Leevi Kääriäinen

Viral RNA Replication in Association with Cellular Membranes

All plus-strand RNA viruses replicate in association with cytoplasmic membranes of infected cells. The RNA replication complex of many virus families is associated with the endoplasmic reticulum membranes, for example, picorna-, flavi-, arteri-, and bromoviruses. However, endosomes and lysosomes (togaviruses), peroxisomes and chloroplasts (tombusviruses), and mitochondria (nodaviruses) are also used as sites for RNA replication. Studies of individual nonstructural proteins, the virus-specific components of the RNA replicase, have revealed that the replication complexes are associated with the membranes and targeted to the respective organelle by the ns proteins rather than RNA. Many ns proteins have hydrophobic sequences and may transverse the membrane like polytopic integral membrane proteins, whereas others interact with membranes monotopically. Hepatitis C virus ns proteins offer examples of polytopic transmembrane proteins (NS2, NS4B), a “tip-anchored” protein attached to the membrane by an amphipathic α-helix (NS5A) and a “tail-anchored” posttranslationally inserted protein (NS5B). Semliki Forest virus nsP1 is attached to the plasma membrane by a specific binding peptide in the middle of the protein, which forms an amphipathic α-helix. Interaction of nsP1 with membrane lipids is essential for its capping enzyme activities. The other soluble replicase proteins are directed to the endo-lysosomal membranes only as part of the initial polyprotein. Poliovirus ns proteins utilize endoplasmic reticulum membranes from which vesicles are released in COPII coats. However, these vesicles are not directed to the normal secretory pathway, but accumulate in the cytoplasm. In many cases the replicase proteins induce membrane invaginations or vesicles, which function as protective environments for RNA replication.

Biogenesis of the Semliki Forest Virus RNA Replication Complex

ABSTRACT The nonstructural (ns) proteins nsP1 to -4, the components of Semliki Forest virus (SFV) RNA polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. All ns proteins were associated with large cytoplasmic vacuoles (CPV), the inner surfaces of which were covered by small invaginations, or spherules, typical of alphavirus infection. All ns proteins were localized by immuno-electron microscopy (EM) to the limiting membranes of CPV and to the spherules, together with newly labeled viral RNA. Along with earlier observations by EM-autoradiography (P. M. Grimley, I. K. Berezesky, and R. M. Friedman, J. Virol. 2:326–338, 1968), these results suggest that individual spherules represent template-associated RNA polymerase complexes. Immunoprecipitation of radiolabeled ns proteins showed that each antiserum precipitated the other three ns proteins, implying that they functioned as a complex. Double labeling with organelle-specific and anti-ns-protein antisera showed that CPV were derivatives of late endosomes and lysosomes. Indeed, CPV frequently contained endocytosed bovine serum albumin-coated gold particles, introduced into the medium at different times after infection. With time, increasing numbers of spherules were also observed on the cell surfaces; they were occasionally released into the medium, probably by secretory lysosomes. We suggest that the spherules arise by primary assembly of the RNA replication complexes at the plasma membrane, guided there by nsP1, which has affinity to lipids specific for the cytoplasmic leaflet of the plasma membrane. Endosomal recycling and fusion of CPV with the plasma membrane can circulate spherules between the plasma membrane and the endosomal-lysosomal compartment.

Secretion of Interferon by Bacillus subtilis

Bacillus subtilis was transformed with a hybrid gene in which the sequence encoding the alpha-amylase signal peptide was joined by a linker to the sequence encoding mature human interferon alpha 2(IFN-alpha 2). The hybrid preprotein was cleaved precisely following the last amino acid of the alpha-amylase signal sequence and was secreted at 0.5--1 mg per liter. IFN-alpha 2, preceded by either one or six amino acids, has the same specific antiviral activity as IFN-alpha 2 itself.

Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity

The replication complexes of all positive strand RNA viruses of eukaryotes are associated with membranes. In the case of Semliki Forest virus (SFV), the main determinant of membrane attachment seems to be the virus‐encoded non‐structural protein NSP1, the capping enzyme of the viral mRNAs, which has guanine‐7‐methyltransferase and guanylyltransferase activities. We show here that both enzymatic activities of SFV NSP1 are inactivated by detergents and reactivated by anionic phospholipids, especially phosphatidylserine. The region of NSP1 responsible for binding to membranes as well as to liposomes was mapped to a short segment, which is conserved in the large alphavirus‐like superfamily of viruses. A synthetic peptide of 20 amino acids from the putative binding site competed with in vitro synthesized NSP1 for binding to liposomes containing phosphatidylserine. These findings suggest a molecular mechanism by which RNA virus replicases attach to intracellular membranes and why they depend on the membranous environment.

