Flavivirus Capsid Protein Research Articles

Identification of a critical role for ZIKV capsid α3 in virus assembly and its genetic interaction with M protein.

Flaviviruses such as Zika and dengue viruses are persistent health concerns in endemic regions worldwide. Efforts to combat the spread of flaviviruses have been challenging, as no antivirals or optimal vaccines are available. Prevention and treatment of flavivirus-induced diseases require a comprehensive understanding of their life cycle. However, several aspects of flavivirus biogenesis, including genome packaging and virion assembly, are not well characterized. In this study, we focused on flavivirus capsid protein (C) using Zika virus (ZIKV) as a model to investigate the role of the externally oriented α3 helix (C α3) without a known or predicted function. Alanine scanning mutagenesis of surface-exposed amino acids on C α3 revealed a critical CN67 residue essential for ZIKV virion production. The CN67A mutation did not affect dimerization or RNA binding of purified C protein in vitro. The virus assembly is severely affected in cells transfected with an infectious cDNA clone of ZIKV with CN67A mutation, resulting in a highly attenuated phenotype. We isolated a revertant virus with a partially restored phenotype by continuous passage of the CN67A mutant virus in Vero E6 cells. Sequence analysis of the revertant revealed a second site mutation in the viral membrane (M) protein MF37L, indicating a genetic interaction between the C and M proteins of ZIKV. Introducing the MF37L mutation on the mutant ZIKV CN67A generated a double-mutant virus phenotypically consistent with the isolated genetic revertant. Similar results were obtained with analogous mutations on C and M proteins of dengue virus, suggesting the critical nature of C α3 and possible C and M residues contributing to virus assembly in other Aedes-transmitted flaviviruses. This study provides the first experimental evidence of a genetic interaction between the C protein and the viral envelope protein M, providing a mechanistic understanding of the molecular interactions involved in the assembly and budding of Aedes-transmitted flaviviruses.

Tick-borne encephalitis virus capsid protein induces translational shutoff as revealed by its structural–biological analysis

Tick-borne encephalitis virus (TBEV) is the most medically relevant tick-transmitted Flavivirus in Eurasia, targeting the host central nervous system and frequently causing severe encephalitis. The primary function of its capsid protein (TBEVC) is to recruit the viral RNA and form a nucleocapsid. Additional functionality of Flavivirus capsid proteins has been documented, but further investigation is needed for TBEVC. Here, we show the first capsid protein 3D structure of a member of the tick-borne flaviviruses group. The structure of monomeric Δ16-TBEVC was determined using high-resolution multidimensional NMR spectroscopy. Based on natural invitro TBEVC homodimerization, the dimeric interfaces were identified by hydrogen deuterium exchange mass spectrometry (MS). Although the assembly of flaviviruses occurs in endoplasmic reticulum-derived vesicles, we observed that TBEVC protein also accumulated in the nuclei and nucleoli of infected cells. In addition, the predicted bipartite nuclear localization sequence in the TBEVC C-terminal part was confirmed experimentally, and we described the interface between TBEVC bipartite nuclear localization sequence and import adapter protein importin-alpha using X-ray crystallography. Furthermore, our coimmunoprecipitation coupled with MS identification revealed 214 interaction partners of TBEVC, including viral envelope and nonstructural NS5 proteins and a wide variety of host proteins involved mainly in rRNA processing and translation initiation. Metabolic labeling experiments further confirmed that TBEVC and other flaviviral capsid proteins are able to induce translational shutoff and decrease of 18S rRNA. These findings may substantially help to design a targeted therapy against TBEV.

Flavivirus Capsid Proteins Inhibit the Interferon Response.

