In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release

Liljeström, Peter; Lusa, Sari; Huylebroeck, Danny; Garoff, Henrik

doi:10.1128/jvi.65.8.4107-4113.1991

Cited by 416 publications

(220 citation statements)

References 57 publications

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“…Other results are consistent with an indirect role in the packing of the virus spike glycoproteins; mutation C39S resulted in defective budding and a revertant contained two additional mutations at the ectodomain of the virus glycoprotein (Ivanova, Lustig, & Schlesinger, 1995). Further, deletion of 6K in a full-length cDNA clone of SFV resulted in a dramatic reduction in virus release (Liljestrom, Lusa, Huylebroeck, & Garoff, 1991), and BHK cells expressing a Δ6K strain produced thermolabile particles, suggesting a disrupted glycoprotein packing (Loewy, Smyth, von Bonsdorff, Liljestrom, & Schlesinger, 1995). Despite these investigations, the specific role of 6K channel activity in these processes is still uncertain.…”

Section: The Alphavirus 6k Proteinsupporting

confidence: 62%

Targeting the Channel Activity of Viroporins

Surya

Torres

2016

Advances in Protein Chemistry and Structural Biology

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Since the discovery that certain small viral membrane proteins, collectively termed as viroporins, can permeabilize host cellular membranes and also behave as ion channels, attempts have been made to link this feature to specific biological roles. In parallel, most viroporins identified so far are virulence factors, and interest has focused toward the discovery of channel inhibitors that would have a therapeutic effect, or be used as research tools to understand the biological roles of viroporin ion channel activity. However, this paradigm is being shifted by the difficulties inherent to small viral membrane proteins, and by the realization that protein-protein interactions and other diverse roles in the virus life cycle may represent an equal, if not, more important target. Therefore, although targeting the channel activity of viroporins can probably be therapeutically useful in some cases, the focus may shift to their other functions in following years. Small-molecule inhibitors have been mostly developed against the influenza A M2 (IAV M2 or AM2). This is not surprising since AM2 is the best characterized viroporin to date, with a well-established biological role in viral pathogenesis combined the most extensive structural investigations conducted, and has emerged as a validated drug target. For other viroporins, these studies are still mostly in their infancy, and together with those for AM2, are the subject of the present review.

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Section: The Alphavirus 6k Proteinsupporting

confidence: 62%

Targeting the Channel Activity of Viroporins

Surya

Torres

2016

Advances in Protein Chemistry and Structural Biology

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“…Alphavirus 6K protein provides a further opportunity for the study of interfacial domain activity in fission: firstly, this small protein's sequence comprises a single PreTM-TMD motif [79], and, secondly, 6K is not required for alphavirus budding, but catalyzes the process [80], which allows recovery of budding defective, but still infectious particles, upon cell transfection with alphaviral replicon.…”

Section: Interfacial Domains In Viral Membrane Fissionmentioning

confidence: 99%

Interfacial pre-transmembrane domains in viral proteins promoting membrane fusion and fission

Lorizate

Huarte

Sáez‐Cirión

et al. 2008

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Membrane fusion and fission underlie two limiting steps of enveloped virus replication cycle: access to the interior of the host-cell (entry) and dissemination of viral progeny after replication (budding), respectively. These dynamic processes proceed mediated by specialized proteins that disrupt and bend the lipid bilayer organization transiently and locally. We introduced Wimley-White membrane-water partitioning free energies of the amino acids as an algorithm for predicting functional domains that may transmit protein conformational energy into membranes. It was found that many viral products possess unusually extended, aromatic-rich pre-transmembrane stretches predicted to stably reside at the membrane interface. Here, we review structure-function studies, as well as data reported on the interaction of representative peptides with model membranes, all of which sustain a functional role for these domains in viral fusion and fission. Since pre-transmembrane sequences also constitute antigenic determinants in a membrane-bound state, we also describe some recent results on their recognition and blocking at membrane interface by neutralizing antibodies.

