Stretch Of Hydrophobic Amino Acids Research Articles

C-terminal amino acids in the type I transmembrane domain of L-type lectin VIP36 affect γ-secretase susceptibility

Regulated intramembrane proteolysis (RIP) is a two-step processing mechanism for transmembrane proteins consisting of ectodomain shedding (shedding), which removes the extracellular domain through juxtamembrane processing and intramembrane proteolysis, which processes membrane-anchored shedding products within the transmembrane domain. RIP irreversibly converts one transmembrane protein into multiple soluble proteins that perform various physiological functions. The only requirement for the substrate of γ-secretase, the major enzyme responsible for intramembrane proteolysis of type I transmembrane proteins, is the absence of a large extracellular domain, and it is thought that γ-secretase can process any type I membrane protein as long as it is shed. In the present study, we showed that the shedding susceptible type I membrane protein VIP36 (36 kDa vesicular integral membrane protein) and its homolog, VIPL, have different γ-secretase susceptibilities in their transmembrane domains. Analysis of the substitution mutants suggested that γ-secretase susceptibility is regulated by C-terminal amino acids in the transmembrane domain. We also compared the transmembrane domains of several shedding susceptible membrane proteins and found that each had a different γ-secretase susceptibility. These results suggest that the transmembrane domain is not simply a stretch of hydrophobic amino acids but is an important element that regulates membrane protein function by controlling the lifetime of the membrane-anchored shedding product.

The mature N-termini of Plasmodium effector proteins confer specificity of export.

Malaria parasites export hundreds of proteins to the cytoplasm of the host red blood cells for their survival. A five amino acid sequence, called the PEXEL motif, is conserved among many exported proteins and is thought to be a signal for export. However, the motif is cleaved inside the endoplasmic reticulum of the parasite, and mature proteins starting from the fourth PEXEL residue travel to the parasite periphery for export. We showed that the PEXEL motif is dispensable for export as long as identical mature proteins can be efficiently produced via alternative means in the ER. We also showed that the exported and non-exported proteins are differentiated at the parasite periphery based on their mature N-termini; however, any discernible export signal within that region remained cryptic. Our study resolves a longstanding paradox in PEXEL protein trafficking.

Modeling Adsorption, Conformation, and Orientation of the Fis1 Tail Anchor at the Mitochondrial Outer Membrane.

Proteins can be targeted to organellar membranes by using a tail anchor (TA), a stretch of hydrophobic amino acids found at the polypeptide carboxyl-terminus. The Fis1 protein (Fis1p), which promotes mitochondrial and peroxisomal division in the yeast Saccharomyces cerevisiae, is targeted to those organelles by its TA. Substantial evidence suggests that Fis1p insertion into the mitochondrial outer membrane can occur without the need for a translocation machinery. However, recent findings raise the possibility that Fis1p insertion into mitochondria might be promoted by a proteinaceous complex. Here, we have performed atomistic and coarse-grained molecular dynamics simulations to analyze the adsorption, conformation, and orientation of the Fis1(TA). Our results support stable insertion at the mitochondrial outer membrane in a monotopic, rather than a bitopic (transmembrane), configuration. Once inserted in the monotopic orientation, unassisted transition to the bitopic orientation is expected to be blocked by the highly charged nature of the TA carboxyl-terminus and by the Fis1p cytosolic domain. Our results are consistent with a model in which Fis1p does not require a translocation machinery for insertion at mitochondria.

Folding and Insertion of Transmembrane Helices at the ER.

In eukaryotic cells, the endoplasmic reticulum (ER) is the entry point for newly synthesized proteins that are subsequently distributed to organelles of the endomembrane system. Some of these proteins are completely translocated into the lumen of the ER while others integrate stretches of amino acids into the greasy 30 Å wide interior of the ER membrane bilayer. It is generally accepted that to exist in this non-aqueous environment the majority of membrane integrated amino acids are primarily non-polar/hydrophobic and adopt an α-helical conformation. These stretches are typically around 20 amino acids long and are known as transmembrane (TM) helices. In this review, we will consider how transmembrane helices achieve membrane integration. We will address questions such as: Where do the stretches of amino acids fold into a helical conformation? What is/are the route/routes that these stretches take from synthesis at the ribosome to integration through the ER translocon? How do these stretches ‘know’ to integrate and in which orientation? How do marginally hydrophobic stretches of amino acids integrate and survive as transmembrane helices?