Zika virus (ZIKV) establishes persistent infections in multiple human tissues, a phenomenon that likely plays a role in its ability to cause congenital birth defects and neurological disease. Multiple nonstructural proteins encoded by ZIKV, in particular NS5, are known to suppress the interferon (IFN) response by attacking different steps in this critical antiviral pathway. Less well known are the potential roles of structural proteins in affecting the host immune response during ZIKV infection. Capsid proteins of flaviviruses are of particular interest because a pool of these viral proteins is targeted to the nuclei during infection and, as such, they have the potential to affect host cell gene expression. In this study, RNA-seq analyses revealed that capsid proteins from six different flaviviruses suppress expression of type I IFN and IFN-stimulated genes. Subsequent interactome and in vitro ubiquitination assays showed that ZIKV capsid protein binds to and prevents activating ubiquitination of RIG-I CARD domains by TRIM25, a host factor that is important for the induction arm of the IFN response. The other flavivirus capsid proteins also interacted with TRIM25, suggesting that these viral proteins may attenuate antiviral signaling pathways at very early stages of infection, potentially even before nonstructural proteins are produced.

Functional Correlation between Subcellular Localizations of Japanese Encephalitis Virus Capsid Protein and Virus Production.

The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at α2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the α2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles.IMPORTANCE This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.

Structural and Functional Properties of the Capsid Protein of Dengue and Related Flavivirus.

Dengue, West Nile and Zika, closely related viruses of the Flaviviridae family, are an increasing global threat, due to the expansion of their mosquito vectors. They present a very similar viral particle with an outer lipid bilayer containing two viral proteins and, within it, the nucleocapsid core. This core is composed by the viral RNA complexed with multiple copies of the capsid protein, a crucial structural protein that mediates not only viral assembly, but also encapsidation, by interacting with host lipid systems. The capsid is a homodimeric protein that contains a disordered N-terminal region, an intermediate flexible fold section and a very stable conserved fold region. Since a better understanding of its structure can give light into its biological activity, here, first, we compared and analyzed relevant mosquito-borne Flavivirus capsid protein sequences and their predicted structures. Then, we studied the alternative conformations enabled by the N-terminal region. Finally, using dengue virus capsid protein as main model, we correlated the protein size, thermal stability and function with its structure/dynamics features. The findings suggest that the capsid protein interaction with host lipid systems leads to minor allosteric changes that may modulate the specific binding of the protein to the viral RNA. Such mechanism can be targeted in future drug development strategies, namely by using improved versions of pep14-23, a dengue virus capsid protein peptide inhibitor, previously developed by us. Such knowledge can yield promising advances against Zika, dengue and closely related Flavivirus.

Crystal Structure of the Japanese Encephalitis Virus Capsid Protein.

Japanese encephalitis (JE) is inflammation and swelling of the brain caused by the JE virus (JEV), a mosquito-borne member of the Flavivirus family. There are around 68,000 JE cases worldwide each year, many of which result in permanent brain damage and death. There is no specific treatment for JE. Here we present the crystal structure of the JEV capsid protein, a potential drug target, at 1.98 Å, and compare it to other flavivirus capsid proteins. The JEV capsid has a helical secondary structure (α helixes 1–4) and a similar protein fold to the dengue virus (DENV), the West Nile virus (WNV), and the Zika virus (ZIKV) capsid proteins. It forms a homodimer by antiparallel pairing with another subunit (‘) through α-helix 1-1’, 2-2’, and 4-4’ interactions. This dimeric form is believed to be the building block of the nucleocapsid. The flexibility of the N-terminal α helix-1 allows the formation of closed and open conformations with possible functional importance. The basic C-terminal pairing of α4-4’ forms a coiled-coil-like structure, indicating possible nucleic acid binding functionality. However, a comparison with other nucleic acid interacting domains indicates that homodimerization would preclude binding. This is the first JEV capsid protein to be described and is an addition to the structural biology of the Flavivirus.

Role of Capsid Anchor in the Morphogenesis of Zika Virus.