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“…To obtain a set of EGFP-fused expression constructs with truncations at the 3/4 site, the sequences corresponding to the truncated 3/4 sites were PCR amplified using specific primers with adaptors and the infectious cDNA clone of SFV, pSP6-SFV4 (27), as a template. The PCR fragments were digested with EcoRV and XhoI and cloned into pET-E2-EGFP, treated with the same enzymes.…”

Section: Bacterial Strains and Expression Vectorsmentioning

confidence: 99%

“…The resulting clones were verified by sequencing the entire transferred fragment and designated pSFV(23/RAGC), pSFV(34/TAGA), pSFV(34/ HAGA), pSFV(34/RSGA), and pSFV(34/RAEV). Capped transcripts were prepared with SP6 polymerase after linearization of the plasmids with SpeI (27) and used for transfection of BHK21 cells by electroporation. The primary virus stock was collected after 24 h at 37°C, titrated, and used in all subsequent experiments.…”

Section: Bacterial Strains and Expression Vectorsmentioning

confidence: 99%

Molecular Determinants of Substrate Specificity for Semliki Forest Virus Nonstructural Protease

Lulla

Tints

et al. 2006

J Virol

100

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The C-terminal cysteine protease domain of Semliki Forest virus nonstructural protein 2 (nsP2) regulates the virus life cycle by sequentially cleaving at three specific sites within the virus-encoded replicase polyprotein P1234. The site between nsP3 and nsP4 (the 3/4 site) is cleaved most efficiently. Analysis of Semliki Forest virus-specific cleavage sites with shuffled N-terminal and C-terminal half-sites showed that the main determinants of cleavage efficiency are located in the region preceding the cleavage site. Random mutagenesis analysis revealed that amino acid residues in positions P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much variation, whereas in the P5 position most residues were permitted. When mutations affecting cleavage efficiency were introduced into the 2/3 and 3/4 cleavage sites, the resulting viruses remained viable but had similar defects in P1234 processing as observed in the in vitro assay. Complete blockage of the 3/4 cleavage was found to be lethal. The amino acid in position P1 had a significant effect on cleavage efficiency, and in this regard the protease markedly preferred a glycine residue over the tyrosine natively present in the 3/4 site. Therefore, the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The protease recognizes at least residues P4 to P1, and the P4 arginine residue plays an important role in the fast cleavage of the 3/4 site. Semliki Forest virus (SFV)is an enveloped positive-strand RNA virus of the genus Alphavirus, family Togaviridae. Alphavirus replication relies on the production of replicase proteins in the form of polyprotein precursor(s), which are then coand posttranslationally processed (40). This finally leads to the generation of individual replicase protein subunits and ensures the formation of proper interactions between the cleavage products of a single polyprotein (35). However, it is not known whether multiple polyproteins are required to build up an individual replication complex. A large body of evidence indicates that the polyprotein expression and cleavage strategy regulates viral RNA replication (20,24,25,37,46). The fulllength nonstructural (ns) polyprotein, designated P1234, is translated directly from the viral genomic RNA and becomes cleaved immediately after or during its synthesis. Its primary cleavage products, polyprotein P123 and nsP4, form the early replication complexes synthesizing negative-strand RNA. P123 is first cleaved to yield nsP1 and P23, giving an intermediate complex, nsP1-P23-nsP4, which is capable of both negativestrand and positive-strand synthesis (20,24,46) but which appears to be very short lived in infected cells. P23 is rapidly cleaved into nsP2 and nsP3, and the replication complex is rearranged into a stable form making exclusively positive-sense RNA with the negative strand as a template.

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In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release

Cited by 416 publications

References 57 publications

Targeting the Channel Activity of Viroporins

Targeting the Channel Activity of Viroporins

Interfacial pre-transmembrane domains in viral proteins promoting membrane fusion and fission

Molecular Determinants of Substrate Specificity for Semliki Forest Virus Nonstructural Protease

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