Immunocytochemical localization of capsid-related particles in subcellular fractions of poliovirus-infected cells

Pfister, Thomas D.; Pasamontes, Luis; Troxler, Monica; Egger, Denise; Bienz, Kurt

doi:10.1016/0042-6822(92)90522-q

Cited by 44 publications

(44 citation statements)

References 48 publications

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“…Picornavirus infection induces rearrangement of intracellular membranes into vesicles, which are associated with nonstructural proteins and viral RNAs (5,38), and these membrane vesicles, called the replication complex, are the site of picornavirus RNA replication (4). In the encapsidation process, it is thought that newly synthesized positive-strand RNAs, which accumulate around the replication complex, are associated with capsid precursors attached to the surface of the replication complex (26,28,38). It has been reported that the poliovirus 2C protein is involved in viral RNA replication (19) and encapsidation (39).…”

Section: Discussionmentioning

confidence: 99%

Aichi Virus Leader Protein Is Involved in Viral RNA Replication and Encapsidation

Sasaki

Nagashima

Taniguchi

2003

J Virol

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Aichi virus, which is associated with human acute gastroenteritis (41), is a member of the genus Kobuvirus of the family Picornaviridae (31, 42). This virus was first isolated in 1989 from a stool specimen from a patient with oyster-associated nonbacterial gastroenteritis in Aichi, Japan (41), and the complete genome sequence was determined in 1998 (42). The single-stranded, positive-sense RNA genome of Aichi virus consists of 8,280 nucleotides (nt) and a poly(A) tract and encodes a polyprotein of 2,432 amino acids (aa) (34,42). One of the features of the genome organization of Aichi virus is that it encodes a leader (L) polypeptide upstream of the capsid coding region.Among picornaviruses besides Aichi virus, aphtho-, cardio-, erbo-, and teschoviruses (10) and porcine enterovirus 8 (18) encode L proteins. The L protein of foot-and-mouth disease virus (FMDV), an aphthovirus, is produced in two forms, Lab and Lb, through initiation of translation at two in-frame AUG codons located 84 nt apart (3). Both forms of the FMDV L protein are papain-like thiol proteinases that cleave at their own C termini (23,30,36). In addition, the FMDV L protein cleaves a eukaryotic translation initiation factor, eIF4G (9), contributing to the shutoff of host cap-dependent protein synthesis. The shutoff of host protein synthesis leads to inhibition of alpha/beta interferon production. The L deletion mutant of FMDV can replicate and spread in non-interferon-responsive baby hamster kidney (BHK) cells (29). However, it can replicate but cannot spread in secondary cells from susceptible animals, such as bovine, ovine, and porcine cells (8). This is associated with the inability of the L deletion mutant to inhibit cap-dependent alpha/beta interferon mRNA translation (7,8). The erbovirus L protein also has autocatalytic activity but does not cleave eIF4GI (15).The cardiovirus L proteins have neither the amino acid sequence motif characteristic of picornavirus proteases nor autocatalytic activity. They are released from a polyprotein by viral protease 3C (27, 33). The L protein of the GDVII strain of Theiler's murine encephalomyelitis virus (TMEV) has been shown to be essential for neurovirulence in mice (6). In addition, it has been reported that L mutants of TMEV and mengovirus exhibit distinct growth properties depending on the host cells: certain L mutants of TMEV and mengovirus can spread in BHK cells but not in mouse L929 cells (1,40,43). For the GDVII strain of TMEV, it has been shown that the L deletion mutant has a defect in the assembly of virions in L929 cells (1), which mainly causes the inability of the mutant to spread in L929 cells. On the other hand, for the DA1 strain of TMEV and mengovirus, the L proteins have been reported to be involved in the inhibition of alpha/beta interferon production by L929 cells (40,44). Furthermore, in the case of encephalomyocarditis virus, it has been suggested that the L protein is involved in internal ribosome entry site-mediated translation of viral RNA (11,17).The Aichi virus L protein does...

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Section: Discussionmentioning

confidence: 99%

Aichi Virus Leader Protein Is Involved in Viral RNA Replication and Encapsidation

Sasaki

Nagashima

Taniguchi

2003

J Virol

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“…As shown in Figure 3, DIMs from infected cells appeared as a homogeneous population of membranous structures of approximately 100 nm in diameter that were either associated to form larger structures (A) or in an isolated form (C). The morphology of these structures closely resembles that of the isolated rosettelike vesicles containing poliovirus replication complexes (22,23). In agreement with our biochemical results showing the presence of poliovirus structural proteins in the DIM fraction (Figure 1), these rosettelike membranes were shown to contain 14S pentamer capsid precursors (23).…”