SUMO Interacting Motifs: Structure and Function.

Small ubiquitin-related modifier (SUMO) is a member of the ubiquitin-related protein family. SUMO modulates protein function through covalent conjugation to lysine residues in a large number of proteins. Once covalently conjugated to a protein, SUMO often regulates that protein’s function by recruiting other cellular proteins. Recruitment frequently involves a non-covalent interaction between SUMO and a SUMO-interacting motif (SIM) in the interacting protein. SIMs generally consist of a four-residue-long hydrophobic stretch of amino acids with aliphatic non-polar side chains flanked on one side by negatively charged amino acid residues. The SIM assumes an extended β-strand-like conformation and binds to a conserved hydrophobic groove in SUMO. In addition to hydrophobic interactions between the SIM non-polar core and hydrophobic residues in the groove, the negatively charged residues in the SIM make favorable electrostatic contacts with positively charged residues in and around the groove. The SIM/SUMO interaction can be regulated by the phosphorylation of residues adjacent to the SIM hydrophobic core, which provide additional negative charges for favorable electrostatic interaction with SUMO. The SUMO interactome consists of hundreds or perhaps thousands of SIM-containing proteins, but we do not fully understand how each SUMOylated protein selects the set of SIM-containing proteins appropriate to its function. SIM/SUMO interactions have critical functions in a large number of essential cellular processes including the formation of membraneless organelles by liquid–liquid phase separation, epigenetic regulation of transcription through histone modification, DNA repair, and a variety of host–pathogen interactions.

Direct binding of ESCRT protein Chm7 to phosphatidic acid-rich membranes at nuclear envelope herniations.

Mechanisms that control nuclear membrane remodeling are essential to maintain the integrity of the nucleus but remain to be fully defined. Here, we identify a phosphatidic acid (PA)-binding capacity in the nuclear envelope (NE)-specific ESCRT, Chm7, in budding yeast. Chm7's interaction with PA-rich membranes is mediated through a conserved hydrophobic stretch of amino acids, which confers recruitment to the NE in a manner that is independent of but required for Chm7's interaction with the LAP2-emerin-MAN1 (LEM) domain protein Heh1 (LEM2). Consistent with the functional importance of PA binding, mutation of this region abrogates recruitment of Chm7 to membranes and abolishes Chm7 function in the context of NE herniations that form during defective nuclear pore complex (NPC) biogenesis. In fact, we show that a PA sensor specifically accumulates within these NE herniations. We suggest that local control of PA metabolism is important for ensuring productive NE remodeling and that its dysregulation may contribute to pathologies associated with defective NPC assembly.

Characterisation of a novel Emaravirus identified in mosaic‐diseased Eurasian aspen (Populus tremula)

