Maturation of hepatitis C virus core protein by signal peptide peptidase is required for virus production

Complete maturation of hepatitis C virus (HCV) core protein requires coordinate cleavage by signal peptidase and an intramembrane protease, signal peptide peptidase. We show that reducing the intracellular levels of signal peptide peptidase lowers the titer of infectious virus released from cells, indicating that it plays an important role in virus production. Proteolysis by the enzyme at a signal peptide between core and the E1 glycoprotein is needed to permit targeting of core to lipid droplets. From mutagenesis studies, introducing mutations into the core-E1 signal peptide delayed the appearance of signal peptide peptidase-processed core until between 48 and 72 h after the beginning of the infectious cycle. Accumulation of mature core at these times coincided with its localization to lipid droplets and a rise in titer of infectious HCV. Therefore, processing of core by signal peptide peptidase is a critical event in the virus life cycle. To study the stage in virus production that may be blocked by interfering with intramembrane cleavage of core, we examined the distribution of viral RNA in cells harboring the core-E1 signal peptide mutant. Results revealed that colocalization of core with HCV RNA required processing of the protein by signal peptide peptidase. Our findings provide new insights into the sequence requirements for proteolysis by signal peptide peptidase. Moreover, they offer compelling evidence for a function for an intramembrane protease to facilitate the association of core with viral genomes, thereby creating putative sites for assembly of nascent virus particles.     

Signal peptide peptidase-catalyzed cleavage of hepatitis C virus core protein is dispensable for virus budding but destabilizes the viral capsid

The capsid of hepatitis C virus (HCV) particles is considered to be composed of the mature form (p21) of core protein. Maturation to p21 involves cleavage of the transmembrane domain of the precursor form (p23) of core protein by signal peptide peptidase (SPP), a cellular protease embedded in the endoplasmic reticulum membrane. Here we have addressed whether SPP-catalyzed maturation to p21 is a prerequisite for HCV particle morphogenesis in the endoplasmic reticulum. HCV structural proteins were expressed by using recombinant Semliki Forest virus replicon in mammalian cells or recombinant baculovirus in insect cells, because these systems have been shown to allow the visualization of HCV budding events and the isolation of HCV-like particles, respectively. Inhibition of SPP-catalyzed cleavage of core protein by either an SPP inhibitor or HCV core mutations not only did not prevent but instead tended to facilitate the observation of viral buds and the recovery of virus-like particles. Remarkably, although maturation to p21 was only partially inhibited by mutations in insect cells, p23 was the only form of core protein found in HCV-like particles. Finally, newly developed assays demonstrated that p23 capsids are more stable than p21 capsids. These results show that SPP-catalyzed cleavage of core protein is dispensable for HCV budding but decreases the stability of the viral capsid. We propose a model in which p23 is the form of HCV core protein committed to virus assembly, and cleavage by SPP occurs during and/or after virus budding to predispose the capsid to subsequent disassembly in a new cell.     

Characterization of the cleavage of signal peptide at the C-terminus of hepatitis C virus core protein by signal peptide peptidase

Production of hepatitis C virus (HCV) core protein requires the cleavages of polyprotein by signal peptidase and signal peptide peptidase (SPP). Cleavage of signal peptide at the C-terminus of HCV core protein by SPP was characterized in this study. The spko mutant (mutate a.a. 189–193 from ASAYQ to PPFPF) is more efficient than the A/F mutant (mutate a.a 189 and 191 from A to F) in blocking the cleavage of signal peptide by signal peptidase. The cleavage efficiency of SPP is inversely proportional to the length of C-terminal extension of the signal peptide: the longer the extension, the less efficiency the cleavage is. Thus, reducing the length of C-terminal extension of signal peptide by signal peptidase cleavage could facilitate further cleavage by SPP. The recombinant core protein fused with signal peptide from the C-terminus of p7 protein, but not those from the C-termini of E1 and E2, could be cleaved by SPP. Therefore, the sequence of the signal peptide is important but not the sole determinant for its cleavage by SPP. Replacement of the HCV core protein E.R.-associated domain (a.a. 120–150) with the E.R.-associated domain (a.a.1–50) of SARS-CoV membrane protein results in the failure of cleavage of this recombinant protein by SPP, though this protein still is E.R.-associated. This result suggests that not only E.R.-association but also specific protein sequence is important for the HCV core protein signal peptide cleavage by SPP. Thus, our results suggest that both sequences of the signal peptide and the E.R.-associated domain are important for the signal peptide cleavage of HCV core protein by SPP. Electronic Supplementary MaterialThe online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Woodchuck hepatitis virus X protein is required for viral infection in vivo.

