scholarly journals Timeliness of Proteolytic Events Is Prerequisite for Efficient Functioning of the Alphaviral Replicase

2018 ◽  
Vol 92 (14) ◽  
Author(s):  
Valeria Lulla ◽  
Liis Karo-Astover ◽  
Kai Rausalu ◽  
Sirle Saul ◽  
Andres Merits ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Mammalian Cells ◽  
Spatial Organization ◽  
Semliki Forest Virus ◽  
Specific Substrate ◽  
Replication Complex ◽  
Polyprotein Processing ◽  
Scissile Bond ◽  
Positive Strand Rna

ABSTRACTPolyprotein processing has an important regulatory role in the life cycle of positive-strand RNA viruses. In the case of alphaviruses, sequential cleavage of the nonstructural polyprotein (ns-polyprotein) at three sites eventually yields four mature nonstructural proteins (nsPs) that continue working in complex to replicate viral genomic RNA and transcribe subgenomic RNA. Recognition of cleavage sites by viral nsP2 protease is guided by short sequences upstream of the scissile bond and, more importantly, by the spatial organization of the replication complex. In this study, we analyzed the consequences of the artificially accelerated processing of the Semliki Forest virus ns-polyprotein. It was found that in mammalian cells, not only the order but also the correct timing of the cleavage events is essential for the success of viral replication. Analysis of the effects of compensatory mutations in rescued viruses as well asin vitrotranslation andtrans-replicase assays corroborated our findings and revealed the importance of the V515 residue in nsP2 for recognizing the P4 position in the nsP1/nsP2 cleavage site. We also extended our conclusions to Sindbis virus by analyzing the properties of the hyperprocessive variant carrying the N614D mutation in nsP2. We conclude that the sequence of the nsP1/nsP2 site in alphaviruses is under selective pressure to avoid the presence of sequences that are recognized too efficiently and would otherwise lead to premature cleavage at this site before completion of essential tasks of RNA synthesis or virus-induced replication complex formation. Even subtle changes in the ns-polyprotein processing pattern appear to lead to virus attenuation.IMPORTANCEThe polyprotein expression strategy is a cornerstone of alphavirus replication. Three sites within the ns-polyprotein are recognized by the viral nsP2 protease and cleaved in a defined order. Specific substrate targeting is achieved by the recognition of the short sequence upstream of the scissile bond and a correct macromolecular assembly of ns-polyprotein. Here, we highlighted the importance of the timeliness of proteolytic events, as an additional layer of regulation of efficient virus replication. We conclude that, somewhat counterintuitively, the cleavage site sequences at the nsP1/nsP2 and nsP2/nsP3 junctions are evolutionarily selected to be recognized by protease inefficiently, to avoid premature cleavages that would be detrimental for the assembly and functionality of the replication complex. Understanding the causes and consequences of viral polyprotein processing events is important for predicting the properties of mutant viruses and should be helpful for the development of better vaccine candidates and understanding potential mechanisms of resistance to protease inhibitors.

scholarly journals Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease

2001 ◽  
Vol 82 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Andres Merits ◽  
Lidia Vasiljeva ◽  
Tero Ahola ◽  
Leevi Kääriäinen ◽  
Petri Auvinen
Keyword(s):  
Mammalian Cells ◽  
Active Sites ◽  
Insect Cells ◽  
Semliki Forest Virus ◽  
Point Mutations ◽  
Proteolytic Processing ◽  
Wild Type ◽  
In Trans ◽  
Polyprotein Precursor

The RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C478A, (P2CA) completely abolished the processing of P12CA34, P12CA3 and P2CA3. Co-expression of P23 and P12CA34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D6A, (P1234DA) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.

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

2000 ◽  
Vol 74 (19) ◽  
pp. 8953-8965 ◽  
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.

scholarly journals Hepatitis C Virus-Like Particle Morphogenesis

2002 ◽  
Vol 76 (8) ◽  
pp. 4073-4079 ◽  
Author(s):  
Emmanuelle Blanchard ◽  
Denys Brand ◽  
Sylvie Trassard ◽  
Alain Goudeau ◽  
Philippe Roingeard
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mammalian Cells ◽  
Semliki Forest Virus ◽  
Virus Like Particle ◽  
Genes Encoding ◽  
Host Cell Interactions ◽  
Viral Morphogenesis ◽  
First Time

ABSTRACT Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.

Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells

1994 ◽  
Vol 107 (12) ◽  
pp. 3623-3633 ◽  
Author(s):  
J. Jantti ◽  
S. Keranen ◽  
J. Toikkanen ◽  
E. Kuismanen ◽  
C. Ehnholm ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Signal Sequence ◽  
Insertion Site ◽  
Membrane Insertion ◽  
Membrane Anchor

Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20 degrees C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37 degrees C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.

Expression of Hepatitis A Virus Precursor Protein P3 in Vivo and in Vitro: Polyprotein Processing of the 3CD Cleavage Site

Virology ◽  
1994 ◽  
Vol 198 (2) ◽  
pp. 524-533 ◽  
Author(s):  
Michael Tesar ◽  
Inga Pak ◽  
Xi-Yu Jia ◽  
Oliver C. Richards ◽  
Donald F. Summers ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Precursor Protein ◽  
Polyprotein Processing

scholarly journals Mitochondrion-Enriched Anionic Phospholipids Facilitate Flock House Virus RNA Polymerase Membrane Association

2009 ◽  
Vol 83 (9) ◽  
pp. 4498-4507 ◽  
Author(s):  
Kenneth A. Stapleford ◽  
Doron Rapaport ◽  
David J. Miller
Keyword(s):  
Rna Polymerase ◽  
Protein A ◽  
Rna Replication ◽  
Mitochondrial Outer Membrane ◽  
Membrane Association ◽  
Flock House Virus ◽  
Replication Complex ◽  
Anionic Phospholipids ◽  
Positive Strand Rna

ABSTRACT One characteristic of all positive-strand RNA viruses is the necessity to assemble viral RNA replication complexes on host intracellular membranes, a process whose molecular details are poorly understood. To study viral replication complex assembly we use the established model system of Flock House virus (FHV), which assembles its replication complexes on the mitochondrial outer membrane. The FHV RNA-dependent RNA polymerase, protein A, is the only viral protein necessary for genome replication in the budding yeast Saccharomyces cerevisiae. To examine the host components involved in protein A-membrane interactions, an initial step of FHV RNA replication complex assembly, we established an in vitro protein A membrane association assay. Protein A translated in vitro rapidly and specifically associated with mitochondria isolated from yeast, insect, and mammalian cells. This process was temperature dependent but independent of protease-sensitive mitochondrial outer membrane components or the host mitochondrial import machinery. Furthermore, lipid-binding studies revealed that protein A preferentially bound to specific anionic phospholipids, in particular the mitochondrion-specific phospholipid cardiolipin. These studies implicate membrane phospholipids as important host determinants for FHV RNA polymerase membrane association and provide evidence for the involvement of host phospholipids in positive-strand RNA virus membrane-specific targeting.

scholarly journals Polyprotein Processing as a Determinant for in Vitro Activity of Semliki Forest Virus Replicase

Viruses ◽  
2017 ◽  
Vol 9 (10) ◽  
pp. 292 ◽  
Author(s):  
Maija K. Pietilä ◽  
Irina C. Albulescu ◽  
Martijn J. van Hemert ◽  
Tero Ahola
Keyword(s):  
Semliki Forest Virus ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Polyprotein Processing

scholarly journals Role of the Matrix-Capsid Cleavage Site Polymorphism S124V of HIV-1 Sub-subtype A2 in Gag Polyprotein Processing

2020 ◽  
Author(s):  
Carla Mavian ◽  
Roxana M Coman ◽  
Ben M Dunn ◽  
Maureen M Goodenow
Keyword(s):  
Cleavage Site ◽  
Viral Fitness ◽  
Cleavage Sites ◽  
Polyprotein Processing ◽  
Gag Polyprotein ◽  
Processing Rate ◽  
Subtype B ◽  
The Matrix ◽  
Hiv 1

