Regulation of the Sequential Processing of Semliki Forest Virus Replicase Polyprotein

Self Cite

111

Chikungunya virus (CHIKV) (genus Alphavirus) has a positive-sense RNA genome. CHIKV nonstructural protein 2 (nsP2) proteolytically processes the viral nonstructural polyprotein, possesses nucleoside triphosphatase (NTPase), RNA triphosphatase, and RNA helicase activities, and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a noncytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG and either an E116K (EK) substitution or a GEEGS sequence insertion after residue T648 (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities, and abolished RNA stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a noncytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels. IMPORTANCECHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the establishment of experimental systems that can be used to conduct virus replication studies at a lower biosafety level. We applied a functional selection approach to develop, for the first time, a noncytotoxic CHIKV replicon capable of persisting in human cell lines. We anticipate that this safe and efficient research tool will be valuable for screening CHIKV replication inhibitors and for identifying and analyzing host factors involved in viral replication. We also analyzed, from virological and protein biochemistry perspectives, the functional defects caused by mutations conferring noncytotoxic phenotypes; we found that all known enzymatic activities of CHIKV nsP2, as well as its RNAbinding capability, were compromised by these mutations, which led to a reduced capacity for replication. C hikungunya virus (CHIKV) is a mosquito-transmitted OldWorld alphavirus belonging to the Togaviridae family. CHIKV infection is characterized by acute illness associated with fever, skin rash, and arthralgia. Symptoms typically resolve within weeks, but frequently CHIKV in...

Section: Hikungunya Virus (Chikv) Is a Mosquito-transmitted Oldmentioning

confidence: 99%

Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2

Utt

Das

Varjak

et al. 2015

Self Cite

111

“…2A and B). Notably, the nsP2 amino terminus has been previously proposed as a protease cofactor (27,28).…”

Section: For Details)mentioning

confidence: 99%

Venezuelan Equine Encephalitis Virus nsP2 Protein Regulates Packaging of the Viral Genome into Infectious Virions

Kim

Atasheva

Frolova

et al. 2013

Alphaviruses are one of the most geographically widespread and yet often neglected group of human and animal pathogens. They are capable of replicating in a wide variety of cells of both vertebrate and insect origin and are widely used for the expression of heterologous genetic information both in vivo and in vitro. In spite of their use in a range of research applications and their recognition as a public health threat, the biology of alphaviruses is insufficiently understood. In this study, we examined the evolution process of one of the alphaviruses, Venezuelan equine encephalitis virus (VEEV), to understand its adaptation mechanism to the inefficient packaging of the viral genome in response to serial mutations introduced into the capsid protein.The new data derived from this study suggest that strong alterations in the ability of capsid protein to package the viral genome leads to accumulation of adaptive mutations, not only in the capsid-specific helix I but also in the nonstructural protein nsP2. The nsP2-specific mutations were detected in the protease domain and in the amino terminus of the protein, which was previously proposed to function as a protease cofactor. These mutations increased infectious virus titers, demonstrated a strong positive impact on viral RNA replication, mediated the development of a more cytopathic phenotype, and made viruses capable of developing a spreading infection. The results suggest not only that packaging of the alphavirus genome is determined by the presence of packaging signals in the RNA and positively charged amino acids in the capsid protein but also that nsP2 is either directly or indirectly involved in the RNA encapsidation process.

“…New mutants were made with the QuikChange XL site-directed mutagenesis kit (Stratagene). Polyprotein construct P12 CA 3 expressing a protease-defective mutant precursor from the T7 promoter has been described (49). Mutations R253E and W259A were cloned into the precursor in EcoRV-DraIII fragments.…”

Section: Methodsmentioning

confidence: 99%

“…To prevent the self-cleavage of the polyprotein, we used a protease active site mutant P12 CA 3 (49). Previous studies showed that P12 CA 3 targets differently from nsP1: the precursor was not found on the plasma membrane but instead resided in numerous punctuate structures in the cytoplasm, some of which were endo/ lysosomal vesicles (39).…”

Section: Vol 81 2007 Membrane Binding Mutants Of Sfv Nsp1 875mentioning

confidence: 99%

“…Thus, the regulated slow processing of the replicase polyprotein enables both minus-strand RNA synthesis and the correct membrane targeting of the complex. The rate of P1234 proteolysis and the order of cleavages are finely tuned (49) to yield first the negative-strand polymerase complex and then the stable positive-strand polymerase which consists of fully processed nsP1 to -4. nsP1 (537 amino acids [aa]) is the sole membrane anchor of the replication complex, since the other replication proteins have no independent membrane affinity in cells (39). nsP1 also catalyzes the methyltransferase and guanylyltransferase reactions needed in the capping of viral mRNAs (1,23,29), and the membrane binding activates nsP1 as an enzyme (3).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Role of the Amphipathic Peptide of Semliki Forest Virus Replicase Protein nsP1 in Membrane Association and Virus Replication

Spuul

Salonen

Merits

et al. 2007

Self Cite

101

108

Semliki Forest virus RNA replication takes place in association with specific cytoplasmic vacuoles, derived from the endosomal apparatus. Of the four virus-encoded replicase proteins, nsP1 serves as the membrane anchor of the replication complex. An amphipathic peptide segment, G 245 STLYTESRKLLRSWHLPSV 264 , has been implicated in the membrane binding of nsP1. nsP1 variants with changes within the peptide were studied after protein expression and in the context of virus infection. Proteins with mutations R253E and W259A accumulated in the cytoplasm and were very poorly palmitoylated. The same mutations also drastically affected the localization of the precursor polyprotein P123, and they were lethal when introduced into the virus genome. Mutations R253A and L255A؉L256A partially changed the localization of nsP1, and the respective viruses acquired compensatory changes. L255A؉L256A only yielded virus encoding L255A؉L256V, indicating the importance of a hydrophobic residue in the central 256 position. When fused to green fluorescent protein, the peptide was required in at least two tandem copies to effect a change in localization, but even then the fusion protein was associated with membranes in a nonspecific manner. Thus, the amphipathic peptide is a crucial element for the membrane association of nsP1 and the replication complex. It provides essential affinity for membranes, and other regions of nsP1 also appear to contribute to the localization of the protein.