The X gene of the mammalian hepadnaviruses is believed to encode a protein of 17 kDa which has been shown to transactivate a wide range of viral and cellular promoters. The necessity for X gene expression during the viral life cycle in vivo has recently been suggested (H.-S. Chen, S. Kaneko, R. Girones, R. W. Anderson, W. E. Hornbuckle, B. C. Tennant, P. J. Cote, J. L. Gerin, R. H. Purcell, and R. H. Miller, J. Virol. 67:1218-1226, 1993). We have independently constructed two variants of woodchuck hepatitis virus (WHV) with mutations in the X coding region. Transient transfection of two different hepatoma cell lines showed that these WHV X gene mutants were competent for virus replication in vitro. To determine whether X expression was required for viral replication in vivo, we injected mutant and wild-type genomes into the livers of susceptible woodchucks. While the wild-type WHV genomes were infectious in all animals examined, the mutant genomes did not initiate a WHV infection in woodchucks. These results indicate that the X gene of the hepadnaviruses plays a major role in viral replication in vivo.     

The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly.

Assembly of replication-competent hepatitis B virus (HBV) nucleocapsids requires the interaction of the core protein, the P protein, and the RNA pregenome. The core protein contains an arginine-rich C-terminal domain which is dispensable for particle formation in heterologous expression systems. Using transient expression in HuH7 cells of a series of C-terminally truncated core proteins, I examined the functional role of this basic region in the context of a complete HBV genome. All variants containing at least the 144 N-terminal amino acids were assembly competent, but efficient pregenome encapsidation was observed only with variants consisting of 164 or more amino acids. These data indicate that one function of the arginine-rich region is to provide the interactions between core protein and RNA pregenome. However, in cores from the variant ending with amino acid 164, the production of complete positive-strand DNA was drastically reduced. Moreover, almost all positive-strand DNA originated from in situ priming, whereas in wild-type particles, this type of priming not supporting the formation of relaxed circular DNA (RC-DNA) accounted for about one half of the positive strands. Further C-terminal residues to position 173 restored RC-DNA formation, and the corresponding variant did not differ from the full-length core protein in all assays used. The observation that RNA encapsidation and formation of RC-DNA can be genetically separated suggests that the core protein, via its basic C-terminal region, also acts as an essential auxiliary component in HBV replication, possibly like a histone, or like a single-stranded-DNA-binding protein. In contrast to their importance for HBV replication, sequences beyond amino acid 164 were not required for the formation of enveloped virions. Since particles from variant 164 did not contain mature DNA genomes, a genome maturation signal is apparently not required for HBV nucleocapsid envelopment.     

Cellular receptor traffic is essential for productive duck hepatitis B virus infection

We have investigated the mechanism of duck hepatitis B virus (DHBV) entry into susceptible primary duck hepatocytes (PDHs), using mutants of carboxypeptidase D (gp180), a transmembrane protein shown to act as the primary cellular receptor for avian hepatitis B virus uptake. The variant proteins were abundantly produced from recombinant adenoviruses and tested for the potential to functionally outcompete the endogenous wild-type receptor. Overexpression of wild-type gp180 significantly enhanced the efficiency of DHBV infection in PDHs but did not affect ongoing DHBV replication, an observation further supporting gp180 receptor function. A gp180 mutant deficient for endocytosis abolished DHBV infection, indicating endocytosis to be the route of hepadnaviral entry. With further gp180 variants, carrying mutations in the cytoplasmic domain and characterized by an accelerated turnover, the ability of gp180 to function as a DHBV receptor was found to depend on a wild-type-like sorting phenotype which largely avoids transport toward the endolysosomal compartment. Based on these data, we propose a model in which a distinct intracellular DHBV traffic to the endosome, but not beyond, is a prerequisite for completion of viral entry, i.e., for fusion and capsid release. Furthermore, the deletion of the two enzymatically active carboxypeptidase domains of gp180 did not lead to a loss of receptor function.     

