouse hepatitis coronavirus (MHV), it has been suggested that the nucleocapsid protein (N) is involved in viral RNA synthesis. As described for MHV, we established that the EAV N protein colocalizes with the viral replication complex, suggesting a role in RNA synthesis. Using an infectious cDNA clone, point mutations and deletions were engineered in the EAV genome to disrupt the expression of each of the structural genes. All structural proteins, including N, were found to be dispensable for genome replication and subgenomic mRNA transcription. We also constructed a mutant in which translation of the intraleader ORF was disrupted. This mutant had a wild-type phenotype, indicating that, at least in cell culture, the product of this ORF does not play a role in the EAV replication cycle.

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The Transformation of Enterovirus Replication Structures: a Three-Dimensional Study of Single- and Double-Membrane Compartments

mBio ◽

10.1128/mbio.00166-11 ◽

2011 ◽

Vol 2 (5) ◽

Cited By ~ 86

Author(s):

Ronald W. A. L. Limpens ◽

Hilde M. van der Schaar ◽

Darshan Kumar ◽

Abraham J. Koster ◽

Eric J. Snijder ◽

...

Keyword(s):

Rna Synthesis ◽

Electron Tomography ◽

Three Dimensional ◽

Membrane Vesicles ◽

Viral Rna ◽

Membrane Structures ◽

Double Membrane ◽

Single Membrane ◽

Membrane Tubules ◽

Positive Strand Rna

ABSTRACTAll positive-strand RNA viruses induce membrane structures in their host cells which are thought to serve as suitable microenvironments for viral RNA synthesis. The structures induced by enteroviruses, which are members of the familyPicornaviridae, have so far been described as either single- or double-membrane vesicles (DMVs). Aside from the number of delimiting membranes, their exact architecture has also remained elusive due to the limitations of conventional electron microscopy. In this study, we used electron tomography (ET) to solve the three-dimensional (3-D) ultrastructure of these compartments. At different time points postinfection, coxsackievirus B3-infected cells were high-pressure frozen and freeze-substituted for ET analysis. The tomograms showed that during the exponential phase of viral RNA synthesis, closed smooth single-membrane tubules constituted the predominant virus-induced membrane structure, with a minor proportion of DMVs that were either closed or connected to the cytosol in a vase-like configuration. As infection progressed, the DMV number steadily increased, while the tubular single-membrane structures gradually disappeared. Late in infection, complex multilamellar structures, previously unreported, became apparent in the cytoplasm. Serial tomography disclosed that their basic unit is a DMV, which is enwrapped by one or multiple cisternae. ET also revealed striking intermediate structures that strongly support the conversion of single-membrane tubules into double-membrane and multilamellar structures by a process of membrane apposition, enwrapping, and fusion. Collectively, our work unravels the sequential appearance of distinct enterovirus-induced replication structures, elucidates their detailed 3-D architecture, and provides the basis for a model for their transformation during the course of infection.IMPORTANCEPositive-strand RNA viruses hijack specific intracellular membranes and remodel them into special structures that support viral RNA synthesis. The ultrastructural characterization of these “replication structures” is key to understanding their precise role. Here, we resolved the three-dimensional architecture of enterovirus-induced membranous compartments and their transformation in time by applying electron tomography to cells infected with coxsackievirus B3 (CVB3). Our results show that closed single-membrane tubules are the predominant initial virus-induced structure, whereas double-membrane vesicles (DMVs) become increasingly abundant at the expense of these tubules as infection progresses. Additionally, more complex multilamellar structures appear late in infection. Based on compelling intermediate structures in our tomograms, we propose a model for transformation from the tubules to DMVs and multilamellar structures via enwrapping events. Our work provides an in-depth analysis of the development of an unsuspected variety of distinct replication structures during the course of CVB3 infection.

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Chapter 2a: Virology

Tick-borne encephalitis - The Book ◽

10.33442/26613980_2a-3 ◽

2020 ◽

Keyword(s):

Host Cell ◽

Secretory Pathway ◽

Virus Assembly ◽

Close Association ◽

Spherical Particles ◽

Structural Proteins ◽

Membrane Structures ◽

Vesicle Membrane ◽

Reading Frame ◽

Tbe Virus

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Open Reading Frame 1a-Encoded Subunits of the Arterivirus Replicase Induce Endoplasmic Reticulum-Derived Double-Membrane Vesicles Which Carry the Viral Replication Complex

Journal of Virology ◽

10.1128/jvi.73.3.2016-2026.1999 ◽

1999 ◽

Vol 73 (3) ◽

pp. 2016-2026 ◽

Cited By ~ 220

Author(s):

Ketil W. Pedersen ◽

Yvonne van der Meer ◽

Norbert Roos ◽

Eric J. Snijder

Keyword(s):