AbstractSince Emaraviruses have been discovered in 2007 several new species were detected in a range of host plants. Five genome segments of a novel Emaravirus from mosaic‐diseased Eurasian aspen (Populus tremula) have been completely determined. The monocistronic, segmented ssRNA genome of the virus shows a genome organisation typical for Emaraviruses encoding the viral RNA‐dependent RNA polymerase (RdRP, 268.2 kDa) on RNA1 (7.1 kb), a glycoprotein precursor (GPP, 73.5 kDa) on RNA2 (2.3 kb), the viral nucleocapsid protein (N, 35.6 kDa) on RNA3 (1.6 kb), and a putative movement protein (MP, 41.0 kDa) on RNA4 (1.6 kb). The fifth identified genome segment (RNA5, 1.3 kb) encodes a protein of unknown function (P28, 28.1 kDa). We discovered that it is distantly related to proteins encoded by Emaraviruses, such as P4 of European mountain ash ringspot‐associated virus. All proteins from this group contain a central hydrophobic region with a conserved secondary structure and a hydrophobic amino acid stretch, bordered by two highly conserved positions, thus clearly representing a new group of homologues of Emaraviruses. The virus identified in Eurasian aspen is closely associated with observed leaf symptoms, such as mottle, yellow blotching, variegation and chloroses along veins. All five viral RNAs were regularly detectable by RT‐PCR in mosaic‐diseased P. tremula in Norway, Finland and Sweden (Fennoscandia). Observed symptoms and testing of mosaic‐diseased Eurasian aspen by virus‐specific RT‐PCR targeting RNA3 and RNA4 confirmed a wide geographic distribution of the virus in Fennoscandia. We could demonstrate that the mosaic‐disease is graft‐transmissible and confirmed that the virus is the causal agent by detection in symptomatic, graft‐inoculated seedlings used as rootstocks as well as in the virus‐infected scions used for graft‐inoculation. Owing to these characteristics, the virus represents a novel species within the genus Emaravirus and was tentatively denominated aspen mosaic‐associated virus.

Molecular Characterization and Taurine Regulation of Taurine Transporter in Goldfish

AbstractTaurine transporter (TauT) is a sodium‐ and chloride‐dependent transport carrier, and it plays a key role in the transportation of taurine. At present, the many functions of TauT in fish remain obscure. In this study, a cDNA clone for encoding a taurine transporter was isolated from Goldfish Carassius auratus. The nucleotide sequence predicted 625 amino acids with significant homology to the TauT gene of Zebrafish Danio rerio. Hydropathic analysis revealed stretches of hydrophobic amino acids that were indicative of 12 transmembrane domains that were similar to those proposed for other cloned TauT genes. The results of a reverse transcription polymerase chain reaction (RT‐PCR) analysis demonstrated that TauT broadly expressed in all of the selected tissues and developmental stages in Goldfish but with prominent differential expression levels. The expression of TauT in Goldfish has diurnal changes. The highest expression was at 1200 hours, and the lowest was at 2100 hours. Moreover, taurine treatments showed similar stimulatory effects on the expression of TauT in the intestines of Goldfish both in vitro and in vivo. Overall, these results may provide new insights into the regulation of TauT expression in fish, which has potential applications for improving fish dietary formulations.

Identification of the retinoschisin-binding site on the retinal Na/K-ATPase.

X-linked juvenile retinoschisis (XLRS) is a hereditary retinal dystrophy, caused by mutations in the RS1 gene which encodes the secreted protein retinoschisin. In recent years, several molecules have been proposed to interact with retinoschisin, including the retinal Na/K-ATPase, L-voltage gated Ca2+ channels, and specific sugars. We recently showed that the retinal Na/K-ATPase consisting of subunits ATP1A3 and ATP1B2 is essential for anchoring retinoschisin to plasma membranes and identified the glycosylated ATP1B2 subunit as the direct interaction partner for retinoschisin. We now aimed to precisely map the retinoschisin binding domain(s) in ATP1B2. In general, retinoschisin binding was not affected after selective elimination of individual glycosylation sites via site-directed mutagenesis as well as after full enzymatic deglycosylation of ATP1B2. Applying the interface prediction tool PresCont, two putative protein-protein interaction patches (“patch I” and “patch II”) consisting each of four hydrophobic amino acid stretches on the ATP1B2 surface were identified. These were consecutively altered by site-directed mutagenesis. Functional assays with the ATP1B2 patch mutants identified patch II and, specifically, the associated amino acid at position 240 (harboring a threonine in ATP1B2) as crucial for retinoschisin binding to ATP1B2. These and previous results led us to suggest an induced-fit binding mechanism for the interaction between retinoschisin and the Na/K-ATPase, which is dependent on threonine 240 in ATP1B2 allowing the accommodation of hyperflexible retinoschisin spikes by the associated protein-protein interaction patch on ATP1B2.

A Novel PEGylated Block Copolymer in New Age Therapeutics for Alzheimer's Disease.

