Ivo C. Lorenz

Folding and Dimerization of Tick-Borne Encephalitis Virus Envelope Proteins prM and E in the Endoplasmic Reticulum

ABSTRACT Flavivirus envelope proteins are synthesized as part of large polyproteins that are co- and posttranslationally cleaved into their individual chains. To investigate whether the interaction of neighboring proteins within the precursor protein is required to ensure proper maturation of the individual components, we have analyzed the folding of the flavivirus tick-borne encephalitis (TBE) virus envelope glycoproteins prM and E by using a recombinant plasmid expression system and virus-infected cells. When expressed in their polyprotein context, prM and E achieved their native folded structures with half-times of approximately 4 min for prM and about 15 min for E. They formed heterodimeric complexes within a few minutes after synthesis that were required for the final folding of E but not for that of prM. Heterodimers could also be formed in trans when these proteins were coexpressed from separate constructs. When expressed without prM, E could form disulfide bonds but did not express a specific conformational epitope and remained sensitive to reduction by dithiothreitol. This is consistent with a chaperone-like role for prM in the folding of E. PrM was able to achieve its native folded structure without coexpression of E, but signal sequence cleavage at the N terminus was delayed. Our results show that prM is an especially rapidly folding viral glycoprotein, that polyprotein cleavage and folding of the TBE virus envelope proteins occurs in a coordinated sequence of processing steps, and that proper and efficient maturation of prM and E can only be achieved by cosynthesis of these two proteins.

Structure of the catalytic domain of the hepatitis C virus NS2-3 protease.

Hepatitis C virus is a major global health problem affecting an estimated 170 million people worldwide. Chronic infection is common and can lead to cirrhosis and liver cancer. There is no vaccine available and current therapies have met with limited success. The viral RNA genome encodes a polyprotein that includes two proteases essential for virus replication. The NS2-3 protease mediates a single cleavage at the NS2/NS3 junction, whereas the NS3-4A protease cleaves at four downstream sites in the polyprotein. NS3-4A is characterized as a serine protease with a chymotrypsin-like fold, but the enzymatic mechanism of the NS2-3 protease remains unresolved. Here we report the crystal structure of the catalytic domain of the NS2-3 protease at 2.3 Å resolution. The structure reveals a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer, and the nucleophilic cysteine by the other. The carboxy-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. Proteolysis through formation of a composite active site occurs in the context of the viral polyprotein expressed in mammalian cells. These features offer unexpected insights into polyprotein processing by hepatitis C virus and new opportunities for antiviral drug design.

Hepatitis C Virus NS2 Protein Contributes to Virus Particle Assembly via Opposing Epistatic Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes

ABSTRACT The hepatitis C virus NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-NS4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus assembly.

Intracellular Assembly and Secretion of Recombinant Subviral Particles from Tick-Borne Encephalitis Virus

ABSTRACT It is believed that flavivirus assembly occurs by intracellular budding of the nucleocapsid into the lumen of the endoplasmic reticulum (ER). Recombinant expression of tick-borne encephalitis (TBE) virus envelope proteins prM and E in mammalian cells leads to their incorporation into enveloped recombinant subviral particles (RSPs), which have been used as a model system for studying assembly and entry processes and are also promising vaccine candidates. In this study, we analyzed the formation and secretion of TBE virus RSPs and of a membrane anchor-free E homodimer in mammalian cells. Immunofluorescence microscopy showed that E was accumulated in the lumen of the ER. RSPs were observed by electron microscopy in the rough and smooth ER and in downstream compartments of the secretory pathway. About 75% of the particles appeared to be of the size expected for RSPs (about 30 nm in diameter), but a number of larger particles and tubular structures were also observed in these compartments. Secretion of membrane anchor-free E dimers was detected 30 min after synthesis of prM and E, and secretion of RSPs was detected 1 h after synthesis of prM and E. We also found that the presence of the single N-linked oligosaccharide side chain on the E protein and its trimming by glucosidases was necessary for secretion of RSPs and truncated E dimers. Our results suggest that incorporation of prM and E into RSPs occurs at the ER membrane without other viral elements being required, followed by rapid transport along the compartments of the secretory pathway and secretion. Moreover, the carbohydrate side chain of E is involved in at least one assembly or transport step.

MEF2B mutations lead to deregulated expression of the oncogene BCL6 in diffuse large B cell lymphoma.

MEF2B encodes a transcriptional activator and is mutated in ∼11% of diffuse large B cell lymphomas (DLBCLs) and ∼12% of follicular lymphomas (FLs). Here we found that MEF2B directly activated the transcription of the proto-oncogene BCL6 in normal germinal-center (GC) B cells and was required for DLBCL proliferation. Mutation of MEF2B resulted in enhanced transcriptional activity of MEF2B either through disruption of its interaction with the corepressor CABIN1 or by rendering it insensitive to inhibitory signaling events mediated by phosphorylation and sumoylation. Consequently, the transcriptional activity of Bcl-6 was deregulated in DLBCLs with MEF2B mutations. Thus, somatic mutations of MEF2B may contribute to lymphomagenesis by deregulating BCL6 expression, and MEF2B may represent an alternative target for blocking Bcl-6 activity in DLBCLs.

