Miniaturization and module integration of an infrared spectrometer

Merits

Golubtsov

et al. 2003

113

The replication of most positive-strand RNA viruses and retroviruses is regulated by proteolytic processing. Alphavirus replicase proteins are synthesized as a polyprotein, called P1234, which is cleaved into nsP1, nsP2, nsP3, and nsP4 by the carboxyl-terminal protease domain of nsP2. The cleavage intermediate P123؉nsP4 synthesizes minus-strand copies of the viral RNA genome, whereas the completely processed complex is required for plus-strand synthesis. To understand the mechanisms responsible for this sequential proteolysis, we analyzed in vitro translated Semliki Forest virus polyproteins containing noncleavable processing sites or various deletions. Processing of each of the three sites in vitro required a different type of activity. Site 3/4 was cleaved in trans by nsP2, its carboxyl-terminal fragment Pro39, and by all polyprotein proteases. Site 1/2 was cleaved in cis with a half-life of about 20 -30 min. Site 2/3 was cleaved rapidly in trans but only after release of nsP1 from the polyprotein exposing an "activator" sequence present in the amino terminus of nsP2. Deletion of amino-terminal amino acids of nsP2 or addition of extra amino acid residues to its amino terminus specifically inhibited the protease activity that processes the 2/3 site. This sequence of delayed processing of P1234 would explain the accumulation of P123 plus nsP4, the early short-lived minus-strand replicase. The polyprotein stage would allow correct assembly and membrane association of the RNA-polymerase complex. Late in infection free nsP2 would cleave at site 2/3 yielding P12 and P34, the products of which, nsP1-4, are distributed to the plasma membrane, nucleus, cytoplasmic aggregates, and proteasomes, respectively.

Section: Three Activities Involved In the Processing Of Sfv Ns Polyprmentioning

confidence: 72%

Section: Three Activities Involved In the Processing Of Sfv Ns Polyprmentioning

confidence: 99%

Regulation of the Sequential Processing of Semliki Forest Virus Replicase Polyprotein

Merits

Golubtsov

et al. 2003

113

“…Nsp4 is the catalytic component of the RNA polymerase (7,8), whereas the functions of the phosphoprotein Nsp3 (9) are poorly defined (10, 11). Nsp2 has several distinct functions.…”

Section: Semliki Forest Virus (Sfv)mentioning

confidence: 99%

“…The parental 42 S RNA is copied to complementary minus-strands, which in turn are used as templates for the synthesis of new 42 S RNA plus-strands and subgenomic 26 S mRNAs. During plus-strand synthesis, the 5Ј ends of the 42 S and 26 S RNAs become modified with covalently attached m 7 GpppA (the cap0 structure) (3)(4)(5). Capping of the RNAs is believed to be obligatory also for the replication of Alphavirus, since a point mutation specifically destroying the guanylyltransferase activity of Nsp1 is lethal for the virus (6).…”