The amyloid cascade hypothesis dealing with the senile plaques is until date thought to be one of the causative pathways leading to the pathophysiology of Alzheimer's disease (AD). Though many aggregation inhibitors of misfolded amyloid beta (Aβ42) peptide have failed in clinical trials, there are some positive aspects of the designed therapeutic peptides for diseases involving proteinaceous aggregation. Here, we evaluated a smart design of side chain tripeptide (Leu-Val-Phe)-based polymeric inhibitor addressing the fundamental hydrophobic amino acid stretch "Lys-Leu-Val-Phe-Phe-Ala" (KLVFFA) of the Aβ42 peptide. The in vitro analyses performed through the thioflavin T (ThT) fluorescence assay, infrared spectroscopy, isothermal calorimetry, cytotoxicity experiments, and so on evinced a promising path towards the development of new age AD therapeutics targeting the inhibition of misfolded Aβ42 peptide fibrillization. The in silico simulations done contoured the mechanism of drug action of the present block copolymer as the competitive inhibition of aggregate-prone hydrophobic stretch of Aβ42. Graphical abstract The production of misfolded Aβ42 peptide from amyloid precursor protein initiates amyloidosis pathway which ends with the deposition of fibrils via the oligomerization and aggregation of Aβ42 monomers. The side chain tripeptide-based PEGylated polymer targets these Aβ42 monomers and oligomers inhibiting their aggregation. This block copolymer also binds and helps degrading the preformed fibrils of Aβ42.

Triacylglycerol mimetics regulate membrane interactions of glycogen branching enzyme: implications for therapy

Adult polyglucosan body disease (APBD) is a neurological disorder characterized by adult-onset neurogenic bladder, spasticity, weakness, and sensory loss. The disease is caused by aberrant glycogen branching enzyme (GBE) (GBE1Y329S) yielding less branched, globular, and soluble glycogen, which tends to aggregate. We explore here whether, despite being a soluble enzyme, GBE1 activity is regulated by protein-membrane interactions. Because soluble proteins can contact a wide variety of cell membranes, we investigated the interactions of purified WT and GBE1Y329S proteins with different types of model membranes (liposomes). Interestingly, both triheptanoin and some triacylglycerol mimetics (TGMs) we have designed (TGM0 and TGM5) markedly enhance GBE1Y329S activity, possibly enough for reversing APBD symptoms. We show that the GBE1Y329S mutation exposes a hydrophobic amino acid stretch, which can either stabilize and enhance or alternatively, reduce the enzyme activity via alteration of protein-membrane interactions. Additionally, we found that WT, but not Y329S, GBE1 activity is modulated by Ca2+ and phosphatidylserine, probably associated with GBE1-mediated regulation of energy consumption and storage. The thermal stabilization and increase in GBE1Y329S activity induced by TGM5 and its omega-3 oil structure suggest that this molecule has a considerable therapeutic potential for treating APBD.

Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis.

To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis. The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3mg/l respectively. Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

Global profiling of SRP interaction with nascent polypeptides.

Signal recognition particle (SRP) is a universally conserved protein-RNA complex that mediates co-translational protein translocation and membrane insertion by targeting translating ribosomes to membrane translocons. The existence of parallel co- and post-translational transport pathways, however, raises the question of the cellular substrate pool of SRP and the molecular basis of substrate selection. Here we determine the binding sites of bacterial SRP within the nascent proteome of Escherichia coli at amino acid resolution, by sequencing messenger RNA footprints of ribosome-nascent-chain complexes associated with SRP. SRP, on the basis of its strong preference for hydrophobic transmembrane domains (TMDs), constitutes a compartment-specific targeting factor for nascent inner membrane proteins (IMPs) that efficiently excludes signal-sequence-containing precursors of periplasmic and outer membrane proteins. SRP associates with hydrophobic TMDs enriched in consecutive stretches of hydrophobic and bulky aromatic amino acids immediately on their emergence from the ribosomal exit tunnel. By contrast with current models, N-terminal TMDs are frequently skipped and TMDs internal to the polypeptide sequence are selectively recognized. Furthermore, SRP binds several TMDs in many multi-spanning membrane proteins, suggesting cycles of SRP-mediated membrane targeting. SRP-mediated targeting is not accompanied by a transient slowdown of translation and is not influenced by the ribosome-associated chaperone trigger factor (TF), which has a distinct substrate pool and acts at different stages during translation. Overall, our proteome-wide data set of SRP-binding sites reveals the underlying principles of pathway decisions for nascent chains in bacteria, with SRP acting as the dominant triaging factor, sufficient to separate IMPs from substrates of the SecA-SecB post-translational translocation and TF-assisted cytosolic protein folding pathways.