AbstractSubtype C and A HIV-1 strains dominate the epidemic in Africa and Asia, while sub-subtype A2 is found at low frequency only in West Africa. To relate Gag processing in vitro with viral fitness, viral protease (PR) enzymatic activity and in vitro Gag processing were evaluated. The rate of sub-subtype A2 Gag polyprotein processing, as production of the p24 protein, was reduced compared to subtype B or C independent of PR subtype, indicating that subtype A2 Gag qualitatively differed from other subtypes. Introduction of subtype B matrix-capsid cleavage site in sub-subtype A2 Gag only partially restored the processing rate. Unique amino acid polymorphism V124S at the matrix-capsid cleavage site, together with other polymorphisms at non-cleavage sites, are differentially influencing the processing of Gag polyproteins. This genetic polymorphisms landscape defining HIV-1 sub-subtypes, subtypes and recombinant forms are determinants of viral fitness and frequency in the HIV-1 infected population.Graphical AbstractHighlightsThe polymorphism at matrix-capsid cleavage site, together with non-cleavage sites polymorphisms, direct the processing rate of the substrate, not the intrinsic activity of the enzyme.The less prevalent and less infectious sub-subtype A2 harbors the matrix-capsid cleavage site polymorphism that we report as a limiting factor for gag processing.Sub-subtype A2 Gag polyprotein processing rate is independent of the PR subtype.

scholarly journals The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication

Viruses ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 483 ◽  
Author(s):  
Tania Quirin ◽  
Yu Chen ◽  
Maija Pietilä ◽  
Deyin Guo ◽  
Tero Ahola
Keyword(s):  
Mammalian Cells ◽  
Semliki Forest Virus ◽  
Protein A ◽  
Flock House Virus ◽  
Fluorescent Marker ◽  
Capping Enzyme ◽  
Replication Systems ◽  
High Level ◽  
Rna Capping

The nodavirus flock house virus (FHV) and the alphavirus Semliki Forest virus (SFV) show evolutionarily intriguing similarities in their replication complexes and RNA capping enzymes. In this study, we first established an efficient FHV trans-replication system in mammalian cells, which disjoins protein expression from viral RNA synthesis. Following transfection, FHV replicase protein A was associated with mitochondria, whose outer surface displayed pouch-like invaginations with a ‘neck’ structure opening towards the cytoplasm. In mitochondrial pellets from transfected cells, high-level synthesis of both genomic and subgenomic RNA was detected in vitro and the newly synthesized RNA was of positive polarity. Secondly, we initiated the study of the putative RNA capping enzyme domain in protein A by mutating the conserved amino acids H93, R100, D141, and W215. RNA replication was abolished for all mutants inside cells and in vitro except for W215A, which showed reduced replication. Transfection of capped RNA template did not rescue the replication activity of the mutants. Comparing the efficiency of SFV and FHV trans-replication systems, the FHV system appeared to produce more RNA. Using fluorescent marker proteins, we demonstrated that both systems could replicate in the same cell. This work may facilitate the comparative analysis of FHV and SFV replication.

scholarly journals Molecular Determinants of Substrate Specificity for Semliki Forest Virus Nonstructural Protease

2006 ◽  
Vol 80 (11) ◽  
pp. 5413-5422 ◽  
Author(s):  
Aleksei Lulla ◽  
Valeria Lulla ◽  
Kairit Tints ◽  
Tero Ahola ◽  
Andres Merits
Keyword(s):  
Life Cycle ◽  
Amino Acid ◽  
Cleavage Site ◽  
Semliki Forest Virus ◽  
Nonstructural Protein ◽  
Cleavage Sites ◽  
Vitro Assay ◽  
Virus Life Cycle ◽  
Cleavage Efficiency

ABSTRACT The C-terminal cysteine protease domain of Semliki Forest virus nonstructural protein 2 (nsP2) regulates the virus life cycle by sequentially cleaving at three specific sites within the virus-encoded replicase polyprotein P1234. The site between nsP3 and nsP4 (the 3/4 site) is cleaved most efficiently. Analysis of Semliki Forest virus-specific cleavage sites with shuffled N-terminal and C-terminal half-sites showed that the main determinants of cleavage efficiency are located in the region preceding the cleavage site. Random mutagenesis analysis revealed that amino acid residues in positions P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much variation, whereas in the P5 position most residues were permitted. When mutations affecting cleavage efficiency were introduced into the 2/3 and 3/4 cleavage sites, the resulting viruses remained viable but had similar defects in P1234 processing as observed in the in vitro assay. Complete blockage of the 3/4 cleavage was found to be lethal. The amino acid in position P1′ had a significant effect on cleavage efficiency, and in this regard the protease markedly preferred a glycine residue over the tyrosine natively present in the 3/4 site. Therefore, the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The protease recognizes at least residues P4 to P1′, and the P4 arginine residue plays an important role in the fast cleavage of the 3/4 site.

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