RESUMO
Classical swine fever virus (CSFV) Core protein is involved in virus RNA protection, transcription regulation and virus virulence. To discover additional Core protein functions a yeast two-hybrid system was used to identify host proteins that interact with Core. Among the identified host proteins, the osteosarcoma amplified 9 protein (OS9) was further studied. Using alanine scanning mutagenesis, the OS9 binding site in the CSFV Core protein was identified, between Core residues (90)IAIM(93), near a putative cleavage site. Truncated versions of Core were used to show that OS9 binds a polypeptide representing the 12 C-terminal Core residues. Cells transfected with a double-fluorescent labeled Core construct demonstrated that co-localization of OS9 and Core occurred only on unprocessed forms of Core protein. A recombinant CSFV containing Core protein where residues (90)IAIM(93) were substituted by alanines showed no altered virulence in swine, but a significant decreased ability to replicate in cell cultures.
Assuntos
Vírus da Febre Suína Clássica/metabolismo , Peste Suína Clássica/metabolismo , Degradação Associada com o Retículo Endoplasmático , Proteínas de Neoplasias/metabolismo , Proteínas do Core Viral/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Peste Suína Clássica/genética , Peste Suína Clássica/virologia , Vírus da Febre Suína Clássica/química , Vírus da Febre Suína Clássica/genética , Vírus da Febre Suína Clássica/patogenicidade , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Ligação Proteica , Suínos , Técnicas do Sistema de Duplo-Híbrido , Proteínas do Core Viral/química , Proteínas do Core Viral/genética , VirulênciaRESUMO
E2, along with E(rns) and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, (818)CPIGWTGVIEC(828), containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a ß-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP (818)CPIGWTGVIEC(828) indicates a membrane fusion activity and a critical role in virus replication.
Assuntos
Vírus da Febre Suína Clássica/fisiologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Substituição de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Vírus da Febre Suína Clássica/genética , Lipossomos/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica , Genética Reversa , Suínos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Here we show that IQGAP1, a cellular protein that plays a pivotal role as a regulator of the cytoskeleton interacts with Classical Swine Fever Virus (CSFV) Core protein. Sequence analyses identified residues within CSFV Core protein (designated as areas I, II, III and IV) that maintain homology to regions within the matrix protein of Moloney Murine Leukemia Virus (MMLV) that mediate binding to IQGAP1 [EMBO J, 2006 25:2155]. Alanine-substitution within Core regions I, II, III and IV identified residues that specifically mediate the Core-IQGAP1 interaction. Recombinant CSFV viruses harboring alanine substitutions at residues (207)ATI(209) (I), (210)VVE(212) (II), (213)GVK(215) (III), or (232)GLYHN(236) (IV) have defective growth in primary swine macrophage cultures. In vivo, substitutions of residues in areas I and III yielded viruses that were completely attenuated in swine. These data shows that the interaction of Core with an integral component of cytoskeletal regulation plays a role in the CSFV cycle.
Assuntos
Vírus da Febre Suína Clássica/patogenicidade , Interações Hospedeiro-Patógeno , Mapeamento de Interação de Proteínas , Proteínas do Core Viral/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Células Cultivadas , Peste Suína Clássica/patologia , Peste Suína Clássica/virologia , Vírus da Febre Suína Clássica/genética , Macrófagos/virologia , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/genética , Mutagênese Sítio-Dirigida , Ligação Proteica , Homologia de Sequência de Aminoácidos , Suínos , Proteínas do Core Viral/genética , VirulênciaRESUMO
Here we have identified host cell proteins involved with the cellular SUMOylation pathway, SUMO-1 (small ubiquitin-like modifier) and UBC9, a SUMO-1 conjugating enzyme that interact with classical swine fever virus (CSFV) Core protein. Five highly conserved lysine residues (K179, K180, K220, K221, and K246) within the CSFV Core were identified as putative SUMOylation sites. Analysis of these interactions showed that K179A, K180A, and K221A substitutions disrupt Core-SUMO-1 binding, while K220A substitution precludes Core-UBC9 binding. In vivo, Core mutant viruses (K179A, K180A, K220A, K221A) and (K220A, K221A) harboring those substitutions were attenuated in swine. These data shows a clear correlation between the disruption of Core protein binding to SUMO-1 and UBC9 and CSFV attenuation. Overall, these data suggest that the interaction of Core with the cellular SUMOylation pathway plays a significant role in the CSFV growth cycle in vivo.
