RESUMO
The protein aggregation is one of the major challenges of the biotechnological industry, especially in the areas of development and commercialization of successful protein-based drug products. The inherent high aggregation tendency of proteins during various manufacturing processes, storage, and administration has significant impact upon the product quality, safety and efficacy. We have developed an interesting protein purification approach that separates the functionally active protein from inactive aggregates using a detergent concentration gradient. The C-terminally His tagged nucleocapsid protein of Crimean Congo Hemorrhagic fever virus (CCHFV) has high aggregation tendency and rapidly precipitates upon purification by NiNTA chromatography. Using the new purification approach reported here, the freshly purified protein by NiNTA chromatography was further processed using a detergent gradient. In this new purification approach the active protein is retained in the low detergent concentration zone while the inactive aggregates are promptly removed by their rapid migration to the high detergent concentration zone. The method prevented further aggregation and retained the RNA binding activity in the native protein despite numerous freeze thaw cycles. This simple approach prevents protein aggregation by rapidly separating the preformed early aggregates and creating the appropriate microenvironment for correctly folded proteins to retain their biological activity. It will be of potential importance to the biotechnological industry and other fields of protein biochemistry that routinely face the challenges of protein aggregation.
Assuntos
Detergentes/química , Proteínas do Nucleocapsídeo/isolamento & purificação , Agregados Proteicos/fisiologia , Biotecnologia , Cromatografia de Afinidade/métodos , Técnicas Genéticas , Vírus da Febre Hemorrágica da Crimeia-Congo/metabolismo , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Dobramento de Proteína , Proteínas/química , Proteínas/isolamento & purificação , RNARESUMO
Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus that causes severe hemorrhagic fever with a mortality rate of up to 30% in certain outbreaks worldwide. The virus has wide endemic distribution. There is no effective antiviral therapeutic or FDA approved vaccine for this zoonotic viral illness. The multifunctional CCHFV nucleocapsid protein (N protein) plays a crucial role in the establishment of viral infection and is an important structural component of the virion. Here we show that CCHFV N protein has a distant RNA-binding site in the stalk domain that specifically recognizes the vRNA panhandle, formed by the base pairing of complementary nucleotides at the 5' and 3' termini of the vRNA genome. Using multiple approaches, including filter-bonding analysis, GFP reporter assay, and biolayer interferometry we observed an N protein-panhandle interaction both in vitro and in vivo The purified WT CCHFV N protein and the stalk domain also recognize the vRNA panhandle of hazara virus, another Nairovirus in the family Bunyaviridae, demonstrating the genus-specific nature of N protein-panhandle interaction. Another RNA-binding site was identified at the head domain of CCHFV N protein that nonspecifically recognizes the single strand RNA (ssRNA) of viral or nonviral origin. Expression of CCHFV N protein stalk domain active in panhandle binding, dramatically inhibited the hazara virus replication in cell culture, illustrating the role of N protein-panhandle interaction in Nairovirus replication. Our findings reveal the stalk domain of N protein as a potential target in therapeutic interventions to manage CCHFV disease.
Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo/fisiologia , Febre Hemorrágica da Crimeia/virologia , Proteínas do Nucleocapsídeo/metabolismo , RNA/metabolismo , Sítios de Ligação , Vírus da Febre Hemorrágica da Crimeia-Congo/química , Febre Hemorrágica da Crimeia/metabolismo , Humanos , Modelos Moleculares , Nairovirus/química , Nairovirus/fisiologia , Proteínas do Nucleocapsídeo/química , Domínios Proteicos , Replicação ViralRESUMO
Crimean Congo hemorrhagic fever, a zoonotic viral disease, has high mortality rate in humans. There is currently no vaccine for Crimean Congo hemorrhagic fever virus (CCHFV) and chemical interventions are limited. The three negative sense genomic RNA segments of CCHFV are specifically encapsidated by the nucleocapsid protein into three ribonucleocapsids, which serve as templates for the viral RNA dependent RNA polymerase. Here we demonstrate that CCHFV nucleocapsid protein has two distinct binding modes for double and single strand RNA. In the double strand RNA binding mode, the nucleocapsid protein preferentially binds to the vRNA panhandle formed by the base pairing of complementary nucleotides at the 5' and 3' termini of viral genome. The CCHFV nucleocapsid protein does not have RNA helix unwinding activity and hence does not melt the duplex vRNA panhandle after binding. In the single strand RNA binding mode, the nucleocapsid protein does not discriminate between viral and non-viral RNA molecules. Binding of both vRNA panhandle and single strand RNA induce a conformational change in the nucleocapsid protein. Nucleocapsid protein remains in a unique conformational state due to simultaneously binding of structurally distinct vRNA panhandle and single strand RNA substrates. Although the role of dual RNA binding modes in the virus replication cycle is unknown, their involvement in the packaging of viral genome and regulation of CCHFV replication in conjunction with RdRp and host derived RNA regulators is highly likely.
Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo , Conformação de Ácido Nucleico , Proteínas do Nucleocapsídeo , RNA Viral , Proteínas de Ligação a RNA , Vírus da Febre Hemorrágica da Crimeia-Congo/química , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/metabolismo , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Ligação Proteica , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Hantaviruses are zoonotic category-A pathogens that cause highly fatal diseases in humans. The hantaviral genome encodes three viral proteins: RNA-dependent RNA polymerase (RdRp or L protein), nucleocapsid protein (N), and a glycoprotein precursor (GPC), which is post-translationally cleaved into two surface glycoproteins Gn and Gc. The cytoplasmic tail of Gn interferes with interferon signaling pathways. N is a multifunctional molecule that was shown to be involved in the transcription and translation of viral proteins. N binds to the host mRNA caps and protects the degradation of mRNA 5' termini, which are later snatched and used as primers by the viral RdRp during transcription initiation. N also seems to lure the host translation machinery for the preferential translation of viral transcripts. Moreover, N was shown to delay the induction of cellular apoptosis and facilitate the transport and localization of viral ribonucleoproteins (RNPs) by exploiting the cellular cytoskeleton and SUMOlyation machinery. Therefore, with their limited protein coding capacity, hantaviruses have evolved several strategies to modulate cellular pathways for their efficient replication.