The flavivirus capsid protein (C) is separated from the downstream premembrane (PrM) protein by a hydrophobic sequence named capsid anchor (Ca). During polyprotein processing, Ca is sequentially cleaved by the viral NS2B/NS3 protease on the cytosolic side and by signal peptidase on the luminal side of the endoplasmic reticulum (ER). To date, Ca is considered important mostly for directing translocation of PrM into the ER lumen. In this study, the role of Ca in the assembly and secretion of Zika virus was investigated using a pseudovirus-based approach. Our results show that, while Ca-mediated anchoring of C to the ER membrane is not needed for the production of infective particles, Ca expression in cis with respect to PrM is strictly required to allow proper assembly of infectious particles. Finally, we show that the presence of heterologous, but not homologous, Ca induces degradation of E through the autophagy/lysosomal pathway.IMPORTANCE The capsid anchor (Ca) is a single-pass transmembrane domain at the C terminus of the capsid protein (C) known to function as a signal for the translocation of PrM into the ER lumen. The objective of this study was to further examine the role of Ca in Zika virus life cycle, whether involved in the formation of nucleocapsid through association with C or in the formation of viral envelope. In this study, we show that Ca has a function beyond the one of translocation signal, controlling protein E stability and therefore its availability for assembly of infectious particles.

Zika Virus Hijacks Stress Granule Proteins and Modulates the Host Stress Response.

Zika virus (ZIKV), a member of the Flaviviridae family, has recently emerged as an important human pathogen with increasing economic and health impact worldwide. Because of its teratogenic nature and association with the serious neurological condition Guillain-Barré syndrome, a tremendous amount of effort has focused on understanding ZIKV pathogenesis. To gain further insights into ZIKV interaction with host cells, we investigated how this pathogen affects stress response pathways. While ZIKV infection induces stress signaling that leads to phosphorylation of eIF2α and cellular translational arrest, stress granule (SG) formation was inhibited. Further analysis revealed that the viral proteins NS3 and NS4A are linked to translational repression, whereas expression of the capsid protein, NS3/NS2B-3, and NS4A interfered with SG formation. Some, but not all, flavivirus capsid proteins also blocked SG assembly, indicating differential interactions between flaviviruses and SG biogenesis pathways. Depletion of the SG components G3BP1, TIAR, and Caprin-1, but not TIA-1, reduced ZIKV replication. Both G3BP1 and Caprin-1 formed complexes with capsid, whereas viral genomic RNA stably interacted with G3BP1 during ZIKV infection. Taken together, these results are consistent with a scenario in which ZIKV uses multiple viral components to hijack key SG proteins to benefit viral replication.IMPORTANCE There is a pressing need to understand ZIKV pathogenesis in order to advance the development of vaccines and therapeutics. The cellular stress response constitutes one of the first lines of defense against viral infection; therefore, understanding how ZIKV evades this antiviral system will provide key insights into ZIKV biology and potentially pathogenesis. Here, we show that ZIKV induces the stress response through activation of the UPR (unfolded protein response) and PKR (protein kinase R), leading to host translational arrest, a process likely mediated by the viral proteins NS3 and NS4A. Despite the activation of translational shutoff, formation of SG is strongly inhibited by the virus. Specifically, ZIKV hijacks the core SG proteins G3BP1, TIAR, and Caprin-1 to facilitate viral replication, resulting in impaired SG assembly. This process is potentially facilitated by the interactions of the viral RNA with G3BP1 as well as the viral capsid protein with G3BP1 and Caprin-1. Interestingly, expression of capsid proteins from several other flaviviruses also inhibited SG formation. Taken together, the present study provides novel insights into how ZIKV modulates cellular stress response pathways during replication.

Flavivirus Infection Impairs Peroxisome Biogenesis and Early Antiviral Signaling.