Granzyme H of cytotoxic lymphocytes is required for clearance of the hepatitis B virus through cleavage of the hepatitis B virus X protein

The granule exocytosis pathway of cytotoxic lymphocytes plays critical roles in eradication of intracellular viruses. However, how hepatitis B virus (HBV) is cleared has not been defined. To clarify immune mechanisms underlying inhibition of the HBV replication, the relationship between granzyme H (GzmH) and HBV clearance was investigated. In this study, we found that the granule exocytosis pathway can inhibit HBV replication without induction of cytolysis of the infected cells. GzmH is essential for HBV eradication. The HBx protein (HBx), required for the replication of HBV, is cleaved at Met(79) by GzmH. GzmH inhibitor can abolish GzmH- and lymphokine-activated killer cell-mediated HBx degradation and HBV clearance. An HBx-deficient HBV is resistant to GzmH- and lymphokine-activated killer cell-mediated viral clearance. Adoptive transfer of GzmH-overexpressing NK cells into HBV carrier mice facilitates in vivo HBV eradication. Importantly, low GzmH expression in cytotoxic lymphocytes of individuals is susceptible to HBV infection and hepatocellular carcinoma. These results indicate that GzmH might be detected as a potential parameter for diagnosis of HBV infection and hepatocellular carcinoma.     

Anchor peptide of transferrin-binding protein B is required for interaction with transferrin-binding protein A

Gram-negative bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae, and Neisseriaceae families rely on an iron acquisition system that acquires iron directly from host transferrin (Tf). The process is mediated by a surface receptor composed of transferrin-binding proteins A and B (TbpA and TbpB). TbpA is an integral outer membrane protein that functions as a gated channel for the passage of iron into the periplasm. TbpB is a surface-exposed lipoprotein that facilitates the iron uptake process. In this study, we demonstrate that the region encompassing amino acids 7-40 of Actinobacillus pleuropneumoniae TbpB is required for forming a complex with TbpA and that the formation of the complex requires the presence of porcine Tf. These results are consistent with a model in which TbpB is responsible for the initial capture of iron-loaded Tf and subsequently interacts with TbpA through the anchor peptide. We propose that TonB binding to TbpA initiates the formation of the TbpB-TbpA complex and transfer of Tf to TbpA.     

nef gene is required for robust productive infection by simian immunodeficiency virus of T-cell-rich paracortex in lymph nodes

The pathogenesis of AIDS virus infection in a nonhuman primate AIDS model was studied by comparing plasma viral loads, CD4(+) T-cell subpopulations in peripheral blood mononuclear cells, and simian immunodeficiency virus (SIV) infection in lymph nodes for rhesus macaques infected with a pathogenic molecularly cloned SIVmac239 strain and those infected with its nef deletion mutant (Deltanef). In agreement with many reports, whereas SIVmac239 infection induced AIDS and depletion of memory CD4(+) T cells in 2 to 3 years postinfection (p.i.), Deltanef infection did not induce any manifestation associated with AIDS up to 6.5 years p.i. To explore the difference in SIV infection in lymphoid tissues, we biopsied lymph nodes at 2, 8, 72, and 82 weeks p.i. and analyzed them by pathological techniques. Maximal numbers of SIV-infected cells (SIV Gag(+), Env(+), and RNA(+)) were detected at 2 weeks p.i. in both the SIVmac239-infected animals and the Deltanef-infected animals. In the SIVmac239-infected animals, most of the infected cells were localized in the T-cell-rich paracortex, whereas in the Deltanef-infected animals, most were localized in B-cell-rich follicles and in the border region between the paracortex and the follicles. Analyses by double staining of CD68(+) macrophages and SIV Gag(+) cells and by double staining of CD3(+) T cells and SIV Env(+) cells revealed that SIV-infected cells were identified as CD4(+) T cells in either the SIVmac239 or the Deltanef infection. Whereas the many functions of Nef protein were reported from in vitro studies, our finding of SIVmac239 replication in the T-cell-rich paracortex in the lymph nodes supports the reported roles of Nef protein in T-cell activation and enhancement of viral infectivity. Furthermore, the abundance of SIVmac239 infection and the paucity of Deltanef infection in the T-cell-rich paracortex accounted for the differences in viral replication and pathogenicity between SIVmac239 and the Deltanef mutant. Thus, our in vivo study indicated that the nef gene enhances SIV replication by robust productive infection in memory CD4(+) T cells in the T-cell-rich region in lymphoid tissues.     