A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein–protein interactions

The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein–protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein–protein interactions of Myb with regulatory proteins.

A Herpesvirus Specific Motif of Epstein-Barr Virus DNA Polymerase Is Required for the Efficient Lytic Genome Synthesis.

Epstein-Barr virus (EBV) is associated with several malignancies, including Burkitt lymphoma and nasopharyngeal carcinoma. To overcome such disorders, understanding the molecular mechanisms of the EBV replication is important. The EBV DNA polymerase (Pol) is one of the essential factors for viral lytic DNA replication. Although it is well known that its C-terminal half, possessing DNA polymerase and 3’-5’ exonuclease activity, is highly conserved among Family B Pols, the NH2-terminal half has yet to be characterized in detail. In this study, we show that a stretch of hydrophobic amino acids within the pre-NH2-terminal domain of EBV Pol plays important role. In addition, we could identify the most essential residue for replication in the motif. These findings will shed light on molecular mechanisms of viral DNA synthesis and will help to develop new herpesviruses treatments.

Amyloid properties of the mouse egg zona pellucida.

The zona pellucida (ZP) surrounding the oocyte is an extracellular fibrillar matrix that plays critical roles during fertilization including species-specific gamete recognition and protection from polyspermy. The mouse ZP is composed of three proteins, ZP1, ZP2, and ZP3, all of which have a ZP polymerization domain that directs protein fibril formation and assembly into the three-dimensional ZP matrix. Egg coats surrounding oocytes in nonmammalian vertebrates and in invertebrates are also fibrillar matrices and are composed of ZP domain-containing proteins suggesting the basic structure and function of the ZP/egg coat is highly conserved. However, sequence similarity between ZP domains is low across species and thus the mechanism for the conservation of ZP/egg coat structure and its function is not known. Using approaches classically used to identify amyloid including conformation-dependent antibodies and dyes, X-ray diffraction, and negative stain electron microscopy, our studies suggest the mouse ZP is a functional amyloid. Amyloids are cross-β sheet fibrillar structures that, while typically associated with neurodegenerative and prion diseases in mammals, can also carry out functional roles in normal cells without resulting pathology. An analysis of the ZP domain from mouse ZP3 and ZP3 homologs from five additional taxa using the algorithm AmylPred 2 to identify amyloidogenic sites, revealed in all taxa a remarkable conservation of regions that were predicted to form amyloid. This included a conserved amyloidogenic region that localized to a stretch of hydrophobic amino acids previously shown in mouse ZP3 to be essential for fibril assembly. Similarly, a domain in the yeast protein α-agglutinin/Sag 1p, that possesses ZP domain-like features and which is essential for mating, also had sites that were predicted to be amyloidogenic including a hydrophobic stretch that appeared analogous to the critical site in mouse ZP3. Together, these studies suggest that amyloidogenesis may be a conserved mechanism for ZP structure and function across billions of years of evolution.