Flaviviruses are significant human pathogens that have an enormous impact on the global health burden. Currently, there are very few vaccines against or therapeutic treatments for flaviviruses, and our understanding of how these viruses cause disease is limited. Evidence suggests that the capsid proteins of flaviviruses play critical nonstructural roles during infection, and therefore, elucidating how these viral proteins affect cellular signaling pathways could lead to novel targets for antiviral therapy. We used affinity purification to identify host cell proteins that interact with the capsid proteins of West Nile and dengue viruses. One of the cellular proteins that formed a stable complex with flavivirus capsid proteins is the peroxisome biogenesis factor Pex19. Intriguingly, flavivirus infection resulted in a significant loss of peroxisomes, an effect that may be due in part to capsid expression. We posited that capsid protein-mediated sequestration and/or degradation of Pex19 results in loss of peroxisomes, a situation that could result in reduced early antiviral signaling. In support of this hypothesis, we observed that induction of the lambda interferon mRNA in response to a viral RNA mimic was reduced by more than 80%. Together, our findings indicate that inhibition of peroxisome biogenesis may be a novel mechanism by which flaviviruses evade the innate immune system during early stages of infection. RNA viruses infect hundreds of millions of people each year, causing significant morbidity and mortality. Chief among these pathogens are the flaviviruses, which include dengue virus and West Nile virus. Despite their medical importance, there are very few prophylactic or therapeutic treatments for these viruses. Moreover, the manner in which they subvert the innate immune response in order to establish infection in mammalian cells is not well understood. Recently, peroxisomes were reported to function in early antiviral signaling, but very little is known regarding if or how pathogenic viruses affect these organelles. We report for the first time that flavivirus infection results in significant loss of peroxisomes in mammalian cells, which may indicate that targeting of peroxisomes is a key strategy used by viruses to subvert early antiviral defenses.

West Nile virus and dengue virus capsid protein negates the antiviral activity of human Sec3 protein through the proteasome pathway

Flavivirus capsid (C) protein is a key structural component of virus particles. The non-structural role of C protein in the pathogenesis of arthropod-borne flaviviruses is not clearly deciphered. This study showed that West Nile virus (WNV) and dengue virus (DENV) utilized C protein to reduce human Sec3p (hSec3p) levels at post-transcriptional level through activation of chymotrypsin-like proteolytic function of 20S proteasome. Mutagenesis studies confirmed amino acids 14, 109-114 of WNV C protein and 13, 102-107 of DENV C protein played an important role in activating the proteolytic function of 20S proteasome. Amino acid residues at 14 (WNV) and 13 (DENV) of C protein were important for C protein-hSec3p binding and physical interaction between C protein and hSec3p was essential to execute hSec3p degradation. Degradation motif required to degrade hSec3p resided between amino acid residues 109-114 of WNV C protein and 102-107 of DENV C protein. Proteasomes, hSec3p binding motif and degradation motif on C protein must be intact for efficient flavivirus production. Clinical isolates of DENV showed more pronounced effect in manipulating the proteasomes and reducing hSec3p levels. This study portrayed the non-structural function of C protein that helped the flavivirus to nullify the antiviral activity of hSec3p by accelerating its degradation and facilitating efficient binding of elongation factor 1α with flaviviral RNA genome.

The Dengue Virus Capsid Protein Inhibitor Peptide Pep14-23 becomes Alpha-Helical upon Binding to Negative Lipids

Dengue virus (DENV) causes a mosquito-borne disease affecting millions of people, which is spreading to temperate regions, including North America and Europe. Currently, there is no effective treatment for DENV infection. The interaction in the host liver of the viral capsid (C) protein with host intracellular lipid droplets (LDs) is crucial for virus formation [1], being previously studied in our labs [2, 3]. DENV C-LDs interaction involves a conserved segment of DENV C intrinsically disordered N-terminus [3], which led to the design of pep14-23, a novel peptide inhibitor of this critical interaction [3].Here, we used bioinformatics tools, combined with circular dichroism (CD) and zeta-potential analysis, to determine the structural parameters and the tendency of DENV C and pep14-23 to interact and bind lipid vesicles. Bioinformatics suggests that the Flavivirus capsid protein N-terminus region, roughly corresponding to pep14-23, has a high α-helical tendency and is likely to interact with lipid systems. CD shows a conversion of pep14-23 to α-helix conformation in the presence of negative phospholipids. Zeta-potential light scattering spectroscopy supports the CD data, showing that the peptide binds strongly to negative lipid vesicles.pep14-23 inhibition mechanism may therefore involve its binding to negative LDs phospholipids and the conversion to an α-helical peptide. This finding contributes to the design of pep14-23 based treatments of DENV and similar Flavivirus infections.