Phosphorylation of the termini of Cauliflower mosaic virus precapsid protein is important for productive infection

Cauliflower mosaic virus (CaMV) coat protein precursor (pre-CP) has 489 amino acids (p57) and is processed by the viral proteinase into three major forms: p44, p39, and p37. The N- and C-terminal extensions of pre-CP are released during maturation by the virus-encoded proteinase. We showed that these extensions are phosphorylated at several sites by host casein kinase II (CKII). We have identified the phosphorylated amino acids using an in vitro phosphorylation assay and tested the effect of mutation of these sites on viral infectivity. Mutation of serines S66, S68, and S72 to alanine in the N-terminal extension abolished phosphorylation of the protein in vitro. Also, mutation of all S and T residues in the C-terminus (450 to 489) made this region insensitive to CKII. Amino acid substitutions also were introduced into a full-length infectious clone of CaMV. Mutated forms of the virus with S66, S68, and S72 substituted with A or D showed a delay in symptom development and affected the infectivity of the virus. However, a mutant with an A substitution of all the S and T residues of the C-terminal extension of CP was not infectious. These results suggest that phosphorylation of the N- and C-termini of CaMV pre-CP plays an important role in the initiation of viral infection.     

HSC90 is required for nascent hepatitis C virus core protein stability in yeast cells

Hepatitis C virus core protein (Core) contributes to HCV pathogenicity. Here, we demonstrate that Core impairs growth in budding yeast. We identify HSP90 inhibitors as compounds that reduce intracellular Core protein level and restore yeast growth. Our results suggest that HSC90 (Hsc82) may function in the protection of the nascent Core polypeptide against degradation in yeast and the C-terminal region of Core corresponding to the organelle-interaction domain was responsible for Hsc82-dependent stability. The yeast system may be utilized to select compounds that can direct the C-terminal region to reduce the stability of Core protein.     

The viral E4 protein is required for the completion of the cottontail rabbit papillomavirus productive cycle in vivo

Expression of the papillomavirus E4 protein correlates with the onset of viral DNA amplification. Using a mutant cottontail rabbit papillomavirus (CRPV) genome incapable of expressing the viral E4 protein, we have shown that E4 is required for the productive stage of the CRPV life cycle in New Zealand White and cottontail rabbits. In these lesions, E4 was not required for papilloma development, but the onset of viral DNA amplification and L1 expression were abolished. Viral genome amplification was partially restored when mutant genomes able to express longer forms of E4 were used. These findings suggest that efficient amplification of the CRPV genome is dependent on the expression of a full-length CRPV E4 protein.     

Cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins

The regulated turnover of endoplasmic reticulum (ER)–resident membrane proteins requires their extraction from the membrane lipid bilayer and subsequent proteasome-mediated degradation. Cleavage within the transmembrane domain provides an attractive mechanism to facilitate protein dislocation but has never been shown for endogenous substrates. To determine whether intramembrane proteolysis, specifically cleavage by the intramembrane-cleaving aspartyl protease signal peptide peptidase (SPP), is involved in this pathway, we generated an SPP-specific somatic cell knockout. In a stable isotope labeling by amino acids in cell culture–based proteomics screen, we identified HO-1 (heme oxygenase-1), the rate-limiting enzyme in the degradation of heme to biliverdin, as a novel SPP substrate. Intramembrane cleavage by catalytically active SPP provided the primary proteolytic step required for the extraction and subsequent proteasome-dependent degradation of HO-1, an ER-resident tail-anchored protein. SPP-mediated proteolysis was not limited to HO-1 but was required for the dislocation and degradation of additional tail-anchored ER-resident proteins. Our study identifies tail-anchored proteins as novel SPP substrates and a specific requirement for SPP-mediated intramembrane cleavage in protein turnover.     