E6 protein of human papillomavirus 16 (HPV16) expressed in Escherichia coli sans a stretch of hydrophobic amino acids, enables purification of GST-ΔE6 in the soluble form and retains the binding ability to p53

Recombinant E6 expressed in Escherichia coli is known to form recalcitrant inclusion bodies even when fused to the soluble GST protein. This study describes the modification of the HPV genotype-16 oncogenic protein E6 in order to obtain it in the soluble form. The modified protein (ΔE6) was expressed in E. coli BL21 as an N-terminal fusion with GST (GST-ΔE6). ΔE6 was constructed by deleting the nucleotide sequences coding for IHDIIL (31–36 a.a), one of the highly hydrophobic peptide stretches, using splicing by overextension polymerase chain reaction (SOE-PCR). The removal of IHDIIL residues rendered the GST-ΔE6 soluble and amenable for purification involving a two step process a preliminary glutathione-GST affinity chromatography followed by gel-filtration chromatography. Evaluation of purified protein fractions by HPLC suggests that GST-ΔE6 exists as a monomer. Further, the ΔE6 in GST-ΔE6 seemed to retain the binding ability to p53 as determined by the glutathione-GST capture ELISA. Purified GST-ΔE6 we reckon, might find use as an essential reagent in immunological assays, in sero-epidemiological studies, and also in studies to delineate the structure and function of HPV16 E6.

Improving the antibacterial activity and selectivity of an ultra short peptide by hydrophobic and hydrophilic amino acid stretches

The principle of amino acid stretches tagged at the C terminal of Luecrocin I, which is an ultra-short antibacterial peptide, by tryptophan and arginine or lysine has been reported. The choice of amino acid type at each stretch position depends on the hydrophobic and hydrophilic regions visualized in the helical wheel pattern of Luecrocin I. Oligopeptide tagging should also consider the properties such as positive charge, hydrophobicity, the content of hydrophobic amino acids, polar angle, the properly hydrophilic and hydrophobic facets. Amidation at C terminal and lysine substitute for arginine can increase selectivity between mammalian cells (hemolytic and MTT assay) and bacterial cells tested. KT2 and RT2 which have 53% hydrophobic residues, 7 positive charges, 160° polar angle, −0.02 (KT2) and −0.04 (RT2) hydrophobicity were effective against S. typhi DMST 22842, S. epidermidis ATCC 12228, E. coli ATCC 25922 and V. cholerae non-O1, non-O139. The SEM images implied that the antibacterial mechanism of RT2 and KT2 may depend on concentration rather than time. Finally, RT2 and KT2 can be new antibacterial agents or may be further developed for alternative antibiotics.

Conformational Selection in Substrate Recognition by Hsp70 Chaperones

Hsp70s are molecular chaperones involved in the folding and assembly of proteins. They recognize hydrophobic amino acid stretches in their substrate binding groove. However, a detailed understanding of substrate specificity is still missing. Here, we use the endoplasmic reticulum-resident Hsp70 BiP to identify binding sites in a natural client protein. Two sites are mutually recognized and form stable Hsp70–substrate complexes. In silico and in vitro analyses revealed an extended substrate conformation as a crucial factor for interaction and show an unexpected plasticity of the substrate binding groove. The basic binding mechanism is conserved among different Hsp70s.

Classical Swine Fever Virus p7 Protein Is a Viroporin Involved in Virulence in Swine

The nonstructural protein p7 of classical swine fever virus (CSFV) is a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa. The protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that are predicted to form transmembrane helices and a cytosolic loop, respectively. Using reverse genetics, partial in-frame deletions of p7 were deleterious for virus growth, demonstrating that CSFV p7 function is critical for virus production in cell cultures. A panel of recombinant mutant CSFVs was created using alanine scanning mutagenesis of the p7 gene harboring sequential three- to six-amino-acid residue substitutions spanning the entire protein. These recombinant viruses allowed the identification of the regions within p7 that are critical for virus production in vitro. In vivo, some of these viruses were partially or completely attenuated in swine relative to the highly virulent parental CSFV Brescia strain, indicating a significant role of p7 in CSFV virulence. Structure-function analyses in model membranes emulating the endoplasmic reticulum lipid composition confirmed that CSFV p7 is a pore-forming protein, and that pore-forming activity resides in the C-terminal transmembrane helix. Therefore, p7 is a viroporin which is clearly involved in the process of CSFV virulence in swine.