Characterization of the Determinants of NS2-3-Independent Virion Morphogenesis of Pestiviruses

Klemens, O.; Dubrau, D.; Tautz, Norbert

doi:10.1128/jvi.01646-15

Cited by 17 publications

(33 citation statements)

References 57 publications

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“…Previous studies showed that polyprotein processing at the NS3/4A site is incomplete in cells infected with YFV or with other flaviviruses. (27,46,47). We also observed that NS3-4A was quickly generated from the sig2A-5 polyprotein, remained stable over time, and was not a precursor to NS3 (Fig.…”

Section: Discussionmentioning

confidence: 55%

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Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

Bozzacco

Andréo

et al. 2016

J Virol

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DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. IMPORTANCEFlaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this report we show that overexpression of the ER-resident cochaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals that DNAJC14 modulation of NS3/4A site processing is an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies to address similarities and/or differences among flaviviruses and to interrogate the precise mechanisms employed for polyprotein processing, a critical step that can ultimately be targeted for novel drug development.

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Section: Discussionmentioning

confidence: 55%

“…1A, 2B, and 3A), along with the cleaved products NS3 and NS4A, suggests an important role for NS3-4A, although its function in the virus life cycle is unknown (27,46,47). Our work demonstrates that cleavage at the NS3/4A junction is a critical step in YFV replication and determines YFV sensitivity to DNAJC14 overexpression.…”

Section: Resultsmentioning

confidence: 77%

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

Bozzacco

Andréo

et al. 2016

J Virol

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“…This virus, for which no infectious cDNA has been established so far, contains, in retrospection, all mutations required for virion morphogenesis in the absence of uncleaved NS2-3. The mutations essential and sufficient for this phenotype could be narrowed down in the context of strain NCP7 to one amino acid exchange in NS2 (E440-V, named 2/EV) and one in NS3 (V132-A, named 3/VA) (23). While these two mutations allowed for the efficient NS2-3-independent virion formation of a monocistronic BVDV encoding a ubiquitin gene between NS2 and NS3, a bicistronic BVDV genome with an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between the genes coding for NS2 and NS3 depends on additional mutations in E2, NS2, and NS5B for efficient packaging (23).…”

mentioning

confidence: 99%

“…The mutations essential and sufficient for this phenotype could be narrowed down in the context of strain NCP7 to one amino acid exchange in NS2 (E440-V, named 2/EV) and one in NS3 (V132-A, named 3/VA) (23). While these two mutations allowed for the efficient NS2-3-independent virion formation of a monocistronic BVDV encoding a ubiquitin gene between NS2 and NS3, a bicistronic BVDV genome with an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between the genes coding for NS2 and NS3 depends on additional mutations in E2, NS2, and NS5B for efficient packaging (23). Further structural and biochemical characterization identified NS3 position 132 to be a crucial determinant in the coordination of the NS3 surface interaction with the NS4A kink region (14).…”

mentioning

confidence: 99%

“…The same applies to the accessory mutations, which are additionally required for high-titer virion formation when an EMCV IRES instead of a ubiquitin-coding sequence is inserted between the NS2 and NS3 genes. For BVDV-1, these accessory mutations comprise the insertion of a lysine in E2 downstream of amino acid 214, the exchange T269-A in NS2, and the mutation E145-G in NS5B (23).…”

mentioning

confidence: 99%

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Determination of Critical Requirements for Classical Swine Fever Virus NS2-3-Independent Virion Formation

Dubrau¹,

Schwindt²,

Klemens³

et al. 2019

J Virol

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For members of the Flaviviridae, it is known that, besides the structural proteins, nonstructural (NS) proteins also play a critical role in virion formation. Pestiviruses, such as bovine viral diarrhea virus (BVDV), rely on uncleaved NS2-3 for virion formation, while its cleavage product, NS3, is selectively active in RNA replication. This dogma was recently challenged by the selection of gain-of-function mutations in NS2 and NS3 which allowed virion formation in the absence of uncleaved NS2-3 in BVDV type 1 (BVDV-1) variants encoding either a ubiquitin (Ubi) (NS2-Ubi-NS3) or an internal ribosome entry site (IRES) (NS2-IRES-NS3) between NS2 and NS3. To determine whether the ability to adapt to NS2-3-independent virion morphogenesis is conserved among pestiviruses, we studied the corresponding NS2 and NS3 mutations (2/T444-V and 3/M132-A) in classical swine fever virus (CSFV). We observed that these mutations were capable of restoring low-level NS2-3-independent virion formation only for CSFV NS2-Ubi-NS3. Interestingly, a second NS2 mutation (V439-D), identified by selection, was essential for high-titer virion production. Similar to previous findings for BVDV-1, these mutations in NS2 and NS3 allowed for low-titer virion production only in CSFV NS2-IRES-NS3. For efficient virion morphogenesis, additional exchanges in NS4A (A48-T) and NS5B (D280-G) were required, indicating that these proteins cooperate in NS2-3-independent virion formation. Interestingly, both NS5B mutations, selected independently for NS2-IRES-NS3 variants of BVDV-1 and CSFV, are located in the fingertip region of the viral RNA-dependent RNA polymerase, classifying this structural element as a novel determinant for pestiviral NS2-3-independent virion formation. Together, these findings will stimulate further mechanistic studies on the genome packaging of pestiviruses. IMPORTANCE For Flaviviridae members, the nonstructural proteins are essential for virion formation and thus exert a dual role in RNA replication and virion morphogenesis. However, it remains unclear how these proteins are functionalized for either process. In wild-type pestiviruses, the NS3/4A complex is selectively active in RNA replication, while NS2-3/4A is essential for virion formation. Mutations recently identified in BVDV-1 rendered NS3/4A capable of supporting NS2-3-independent virion morphogenesis. A comparison of NS3/4A complexes incapable/capable of supporting virion morphogenesis revealed that changes in NS3/NS4A surface interactions are decisive for the gain of function. However, so far, the role of the NS2 mutations as well as the accessory mutations additionally required in the NS2-IRES-NS3 virus variant has not been clarified. To unravel the course of genome packaging, the additional sets of mutations obtained for a second pestivirus species (CSFV) are of significant importance to develop mechanistic models for this complex process.

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Non-structural proteins of bovine viral diarrhea virus

et al. 2022

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Bovine viral diarrhea virus (BVDV) belongs to the family Flaviviridae genus pestivirus. The viral genome is a single-stranded, positive-sense RNA that encodes four structural proteins (i.e., C, Erns, E1, and E2) and eight non-structural proteins (NSPs) (i.e., Npro, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Cattle infected with BVDV exhibit a number of different clinical signs including diarrhea, abortion, and other reproductive disorders which have a serious impact on the cattle industry worldwide. Research on BVDV mainly focuses on its structural protein, however, progress in understanding the functions of the NSPs of BVDV has also been made in recent decades. The knowledge gained on the BVDV non-structural proteins is helpful to more fully understand the viral replication process and the molecular mechanism of viral persistent infection. This review focuses on the functions of BVDV NSPs and provides references for the identification of BVDV, the diagnosis and prevention of Bovine viral diarrhea mucosal disease (BVD-MD), and the development of vaccines.

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Characterization of the Determinants of NS2-3-Independent Virion Morphogenesis of Pestiviruses

Cited by 17 publications

References 57 publications

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

Determination of Critical Requirements for Classical Swine Fever Virus NS2-3-Independent Virion Formation

Non-structural proteins of bovine viral diarrhea virus

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