Section: Association Of Poliovirus Proteins With Detergentinsoluble Mmentioning

confidence: 94%

“…Our current results show that poliovirus structural proteins and some other viral proteins accumulate in membranes displaying the biochemical features of DIMs. The morphology of these membranes, as examined by transmission electron microscopy, closely resembles that of the reported poliovirus replication membranous complexes (22,23). Taking into account that the poliovirus capsid proteins are synthesized as a myristoylated precursor, we have investigated the possible role of the lipid moiety in targeting to DIMs.…”

mentioning

confidence: 88%

The Amino-Terminal Nine Amino Acid Sequence of Poliovirus Capsid VP4 Protein Is Sufficient To Confer N-Myristoylation and Targeting to Detergent-Insoluble Membranes

Martı́n-Belmonte¹,

López-Guerrero²,

Carrasco³

et al. 2000

Biochemistry

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The confinement of membrane proteins by lipid-lipid interactions into specialized detergent-insoluble membrane (DIM) microdomains has been proposed as a general mechanism to recruit selectively lipid-modified proteins and specific transmembrane proteins. Poliovirus capsid VP4 protein and its precursors are myristoylated at the NH(2)-terminal Gly residue. To determine whether poliovirus uses DIMs during its replicative cycle, we isolated DIMs from poliovirus-infected HeLa cells and identified the presence of capsid proteins and their precursors, proteinases 2A and 3C, and other viral proteins involved in poliovirus RNA replication such as protein 2C and the polymerase 3D. The morphology of these DIMs was similar to that of the previously described rosette-like vesicles associated with replication complexes isolated from poliovirus-infected cells. To examine the possible role of the myristoyl moiety in the targeting of poliovirus structural proteins to DIMs, we generated a chimeric protein consisting of the nine amino-terminal amino acids from VP4 fused to the amino terminus of the green fluorescent protein (GFP). The selected VP4 sequence was sufficient to confer N-myristoylation and targeting to DIMs to the GFP chimera. Mutations within this sequence known to affect both myristoylation and poliovirus assembly abrogated the targeting of the GFP chimera. These results indicate that the myristoylated amino-terminal nonapeptide from poliovirus VP4 protein constitutes a signal for incorporation into DIMs.

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“…by discontinuous sucrose gradient centrifugation of a cytoplasmic extract as described (Bienz et al, 1990), except that a 20% sucrose solution was layered on top of the 30 %/45 % sucrose cushions. The vesicular fractions banding on top of each of the sucrose layers were harvested and examined by (immuno-) electron microscopy (IEM) (Bienz et al, 1990;Pfister et al, 1992).…”

Section: Methodsmentioning

confidence: 99%

“…In the infected cell, progeny RNA synthesis and RNA encapsidation have been proposed to be coupled processes, since (i) progeny RNA becomes encapsidated immediately after its synthesis (Baltimore et al, 1966) and (ii) progeny virus (Caliguiri & Compans, 1973) as well as subviral particles (Caliguiri & Mosser, 1971;Pfister et aL, 1992;Yin, 1977) could be detected in subcellular RC-containing fractions.…”

Section: Introductionmentioning

confidence: 99%

Poliovirus subviral particles associated with progeny RNA in the replication complex

Pfister

Egger

Bienz

1995

Journal of General Virology

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The poliovirus replication complex (RC), the site of genomic 36S RNA synthesis, was previously shown to contain subviral particles of 5S protomer and 14S pentamer antigenicity. The present investigation demonstrates that 5S/14S antigenic subviral particles can be cross-linked to viral RNA by UV irradiation of a subcellular fraction containing the poliovirus RC. Each capsid protein of the subviral particles, i.e. VP0, VP1 and VP3, was cross-linked to viral RNA. SDS-PAGE analysis of the cross-linked capsid proteins revealed a bandshift for VP1, whereas VP0 migrated in several bands, which were interpreted to be multimers of VP0 linked by short stretches of RNA. It was found that 36S RNA rather than replicative intermediate RNA was cross-linked to capsid proteins. Our results indicate that encapsidation of poliovirus RNA starts in the RC and is initiated by 14S pentamers.

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Immunocytochemical localization of capsid-related particles in subcellular fractions of poliovirus-infected cells

Cited by 44 publications

References 48 publications

Aichi Virus Leader Protein Is Involved in Viral RNA Replication and Encapsidation

Aichi Virus Leader Protein Is Involved in Viral RNA Replication and Encapsidation

The Amino-Terminal Nine Amino Acid Sequence of Poliovirus Capsid VP4 Protein Is Sufficient To Confer N-Myristoylation and Targeting to Detergent-Insoluble Membranes

Poliovirus subviral particles associated with progeny RNA in the replication complex

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