Characterization of Determinants Important for Hepatitis C Virus p7 Function in Morphogenesis by Using trans-Complementation

ABSTRACT Hepatitis C virus (HCV) p7 is an integral membrane protein that forms ion channels in vitro and that is crucial for the efficient assembly and release of infectious virions. Due to these properties, p7 was included in the family of viroporins that comprises proteins like influenza A virus M2 and human immunodeficiency virus type 1 (HIV-1) vpu, which alter membrane permeability and facilitate the release of infectious viruses. p7 from different HCV isolates sustains virus production with variable efficiency. Moreover, p7 determinants modulate processing at the E2/p7 and the p7/NS2 signal peptidase cleavage sites, and E2/p7 cleavage is incomplete. Consequently, it was unclear if a differential ability to sustain virus production was due to variable ion channel activity or due to alternate processing at these sites. Therefore, we developed a trans-complementation assay permitting the analysis of p7 outside of the HCV polyprotein and thus independently of processing. The rescue of p7-defective HCV genomes was accomplished by providing E2, p7, and NS2, or, in some cases, by p7 alone both in a transient complementation assay as well as in stable cell lines. In contrast, neither influenza A virus M2 nor HIV-1 vpu compensated for defective p7 in HCV morphogenesis. Thus, p7 is absolutely essential for the production of infectious HCV particles. Moreover, our data indicate that p7 can operate independently of an upstream signal sequence, and that a tyrosine residue close to the conserved dibasic motif of p7 is important for optimal virus production in the context of genotype 2a viruses. The experimental system described here should be helpful to investigate further key determinants of p7 that are essential for its structure and function in the absence of secondary effects caused by altered polyprotein processing.

Determinants of the Hepatitis C Virus Nonstructural Protein 2 Protease Domain Required for Production of Infectious Virus

ABSTRACT The hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a dimeric multifunctional hydrophobic protein with an essential but poorly understood role in infectious virus production. We investigated the determinants of NS2 function in the HCV life cycle. On the basis of the crystal structure of the postcleavage form of the NS2 protease domain, we mutated conserved features and analyzed the effects of these changes on polyprotein processing, replication, and infectious virus production. We found that mutations around the protease active site inhibit viral RNA replication, likely by preventing NS2-3 cleavage. In contrast, alterations at the dimer interface or in the C-terminal region did not affect replication, NS2 stability, or NS2 protease activity but decreased infectious virus production. A comprehensive deletion and mutagenesis analysis of the C-terminal end of NS2 revealed the importance of its C-terminal leucine residue in infectious particle production. The crystal structure of the NS2 protease domain shows that this C-terminal leucine is locked in the active site, and mutation or deletion of this residue could therefore alter the conformation of NS2 and disrupt potential protein-protein interactions important for infectious particle production. These studies begin to dissect the residues of NS2 involved in its multiple essential roles in the HCV life cycle and suggest NS2 as a viable target for HCV-specific inhibitors.

Mitotic Raf-1 Is Stimulated Independently of Ras and Is Active in the Cytoplasm

Raf-1 is a Ser/Thr protein kinase that is involved in regulation of proliferation, differentiation, and apoptosis. Recently, we and others showed that Raf-1 is not only activated in mitogenic pathways leading to cell cycle entry but also during mitosis. Transient expression studies in COS cells now demonstrate that, in contrast to growth factor-dependent activation of Raf-1, mitotic activation of Raf-1 is Ras-independent. Dominant negative RasS17N does not interfere with mitotic activation of Raf-1, whereas epidermal growth factor-dependent stimulation of Raf-1 is inhibited. In addition, the Raf-1 mutant RafR89L, which cannot bind to activated Ras, is still stimulated in mitotic cells. Mitotic activation of Raf-1 seems to be partially dependent on tyrosine phosphorylation since the kinase activity of the Raf mutant RafYY340/341FF, which can no longer be activated by Src, is reduced in mitotic cells. Surprisingly, cell fractionation experiments showed that mitotic-activated Raf-1 is predominantly located in the cytoplasm in contrast to the mitogen-activated Raf-1 that is bound to the plasma membrane. In addition, mitotic activation of Raf-1 does not lead to stimulation of the mitogen-activated protein kinase kinase (MAPKK or MEK) and the extracellular signal-regulated protein kinase (ERK). These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ERK pathway. These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ERK pathway.

Neutralizing antibodies are unable to inhibit direct viral cell-to-cell spread of human cytomegalovirus

Infection with human cytomegalovirus (CMV) during pregnancy is the most common cause of congenital disorders, and can lead to severe life-long disabilities with associated high cost of care. Since there is no vaccine or effective treatment, current efforts are focused on identifying potent neutralizing antibodies. A panel of CMV monoclonal antibodies identified from patent applications, was synthesized and expressed in order to reproduce data from the literature showing that anti-glycoprotein B antibodies neutralized virus entry into all cell types and that anti-pentameric complex antibodies are highly potent in preventing virus entry into epithelial cells. It had not been established whether antibodies could prevent subsequent rounds of infection that are mediated primarily by direct cell-to-cell transmission. A thorough validation of a plaque reduction assay to monitor cell-to-cell spread led to the conclusion that neutralizing antibodies do not significantly inhibit plaque formation or reduce plaque size when they are added post-infection.

The Hepatitis C Virus Nonstructural Protein 2 (NS2): An Up-and-Coming Antiviral Drug Target.

Infection with Hepatitis C Virus (HCV) continues to be a major global health problem. To overcome the limitations of current therapies using interferon-α in combination with ribavirin, there is a need to develop drugs that specifically block viral proteins. Highly efficient protease and polymerase inhibitors are currently undergoing clinical testing and will become available in the next few years. However, with resistance mutations emerging quickly, additional enzymatic activities or functions of HCV have to be targeted by novel compounds. One candidate molecule is the nonstructural protein 2 (NS2), which contains a proteolytic activity that is essential for viral RNA replication. In addition, NS2 is crucial for the assembly of progeny virions and modulates various cellular processes that interfere with viral replication. This review describes the functions of NS2 in the life cycle of HCV and highlights potential antiviral strategies involving NS2.