RNA HELICASE ACTIVITY OF SEMLIKI FOREST VIRUS REPLICASE PROTEIN NSP2

Semliki Forest virus replicase protein nsP2 shares sequence homology with several putative NTPases and RNA helicases. NsP2 has RNA‐dependent NTPase activity. Here we expressed polyhistidine‐tagged nsP2 in Escherichia coli, purified it by metal‐affinity chromatography, and used it in RNA helicase assays. RNA helicase CI of plum pox potyvirus was used as a positive control. Unwinding of α‐32P‐labelled partially double‐stranded RNA required nsP2, Mg2+ and NTPs. NsP2 with a mutation, K192N, in the NTP‐binding sequence GVPGSGK192SA could not unwind dsRNA and had no NTPase activity. This is the first demonstration of RNA helicase activity within the large alphavirus superfamily.

Identification of a novel function of the Alphavirus capping apparatus. RNA 5′-triphosphatase activity of Nsp2.

Both genomic and subgenomic RNAs of theAlphavirus have m7G(5′)ppp(5′)N (cap0 structure) at their 5′ end. Previously it has been shown thatAlphavirus-specific nonstructural protein Nsp1 has guanine-7N-methyltransferase and guanylyltransferase activities needed in the synthesis of the cap structure. During normal cap synthesis the 5′ γ-phosphate of the nascent viral RNA chain is removed by a specific RNA 5′-triphosphatase before condensation with GMP, delivered by the guanylyltransferase. Using a novel RNA triphosphatase assay, we show here that nonstructural protein Nsp2 (799 amino acids) of Semliki Forest virus specifically cleaves the γ,β-triphosphate bond at the 5′ end of RNA. The same activity was demonstrated for Nsp2 of Sindbis virus, as well as for the amino-terminal fragment of Semliki Forest virus Nsp2-N (residues 1–470). The carboxyl-terminal part of Semliki Forest virus Nsp2-C (residues 471–799) had no RNA triphosphatase activity. Replacement of Lys-192 by Asn in the nucleotide-binding site completely abolished RNA triphosphatase and nucleoside triphosphatase activities of Semliki Forest virus Nsp2 and Nsp2-N. Here we provide biochemical characterization of the newly found function of Nsp2 and discuss the unique properties of the entire Alphavirus-capping apparatus.

Properly Folded Nonstructural Polyprotein Directs the Semliki Forest Virus Replication Complex to the Endosomal Compartment

ABSTRACT The late RNA synthesis in alphavirus-infected cells, generating plus-strand RNAs, takes place on cytoplasmic vacuoles (CPVs), which are modified endosomes and lysosomes. The cytosolic surface of CPVs consists of regular membrane invaginations or spherules, which are the sites of RNA synthesis (P. Kujala, A. Ikäheimonen, N. Ehsani, H. Vihinen, P. Auvinen, and L. Kääriäinen J. Virol. 75:3873-3884, 2001). To understand how CPVs arise, we have expressed the individual Semliki Forest virus (SFV) nonstructural proteins nsP1 to nsP4 in different combinations, as well as their precursor polyprotein P1234 and its cleavage intermediates. A complex of nsPs was obtained from P123 or P1234, indicating that the precursor stage is essential for the assembly of the polymerase complex. To prevent the processing of the polyprotein and its cleavage intermediates, constructs with the mutation C478A (designated with a superscript CA) in the active site of the protease domain of nsP2 were used. Uncleaved polyproteins containing nsP1 were membrane bound and palmitoylated, and those containing nsP3 were phosphorylated, reflecting properties of authentic nsP1 and nsP3, respectively. Similarly, polyproteins containing nsP1 or nsP2 had enzymatic activities specific for the individual proteins, indicating that they were correctly folded in the precursor state. Uncleaved P12CA was localized almost exclusively to the plasma membrane and filopodia, like nsP1 alone, whereas P12CA3 and P12CA34 were found on cytoplasmic vesicles, some of which contained late endosomal markers. In immunoelectron microscopy these vesicles resembled CPVs in SFV-infected cells. Our results indicate that the nsP1 domain alone is responsible for the membrane association of the nonstructural polyprotein, whereas the nsP1 domain together with the nsP3 domain targets it to the intracellular vesicles.

