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. Ikähei-monen, 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 P12 CA was localized almost exclusively to the plasma membrane and filopodia, like nsP1 alone, whereas P12 CA 3 and P12 CA 34 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.The alphaviruses replicate in the cytoplasm of both invertebrate and vertebrate cells. The virus enters the cell by adsorptive endocytosis, followed by fusion of the virus envelope with endosomal membranes (34). The virus nucleocapsid is disassembled by ribosomes, which have affinity for the capsid protein (52, 63). The capped positive-strand RNA genome of about 11.5 kb is then translated to yield a polyprotein, P1234, of about 2,500 amino acids (aa), the precursor of nonstructural proteins nsP1 to nsP4. The parental 42S RNA genome is copied to complementary minus-strand RNA by a short-lived RNA polymerase consisting of the catalytic subunit nsP4 and polyprotein P123, the initial cleavage products of P1234 (31, 50). By inhibition of cleavage between nsP2 and nsP3, minusstrand RNA synthesis was demonstrated also to occur by the nsP1-P23-nsP4 combination (30).Early work with Semliki Forest virus (SFV) and Sindbis virus showed that the parental RNA was converted into an RNaseresistant, membrane-associated form soon after infection, suggesting that the synthesis of minus strands takes place in association with membranes (8,12,17). The cellular structures associated with the...
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 m 7 GTP or m 7 GDP. GMP, GpppG, and GpppA were poor substrates for the P110 methyltransferase. There was no evidence for further methylation of m 7 GTP when it was used as a substrate for the methyltransferase. P110 was also a guanylyltransferase, which formed a covalent complex, P110-m 7 GMP, in the presence of AdoMet and GTP, because radioactivity from both [␣-32 P]GTP and [ 3 H-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 m 7 GTP, m 7 GDP, et 2 m 7 GMP, and m 2 et 7 GMP 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.Hepatitis E virus (HEV) is an important etiological agent of acute epidemic and sporadic enteric hepatitis affecting millions of people mainly in developing countries. The first confirmed HEV epidemic, due to contamination of drinking water in New Delhi, India, was described in 1955. In addition to large epidemics in India and China, there are annually about 2 million sporadic cases of HEV infections in India alone. The mortality among HEV patients has been 0.5 to 4%, except in the case of pregnant women, for whom the average mortality is 20% (for reviews, see references 22 and 33). Recently, closely related viruses have been isolated from pigs, cows, sheep, goats, and rats, indicating zoonotic HEV infections (13).HEV is a nonenveloped virus with a diameter of 27 to 34 nm (9, 10), which does not replicate in cell cultures (1). The complete nucleotide sequence of the positive-strand RNA genome has been determined for several isolates from different parts of the world (40). (For references, see reference 8.) The HEV genome consists of a 27-nucleotide (nt)-long 5Ј noncoding region followed by an open reading frame (ORF) coding for a nonstructural protein of 1,693 aa residues. ORF2 starts 38 nt downstream of the termination codon of ORF1 and codes for the capsid protein of 660 aa. ORF3 between nt 5105 and 5476 overlaps with ORF2 and codes for a 123-aa-long polypeptide with unknown function. The 3Ј noncoding region is 65 n...
The search for inhibitors of viral replication is dependent on understanding the events taking place at the molecular level during viral infection. All the essential steps during the viral life cycle are potential targets for antiviral drugs. Classical inhibitors of herpesvirus replication cause chain termination during viral DNA replication. Similarly, the HIV reverse transcriptase is the major target of anti-HIV compounds. The broad-spectrum antiviral agent ribavirin affects viral nucleic acid replication by multiple mechanisms. Another major enzyme encoded by many viruses is a protease responsible for the processing of virus-encoded polyproteins. The HIV protease has been very successfully targeted, and hepatitis C virus and rhinovirus protease inhibitors are being actively developed. The complex series of interactions during virus entry is a rapidly emerging and promising target for inhibitors of HIV and many other viruses. New anti-influenza drugs inhibit virus release from infected cells. Several stages of the viral life cycle remain incompletely characterized and are therefore poorly exploited in antiviral strategies. These include, among others, the RNA capping reactions catalyzed by many viruses, as well as the membrane association of replication complexes which is common to all positive-strand RNA viruses.
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