Endoplasmic Reticulum ◽

Rna Synthesis ◽

Membrane Vesicles ◽

Equine Arteritis Virus ◽

Open Reading Frame ◽

Genome Replication ◽

Electron Microscopic ◽

Double Membrane ◽

Reading Frame ◽

Replication Complex

ABSTRACT The replicase of equine arteritis virus (EAV; familyArteriviridae, order Nidovirales) is expressed in the form of two polyproteins (the open reading frame 1a [ORF1a] and ORF1ab proteins). Three viral proteases cleave these precursors into 12 nonstructural proteins, which direct both genome replication and subgenomic mRNA transcription. Immunofluorescence assays showed that most EAV replicase subunits localize to membranes in the perinuclear region of the infected cell. Using replicase-specific antibodies and cryoimmunoelectron microscopy, unusual double-membrane vesicles (DMVs) were identified as the probable site of EAV RNA synthesis. These DMVs were previously observed in cells infected with different arteriviruses but were never implicated in viral RNA synthesis. Extensive electron microscopic analysis showed that they appear to be derived from paired endoplasmic reticulum membranes and that they are most likely formed by protrusion and detachment of vesicular structures with a double membrane. Interestingly, very similar membrane rearrangements were observed upon expression of ORF1a-encoded replicase subunits nsp2 to nsp7 from an alphavirus-based expression vector. Apparently, the formation of a membrane-bound scaffold for the replication complex is a distinct step in the arterivirus life cycle, which is directed by the ORF1a protein and does not depend on other viral proteins and/or EAV-specific RNA synthesis.

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Chapter 2a: Virology

Tick-borne encephalitis - The Book ◽

10.33442/978-981-14-0914-1_2a ◽

2019 ◽

Author(s):

Daniel Růžek ◽

Kentaro Yoshii ◽

Marshall E. Bloom ◽

Ernest A. Gould

Keyword(s):

Host Cell ◽

Secretory Pathway ◽

Virus Assembly ◽

Close Association ◽

Spherical Particles ◽

Structural Proteins ◽

Membrane Structures ◽

Vesicle Membrane ◽

Reading Frame ◽

Tbe Virus

• TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae. • Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). Additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalaya subtype (Himalayan and “178-79” group; TBEV-HIM). • TBEV virions are small enveloped spherical particles about 50 nm in diameter. • The TBEV genome consists of a single-stranded positive sense RNA molecule. • The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs). • The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity. • The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). • TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.

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Role of Structural and Non-Structural Proteins and Therapeutic Targets of SARS-CoV-2 for COVID-19

Cells ◽

10.3390/cells10040821 ◽

2021 ◽

Vol 10 (4) ◽

pp. 821

Author(s):

Rohitash Yadav ◽

Jitendra Kumar Chaudhary ◽

Neeraj Jain ◽

Pankaj Kumar Chaudhary ◽

Supriya Khanra ◽

...

Keyword(s):

Host Cell ◽

Protein S ◽

Structural Similarity ◽

Cell Receptor ◽

Molecular Assembly ◽

The Body ◽

Spike Protein ◽

Structural Proteins ◽

Structural Protein ◽

Novel Coronavirus

Coronavirus belongs to the family of Coronaviridae, comprising single-stranded, positive-sense RNA genome (+ ssRNA) of around 26 to 32 kilobases, and has been known to cause infection to a myriad of mammalian hosts, such as humans, cats, bats, civets, dogs, and camels with varied consequences in terms of death and debilitation. Strikingly, novel coronavirus (2019-nCoV), later renamed as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and found to be the causative agent of coronavirus disease-19 (COVID-19), shows 88% of sequence identity with bat-SL-CoVZC45 and bat-SL-CoVZXC21, 79% with SARS-CoV and 50% with MERS-CoV, respectively. Despite key amino acid residual variability, there is an incredible structural similarity between the receptor binding domain (RBD) of spike protein (S) of SARS-CoV-2 and SARS-CoV. During infection, spike protein of SARS-CoV-2 compared to SARS-CoV displays 10–20 times greater affinity for its cognate host cell receptor, angiotensin-converting enzyme 2 (ACE2), leading proteolytic cleavage of S protein by transmembrane protease serine 2 (TMPRSS2). Following cellular entry, the ORF-1a and ORF-1ab, located downstream to 5′ end of + ssRNA genome, undergo translation, thereby forming two large polyproteins, pp1a and pp1ab. These polyproteins, following protease-induced cleavage and molecular assembly, form functional viral RNA polymerase, also referred to as replicase. Thereafter, uninterrupted orchestrated replication-transcription molecular events lead to the synthesis of multiple nested sets of subgenomic mRNAs (sgRNAs), which are finally translated to several structural and accessory proteins participating in structure formation and various molecular functions of virus, respectively. These multiple structural proteins assemble and encapsulate genomic RNA (gRNA), resulting in numerous viral progenies, which eventually exit the host cell, and spread infection to rest of the body. In this review, we primarily focus on genomic organization, structural and non-structural protein components, and potential prospective molecular targets for development of therapeutic drugs, convalescent plasm therapy, and a myriad of potential vaccines to tackle SARS-CoV-2 infection.