Phosphatidylinositol 3-Kinase-, Actin-, and Microtubule-Dependent Transport of Semliki Forest Virus Replication Complexes from the Plasma Membrane to Modified Lysosomes

ABSTRACT Like other positive-strand RNA viruses, alphaviruses replicate their genomes in association with modified intracellular membranes. Alphavirus replication sites consist of numerous bulb-shaped membrane invaginations (spherules), which contain the double-stranded replication intermediates. Time course studies with Semliki Forest virus (SFV)-infected cells were combined with live-cell imaging and electron microscopy to reveal that the replication complex spherules of SFV undergo an unprecedented large-scale movement between cellular compartments. The spherules first accumulated at the plasma membrane and were then internalized using an endocytic process that required a functional actin-myosin network, as shown by blebbistatin treatment. Wortmannin and other inhibitors indicated that the internalization of spherules also required the activity of phosphatidylinositol 3-kinase. The spherules therefore represent an unusual type of endocytic cargo. After endocytosis, spherule-containing vesicles were highly dynamic and had a neutral pH. These primary carriers fused with acidic endosomes and moved long distances on microtubules, in a manner prevented by nocodazole. The result of the large-scale migration was the formation of a very stable compartment, where the spherules were accumulated on the outer surfaces of unusually large and static acidic vacuoles localized in the pericentriolar region. Our work highlights both fundamental similarities and important differences in the processes that lead to the modified membrane compartments in cells infected by distinct groups of positive-sense RNA viruses.

Virus-Specific mRNA Capping Enzyme Encoded by Hepatitis E Virus

ABSTRACT Hepatitis E virus (HEV), a positive-strand RNA virus, is an important causative agent of waterborne hepatitis. Expression of cDNA (encoding amino acids 1 to 979 of HEV nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kDa protein (P110), a fraction of which was proteolytically processed to an 80-kDa protein. P110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. Immunopurified P110 catalyzed transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP and GDP to yield m7GTP or m7GDP. GMP, GpppG, and GpppA were poor substrates for the P110 methyltransferase. There was no evidence for further methylation of m7GTP when it was used as a substrate for the methyltransferase. P110 was also a guanylyltransferase, which formed a covalent complex, P110-m7GMP, in the presence of AdoMet and GTP, because radioactivity from both [α-32P]GTP and [3H-methyl]AdoMet was found in the covalent guanylate complex. Since both methyltransferase and guanylyltransferase reactions are strictly virus specific, they should offer optimal targets for development of antiviral drugs. Cap analogs such as m7GTP, m7GDP, et2m7GMP, and m2et7GMP inhibited the methyltransferase reaction. HEV P110 capping enzyme has similar properties to the methyltransferase and guanylyltransferase of alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus (a potexvirus) nonstructural protein, indicating there is a common evolutionary origin of these distantly related plant and animal virus families.

Functions of alphavirus nonstructural proteins in RNA replication

Publisher Summary Alphaviruses are enveloped positive-strand RNA viruses transmitted to vertebrate hosts by mosquitoes. Several alphaviruses are pathogenic to humans or domestic animals, causing serious central nervous system infections or milder infections, for example, arthritis, rash, and fever. The structure and replication of Semliki Forest virus (SFV) and Sindbis virus (SIN) have been studied extensively during the past 30 years. Alphaviruses have been important probes in cell biology to study the translation, glycosylation, folding, and transport of membrane glycoproteins, as well as endocytosis and membrane fusion mechanisms. A new organelle, the intermediate compartment, operating between the endoplasmic retieulum and the Golgi complex has been found by the aid of SFV. During the past 10 years, alphavirus replicons have been increasingly used as expression vectors for basic research, for the generation of vaccines, and for the production of recombinant proteins in industrial scale. The main approaches of laboratories in the recent years have been twofold. On one hand, they have discovered and characterized the enzymatic activities of the individual replicase proteins and on the other hand, they have studied the localization, membrane association, and other cell biological aspects of the replication complex.