Limited cross-reactivity of mouse monoclonal antibodies against Dengue virus capsid protein among four serotypes

BackgroundDengue illness is one of the important mosquito-borne viral diseases in tropical and subtropical regions. Four serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4) are classified in the Flavivirus genus of the family Flaviviridae. We prepared monoclonal antibodies against DENV capsid protein from mice immunized with DENV-2 and determined the cross-reactivity with each serotype of DENV and Japanese encephalitis virus.Methods and resultsTo clarify the relationship between the cross-reactivity of monoclonal antibodies and the diversity of these viruses, we examined the situations of flaviviruses by analyses of phylogenetic trees. Among a total of 60 prepared monoclonal antibodies specific for DENV, five monoclonal antibodies stained the nuclei of infected cells and were found to be specific to the capsid protein. Three were specific to DENV-2, while the other two were cross-reactive with DENV-2 and DENV-4. No monoclonal antibodies were cross-reactive with all four serotypes. Phylogenetic analysis of DENV amino acid sequences of the capsid protein revealed that DENV-2 and DENV-4 were clustered in the same branch, while DENV-1 and DENV-3 were clustered in the other branch. However, these classifications of the capsid protein were different from those of the envelope and nonstructural 1 proteins. Phylogenetic distances between the four serotypes of DENV were as different as those of other flaviviruses, such as Japanese encephalitis virus and West Nile virus. Large variations in the DENV serotypes were comparable with the differences between species of flavivirus. Furthermore, the diversity of flavivirus capsid protein was much greater than that of envelope and nonstructural 1 proteins.ConclusionIn this study, we produced specific monoclonal antibodies that can be used to detect DENV-2 capsid protein, but not a cross-reactive one with all serotypes of DENV capsid protein. The high diversity of the DENV capsid protein sequence by phylogenetic analysis supported the low cross-reactivity of monoclonal antibodies against DENV capsid protein.

A novel antagonistic relationship between human Sec3 exocyst and flavivirus capsid protein

A novel antagonistic relationship between human Sec3 exocyst and flavivirus capsid protein

Human Sec3 protein is a novel transcriptional and translational repressor of flavivirus

The Flaviviridae family consists of several medically important pathogens such as West Nile virus (WNV) and Dengue virus (DENV). Flavivirus capsid (C) protein is a key structural component of virus particles. However, the role of C protein in the pathogenesis of arthropod-borne flaviviruses is poorly understood. To examine whether flavivirus C protein can associate with cellular proteins, and contribute to viral pathogenesis, WNV/DENV C protein was screened against a human brain/liver cDNA yeast two-hybrid library. This study identified human Sec3 exocyst protein (hSec3p) as a novel interacting partner of WNV and DENV C protein. Mutagenesis studies showed that the SH2 domain-binding motif of hSec3p binds to the first 15 amino acids of C protein. We report for the first time that hSec3p can modulate virus production by affecting viral RNA transcription and translation through the sequestration of elongation factor 1alpha (EF1alpha). This molecular discovery shed light on the protective role of hSec3p during flavivirus infection. This study also highlighted the antagonistic mechanism adopted by flavivirus C protein that can negatively regulate the formation of hSec3p-EF1alpha complex by sequestering hSec3p to establish successful infection.

Processing of Capsid Protein by Cathepsin L Plays a Crucial Role in Replication of Japanese Encephalitis Virus in Neural and Macrophage Cells