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RNA-Protein Interaction Analysis of SARS-CoV-2 5′ and 3′ Untranslated Regions Reveals a Role of Lysosome-Associated Membrane Protein-2a during Viral Infection

mSystems ◽

10.1128/msystems.00643-21 ◽

2021 ◽

Author(s):

Rohit Verma ◽

Sandhini Saha ◽

Shiv Kumar ◽

Shailendra Mani ◽

Tushar Kanti Maiti ◽

...

Keyword(s):

Interaction Analysis ◽

Rna Virus ◽

Untranslated Regions ◽

Host Proteins ◽

Positive Strand ◽

Replication Complex ◽

Positive Strand Rna Virus ◽

Positive Strand Rna ◽

Viral Genomic Rna

Replication of a positive-strand RNA virus involves an RNA-protein complex consisting of viral genomic RNA, host RNA(s), virus-encoded proteins, and host proteins. Dissecting out individual components of the replication complex will help decode the mechanism of viral replication. 5′ and 3′ UTRs in positive-strand RNA viruses play essential regulatory roles in virus replication.

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Lysis Genes of the Bacillus subtilisDefective Prophage PBSX

Journal of Bacteriology ◽

10.1128/jb.180.8.2110-2117.1998 ◽

1998 ◽

Vol 180 (8) ◽

pp. 2110-2117 ◽

Cited By ~ 37

Author(s):

Susanne Krogh ◽

Steen T. Jørgensen ◽

Kevin M. Devine

Keyword(s):

Functional Analysis ◽

Host Cell ◽

Expression System ◽

Cell Lysis ◽

Lethal Gene ◽

Gram Negative Bacteria ◽

Gene Products ◽

Gram Negative ◽

Gram Positive Bacteria ◽

Exported Protein

ABSTRACT Four genes identified within the late operon of PBSX show characteristics expected of a host cell lysis system; they arexepA, encoding an exported protein; xhlA, encoding a putative membrane-associated protein; xhlB, encoding a putative holin; and xlyA, encoding a putative endolysin. In this work, we have assessed the contribution of each gene to host cell lysis by expressing the four genes in different combinations under the control of their natural promoter located on the chromosome of Bacillus subtilis 168. The results show thatxepA is unlikely to be involved in host cell lysis. Expression of both xhlA and xhlB is necessary to effect host cell lysis of B. subtilis. Expression ofxhlB (encoding the putative holin) together withxlyA (encoding the endolysin) cannot effect cell lysis, indicating that the PBSX lysis system differs from those identified in the phages of gram-negative bacteria. Since host cell lysis can be achieved when xlyA is inactivated, it is probable that PBSX encodes a second endolysin activity which also uses XhlA and XhlB for export from the cell. The chromosome-based expression system developed in this study to investigate the functions of the PBSX lysis genes should be a valuable tool for the analysis of other host cell lysis systems and for expression and functional analysis of other lethal gene products in gram-positive bacteria.

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Remodeling the Endoplasmic Reticulum by Poliovirus Infection and by Individual Viral Proteins: an Autophagy-Like Origin for Virus-Induced Vesicles

Journal of Virology ◽

10.1128/jvi.74.19.8953-8965.2000 ◽

2000 ◽

Vol 74 (19) ◽

pp. 8953-8965 ◽

Cited By ~ 360

Author(s):

David A. Suhy ◽

Thomas H. Giddings ◽

Karla Kirkegaard

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Buoyant Density ◽

Rna Replication ◽

Viral Proteins ◽

Replication Complex ◽

Poliovirus Infection ◽

Endosomal Membranes ◽

Infected Cells ◽

Positive Strand Rna

ABSTRACT All positive-strand RNA viruses of eukaryotes studied assemble RNA replication complexes on the surfaces of cytoplasmic membranes. Infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. Poliovirus-induced membranes did not cofractionate with endoplasmic reticulum (ER), lysosomes, mitochondria, or the majority of Golgi-derived or endosomal membranes in buoyant density gradients, although changes in ionic strength affected ER and virus-induced vesicles, but not other cellular organelles, similarly. When expressed in isolation, two viral proteins of the poliovirus RNA replication complex, 3A and 2C, cofractionated with ER membranes. However, in cells that expressed 2BC, a proteolytic precursor of the 2B and 2C proteins, membranes identical in buoyant density to those observed during poliovirus infection were formed. When coexpressed with 2BC, viral protein 3A was quantitatively incorporated into these fractions, and the membranes formed were ultrastructurally similar to those in poliovirus-infected cells. These data argue that poliovirus-induced vesicles derive from the ER by the action of viral proteins 2BC and 3A by a mechanism that excludes resident host proteins. The double-membraned morphology, cytosolic content, and apparent ER origin of poliovirus-induced membranes are all consistent with an autophagic origin for these membranes.

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