Section: Semliki Forest Virus (Sfv)mentioning

confidence: 99%

Identification of a Novel Function of the AlphavirusCapping Apparatus

Merits

Auvinen

et al. 2000

177

162

Semliki Forest virus (SFV)1 is member of the Alphavirus genus of the Togaviridae family. SFV has a positive-stranded 42 S RNA genome of 11.5 kilobases. RNA replication of SFV takes place in the cytoplasm and is catalyzed by the viral RNA-dependent RNA polymerase, which contains the virusspecific proteins Nsp1-4. These are cleavage products of a large (2342 aa) nonstructural polyprotein P1234. In the RNA polymerase complex all Nsps are in close association with each other (1, 2). The parental 42 S RNA is copied to complementary minus-strands, which in turn are used as templates for the synthesis of new 42 S RNA plus-strands and subgenomic 26 S mRNAs. During plus-strand synthesis, the 5Ј ends of the 42 S and 26 S RNAs become modified with covalently attached m 7 GpppA (the cap0 structure) (3-5). Capping of the RNAs is believed to be obligatory also for the replication of Alphavirus, since a point mutation specifically destroying the guanylyltransferase activity of Nsp1 is lethal for the virus (6).The functions of Alphavirus Nsps in replication have been studied using various genetic and biochemical approaches (1, 2). Nsp4 is the catalytic component of the RNA polymerase (7,8), whereas the functions of the phosphoprotein Nsp3 (9) are poorly defined (10, 11). Nsp2 has several distinct functions. It has nucleoside triphosphatase (NTPase) activity at its aminoterminal half (12), which is vital for the virus replication (13). Nsp2 has RNA helicase activity, which utilizes NTP hydrolysis as the energy source (14). The carboxyl-terminal part of the protein is a papain-like protease responsible for the autocatalytic cleavages of the nonstructural polyprotein (2, 15, 16). The carboxyl-terminal part has a nuclear localization sequence, which is responsible for sequestering of about half of the molecules to the nucleus during infection (17, 18). Furthermore, Nsp2 regulates transcription of the subgenomic 26 S RNA (Ref.19 and references therein). Here we show that Nsp2 has yet an additional activity required for capping of the virus mRNAs.Capping of cellular mRNAs occurs in the nucleus and comprises four different reactions. RNA 5Ј-triphosphatase removes the ␥-phosphate from the 5Ј end of the nascent RNA molecule (pppRNA 3 ppRNA). Guanylyltransferase reacts with a GTP molecule to form a covalent complex with GMP, which is then transferred from guanylyltransferase to the 5Ј end of RNA to form G(5Ј)ppp(5Ј)NpRNA. Methylation by guanine-7N-methyltransferase yields an RNA molecule with the cap0 structure (m 7 GpppNpRNA). Further methylation by nucleoside-2Ј-Omethyltransferase of the riboses of the penultimate and the adjacent nucleotides yields cap1 and cap2 structures, respectively (20,21).Unlike cellular mRNAs, the capping of Alphavirus RNAs takes place in the cytoplasm and is carried out by reactions that differ from the nuclear reactions as follows. (i) Nsp1 catalyzes transfer of the methyl group from S-adenosylmethionine to GTP to yield m 7 GTP (methyltransferase reaction), and (ii) a covalent guanylate complex Nsp1-m 7 GMP i...

“…nsP2 mutant PRDR is not lethal for the virus in cell culture, but SFV carrying this mutation is apathogenic for mouse (21). In addition, the carboxylterminal domain of SFV nsP2 has been implicated in the regulation of the synthesis of the subgenomic mRNA (22)(23)(24).…”

mentioning

confidence: 99%

Site-specific Protease Activity of the Carboxyl-terminal Domain of Semliki Forest Virus Replicase Protein nsP2

Valmu

Kääriäinen

et al. 2001

118

The virus-specific components (nsP1-nsP4) of Semliki Forest virus RNA polymerase are synthesized as a large polyprotein (P1234), which is cleaved by a virus-encoded protease. Based on mutagenesis studies, nsP2 has been implicated as the protease moiety of P1234. Here, we show that purified nsP2 (799 amino acids) and its C-terminal domain Pro39 (amino acids 459 -799) specifically process P1234 and its cleavage intermediates. Analysis of cleavage products of in vitro synthesized P12, P23, and P34 revealed cleavages at sites 1/2, 2/3, and 3/4. The cleavage regions of P1/2, P2/3, and P3/4 were expressed as thioredoxin fusion proteins (Trx12, Trx23, and Trx34), containing ϳ20 amino acids on each side of the cleavage sites. After exposure of these purified fusion proteins to nsP2 or Pro39, the reaction products were analyzed by SDS-polyacrylamide gel electrophoresis, mass spectrometry, and amino-terminal sequencing. The expected amino termini of nsP2, nsP3, and nsP4 were detected. The cleavage at 3/4 site was most efficient, whereas cleavage at 1/2 site required 5000-fold more of Pro39, and 2/3 site was almost resistant to cleavage. The activity of Pro39 was inhibited by N-ethylmaleimide, Zn 2؉ , and Cu 2؉ , but not by EDTA, phenylmethylsulfonyl fluoride, or pepstatin, in accordance with the thiol proteinase nature of nsP2.