ORF66 protein kinase function is required for T-cell tropism of varicella-zoster virus in vivo

Several functions have been attributed to the serine/threonine protein kinase encoded by open reading frame 66 (ORF66) of varicella-zoster virus (VZV), including modulation of the apoptosis and interferon pathways, down-regulation of major histocompatibility complex class I cell surface expression, and regulation of IE62 localization. The amino acid sequence of the ORF66 protein contains a recognizable conserved kinase domain. Point mutations were introduced into conserved protein kinase motifs to evaluate their importance to ORF66 protein functions. Two substitution mutants were generated, including a G102A substitution, which blocked autophosphorylation and altered IE62 localization, and an S250P substitution, which had no effect on either autophosphorylation or IE62 localization. Both kinase domain mutants grew to titers equivalent to recombinant parent Oka (pOka) in vitro. pOka66G102A had slightly reduced growth in skin, which was comparable to the reduction observed when ORF66 translation was prevented by stop codon insertions in pOka66S. In contrast, infection of T-cell xenografts with pOka66G102A was associated with a significant decrease in infectious virus production equivalent to the impaired T-cell tropism found with pOka66S infection of T-cell xenografts in vivo. Disrupting kinase activity with the G102A mutation did not alter IE62 cytoplasmic localization in VZV-infected T cells, suggesting that decreased T-cell tropism is due to other ORF66 protein functions. The G102A mutation reduced the antiapoptotic effects of VZV infection of T cells. These experiments indicate that the T-cell tropism of VZV depends upon intact ORF66 protein kinase function.     

Rubella virus E2 signal peptide is required for perinuclear localization of capsid protein and virus assembly

The rubella virus (RV) structural proteins capsid, E2, and E1 are synthesized as a polyprotein precursor. The signal peptide that initiates translocation of E2 into the lumen of the endoplasmic reticulum remains attached to the carboxy terminus of the capsid protein after cleavage by signal peptidase. Among togaviruses, this feature is unique to RV. The E2 signal peptide has previously been shown to function as a membrane anchor for the capsid protein. In the present study, we demonstrate that this domain is required for RV glycoprotein-dependent localization of the capsid protein to the juxtanuclear region and subsequent virus assembly at the Golgi complex.     

The neuroendocrine protein 7B2 is required for peptide hormone processing in vivo and provides a novel mechanism for pituitary Cushing's disease

The neuroendocrine protein 7B2 has been implicated in activation of prohormone convertase 2 (PC2), an important neuroendocrine precursor processing endoprotease. To test this hypothesis, we created a null mutation in 7B2 employing a novel transposon-facilitated technique and compared the phenotypes of 7B2 and PC2 nulls. 7B2 null mice have no demonstrable PC2 activity, are deficient in processing islet hormones, and display hypoglycemia, hyperproinsulinemia, and hypoglucagonemia. In contrast to the PC2 null phenotype, these mice show markedly elevated circulating ACTH and corticosterone levels, with adrenocortical expansion. They die before 9 weeks of severe Cushing's syndrome arising from pituitary intermediate lobe ACTH hypersecretion. We conclude that 7B2 is indeed required for activation of PC2 in vivo but has additional important functions in regulating pituitary hormone secretion.     

In vivo phosphorylation and protein analysis of hepatitis B virus core antigen.

The C open reading frame of the hepatitis B virus contains two in-frame ATG codons that are separated by the precore region and encodes two major polypeptides that are antigenically distinct and that are probably synthesized from individual mRNAs. The precore region directs the secretion of the e antigen, whereas the core antigen can be expressed in the absence of these sequences. In this report a transient expression system was used to study the hepatitis B virus core antigen. By using a chimeric complex of adenovirus major late promoter-simian virus 40 enhancer sequences, we were able to achieve high levels of core antigen expression in transfected cells, permitting characterization of this protein and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The core polypeptide is a 20.9-kilodalton protein, and we show in this study that it is phosphorylated in vivo. Cell fractionation studies, the results of which are supported by indirect immunofluorescence, localized the phosphocore in the cytosol and the nucleus and indicated that it is associated with the membrane of transfected cells. Results of Triton X-114 solubilization studies indicated that the phosphocore is peripherally associated with cytoplasmic membranes. Expression of the membrane-associated phosphocore occurred in the absence of the precore sequences. The phosphocore also assembled into particles in the absence of other viral gene products or intact DNA.