The flavivirus capsid protein not only is a component of nucleocapsids but also plays a role in viral replication. In this study, we found a small capsid protein in cells infected with Japanese encephalitis virus (JEV) but not in the viral particles. The small capsid protein was shown to be generated by processing with host cysteine protease cathepsin L. An in vitro cleavage assay revealed that cathepsin L cleaves the capsid protein between amino acid residues Lys(18) and Arg(19), which are well conserved among the mosquito-borne flaviviruses. A mutant JEV resistant to the cleavage of the capsid protein by cathepsin L was generated from an infectious cDNA clone of JEV by introducing a substitution in the cleavage site. The mutant JEV exhibited growth kinetics similar to those of the wild-type JEV in monkey (Vero), mosquito (C6/36), and porcine (PK15) cell lines, whereas replication of the mutant JEV in mouse macrophage (RAW264.7) and neuroblastoma (N18) cells was impaired. Furthermore, the neurovirulence and neuroinvasiveness of the mutant JEV to mice were lower than those of the wild-type JEV. These results suggest that the processing of the JEV capsid protein by cathepsin L plays a crucial role in the replication of JEV in neural and macrophage cells, which leads to the pathogenesis of JEV infection.

Functional requirements of the yellow fever virus capsid protein.

Although it is known that the flavivirus capsid protein is essential for genome packaging and formation of infectious particles, the minimal requirements of the dimeric capsid protein for virus assembly/disassembly have not been characterized. By use of a trans-packaging system that involved packaging a yellow fever virus (YFV) replicon into pseudo-infectious particles by supplying the YFV structural proteins using a Sindbis virus helper construct, the functional elements within the YFV capsid protein (YFC) were characterized. Various N- and C-terminal truncations, internal deletions, and point mutations of YFC were analyzed for their ability to package the YFV replicon. Consistent with previous reports on the tick-borne encephalitis virus capsid protein, YFC demonstrates remarkable functional flexibility. Nearly 40 residues of YFC could be removed from the N terminus while the ability to package replicon RNA was retained. Additionally, YFC containing a deletion of approximately 27 residues of the C terminus, including a complete deletion of C-terminal helix 4, was functional. Internal deletions encompassing the internal hydrophobic sequence in YFC were, in general, tolerated to a lesser extent. Site-directed mutagenesis of helix 4 residues predicted to be involved in intermonomeric interactions were also analyzed, and although single mutations did not affect packaging, a YFC with the double mutation of leucine 81 and valine 88 was nonfunctional. The effects of mutations in YFC on the viability of YFV infection were also analyzed, and these results were similar to those obtained using the replicon packaging system, thus underscoring the flexibility of YFC with respect to the requirements for its functioning.

Crystal Structure of the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Dimerization Domain Reveals Evolutionary Linkage between Corona- and Arteriviridae

The causative agent of severe acute respiratory syndrome (SARS) is the SARS-associated coronavirus, SARS-CoV. The nucleocapsid (N) protein plays an essential role in SARS-CoV genome packaging and virion assembly. We have previously shown that SARS-CoV N protein forms a dimer in solution through its C-terminal domain. In this study, the crystal structure of the dimerization domain, consisting of residues 270–370, is determined to 1.75Å resolution. The structure shows a dimer with extensive interactions between the two subunits, suggesting that the dimeric form of the N protein is the functional unit in vivo. Although lacking significant sequence similarity, the dimerization domain of SARS-CoV N protein has a fold similar to that of the nucleocapsid protein of the porcine reproductive and respiratory syndrome virus. This finding provides structural evidence of the evolutionary link between Coronaviridae and Arteriviridae, suggesting that the N proteins of both viruses have a common origin.

Mutagenesis of the NS2B-NS3-mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing.

Analysis of flavivirus polyprotein processing has revealed the presence of a substrate for the virus-encoded NS2B-NS3 protease at the carboxy-terminal end of the C (capsid or core) protein. Cleavage at this site has been implicated in the efficient generation of the amino terminus of prM via signal peptidase cleavage. Yellow fever virus has four basic residues (Arg-Lys-Arg-Arg) in the P1 through P4 positions of this cleavage site. Multiple alanine substitutions were made for these residues in order to investigate the substrate specificity and biological significance of this cleavage. Mutants were analyzed by several methods: (i) a cell-free trans processing assay for direct analysis of NS2B-NS3-mediated cleavage; (ii) a trans processing assay in BHK-21 cells, using a C-prM polyprotein, for analysis of prM production; (iii) an infectivity assay of full-length transcripts to determine plaque-forming ability; and (iv) analysis of proteins expressed from full-length transcripts to assess processing in the context of the complete genome. Mutants that exhibited severe defects in processing in vitro and in vivo were incapable of forming plaques. Mutants that contained two adjacent basic residues within the P1 through P4 region were processed more efficiently in vitro and in vivo, and transcripts bearing these mutations were fully infectious. Furthermore, two naturally occurring plaque-forming revertants were analyzed and shown to have restored protein processing phenotypes in vivo. Finally, the efficient production of prM was shown to be dependent on the proteolytic activity of NS3. These data support a model of two coordinated cleavages, one that generates the carboxy terminus of C and another that generates the amino terminus of prM. A block in the viral protease-mediated cleavage inhibits the production of prM by the signal peptidase, inhibits particle release, and eliminates plaque formation.

A Conserved Internal Hydrophobic Domain Mediates the Stable Membrane Integration of the Dengue Virus Capsid Protein

The mature flavivirus capsid protein (virion C) is commonly thought to be free in the cytoplasm of infected cells and to form a nucleocapsid-like complex with genomic RNA in mature virus particles. There is little sequence conservation among flavivirus virion C proteins, but they are similar in size (e.g., 99 amino acids [aa] for the dengue-4 [DEN4] C) and in bearing a net positive charge. In addition, we noted that C contained a conserved internal hydrophobic segment (spanning aa 45–65 in the DEN4 C). Results ofin vivoexpression andin vitrotranslation of wt and mutant forms of the DEN4 virion C demonstrated that the conserved internal hydrophobic segment in the DEN C functioned as a membrane anchor domain. Signal peptide function of this segment was also suggested by its requirement for the entry of C into membranes. Virion C was integrated in membranes in a “hairpin” conformation; positively charged segments amino- and carboxy-terminal to the hydrophobic signal-anchor segment were accessible to protease digestion in the “cytoplasm.” The net positive charge in the amino-terminal extramembraneous portion of C (aa 1–44) was one determinant of the hairpin membrane orientation; a conserved positively charged residue within the hydrophobic segment (Arg-54 in the DEN4 C) was not.

Formation of the flavivirus envelope: role of the viral NS2B-NS3 protease.

One of the late processing events in the flavivirus replication cycle involves cleavage of the intracellular form of the flavivirus capsid protein (Cint) to the mature virion form (Cvir) lacking the carboxy-terminal stretch of hydrophobic amino acids which serves as a signal peptide for the downstream prM protein. This cleavage event was hypothesized to be effected by a viral protease and to be associated with virion formation. We have proposed a model of flavivirus virion formation in which processing of the C-prM precursor at the upstream signalase site is upregulated by interaction of the NS2B part of the protease with the prM signal peptide or with an adjacent carboxy-terminal region of the capsid protein in the precursor, and processing of Cint by the NS2B-NS3 protease follows the signalase cleavage. Recently, an alternative hypothesis was proposed which suggests a reverse order of these two cleavage events, namely, that cleavage of the C-prM precursor by the NS2B-NS3 protease at the Cint-->Cvir dibasic cleavage site is a prerequisite for the subsequent signalase cleavage of the prM signal peptide. To distinguish between these alternative models, we prepared a series of expression cassettes carrying mutations at the Cint-->Cvir dibasic cleavage site and investigated the effects of these mutations on signalase processing of C-prM and on formation and secretion of prM-E heterodimers. For certain mutated C-prM precursors, namely, for those with Lys-->Gly disruption of the dibasic site, efficient formation of prM was observed upon expression from larger cassettes encoding the viral protease, despite the absence of processing at the Cint-->Cvir cleavage site. Surprisingly, formation and secretion of prM-E heterodimers accompanied by late cleavage of prM was also observed for these cassettes, with an efficiency comparable to that of the wild-type expression cassette. These observations contradict the model in which cleavage of the C-prM precursor at the Cint-->Cvir dibasic site is a prerequisite for signalase cleavage.