Implantation of a toric intraocular lens after repeated radial keratotomy procedures: A case report.

Corneal alterations due to radial keratotomy (RK) complicate intraocular lens calculations, which may explain why there have been few reports of toric intraocular lens (TIOL) implantation after excessive or multiple operations. A 71-year-old male with a history of repeated RKs and at least 30 corneal incisions in each eye was referred for cataract surgery. Preoperatively, the best-corrected distance visual acuity was 0.7 decimal (0.15 logMAR) in the right eye and 0.9 decimal (0.05 logMAR) in the left eye. The refractive errors were -8.00 -3.00 × 80 and -6.00 -3.50 × 80, respectively. The total corneal cylindrical powers (real power; anterior and posterior) were, respectively, -0.90 D and -3.60 D at 9 a.m., compared to -1.60 D and -3.80 D at 1 p.m. Corneal astigmatism in the left eye was considered symmetric and diurnally stable; therefore, an XY1AT6 TIOL (Hoya, Tokyo, Japan; cylindrical power at the plane, +3.75 D) was implanted. A non-toric intraocular lens, the XY1 (Hoya), was implanted in the right eye. Six-month postoperative best-corrected distance visual acuities were 1.2 decimal (-0.08 logMAR) and 1.0 decimal (0.00 logMAR) in the right and left eyes, respectively. Post-operative manifest refractions were +0.00 -3.00 × 70 and -1.00 -2.00 × 85, respectively. The TIOL reduced refractive astigmatism in the left eye; therefore, we believe that even after multiple RKs, the TIOL can be a suitable candidate to correct astigmatism if the corneal astigmatism is diurnally stable and symmetric.

Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication.

In this study, we elucidated the mechanism by which human choline kinase-α (hCKα) interacts with nonstructural protein 5A (NS5A) and phosphatidylinositol-4-kinase IIIα (PI4KIIIα), the lipid kinase crucial for maintaining the integrity of virus-induced membranous webs, and modulates hepatitis C virus (HCV) replication. hCKα activity positively modulated phosphatidylinositol-4-phosphate (PI4P) levels in HCV-expressing cells, and hCKα-mediated PI4P accumulation was abolished by AL-9, a PI4KIIIα-specific inhibitor. hCKα colocalized with NS5A and PI4KIIIα or PI4P; NS5A expression increased hCKα and PI4KIIIα colocalization; and hCKα formed a ternary complex with PI4KIIIα and NS5A, supporting the functional interplay of hCKα with PI4KIIIα and NS5A. PI4KIIIα inactivation by AL-9 or hCKα inactivation by CK37, a specific hCKα inhibitor, impaired the endoplasmic reticulum (ER) localization and colocalization of these three molecules. Interestingly, hCKα knockdown or inactivation inhibited PI4KIIIα-NS5A binding. In an in vitro PI4KIIIα activity assay, hCKα activity slightly increased PI4KIIIα basal activity but greatly augmented NS5A-induced PI4KIIIα activity, supporting the essential role of ternary complex formation in robust PI4KIIIα activation. Concurring with the upregulation of PI4P production and viral replication, overexpression of active hCKα-R (but not the D288A mutant) restored PI4KIIIα and NS5A translocation to the ER in hCKα stable knockdown cells. Furthermore, active PI4KIIIα overexpression restored PI4P production, PI4KIIIα and NS5A translocation to the ER, and viral replication in CK37-treated cells. Based on our results, hCKα functions as an indispensable regulator that bridges PI4KIIIα and NS5A and potentiates NS5A-stimulated PI4KIIIα activity, which then facilitates the targeting of the ternary complex to the ER for viral replication.IMPORTANCE The mechanisms by which hCKα activity modulates the transport of the hCKα-NS5A complex to the ER are not understood. In the present study, we investigated how hCKα interacts with PI4KIIIα (a key element that maintains the integrity of the "membranous web" structure) and NS5A to regulate viral replication. We demonstrated that HCV hijacks hCKα to bridge PI4KIIIα and NS5A, forming a ternary complex, which then stimulates PI4KIIIα activity to produce PI4P. Pronounced PI4P synthesis then redirects the translocation of the ternary complex to the ER-derived, PI4P-enriched membrane for assembly of the viral replication complex and viral replication. Our study provides novel insights into the indispensable modulatory role of hCKα in the recruitment of PI4KIIIα to NS5A and in NS5A-stimulated PI4P production and reveals a new perspective for understanding the impact of profound PI4KIIIα activation on the targeting of PI4KIIIα and NS5A to the PI4P-enriched membrane for viral replication complex formation.

Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation.

UNLABELLED: Hepatitis C virus (HCV) infection reorganizes cellular membranes to create an active viral replication site named the membranous web (MW). The role that human choline kinase-α (hCKα) plays in HCV replication remains elusive. Here, we first showed that hCKα activity, not the CDP-choline pathway, promoted viral RNA replication. Confocal microscopy and subcellular fractionation of HCV-infected cells revealed that a small fraction of hCKα colocalized with the viral replication complex (RC) on the endoplasmic reticulum (ER) and that HCV infection increased hCKα localization to the ER. In the pTM-NS3-NS5B model, NS3-NS5B expression increased the localization of the wild-type, not the inactive D288A mutant, hCKα on the ER, and hCKα activity was required for effective trafficking of hCKα and NS5A to the ER. Coimmunoprecipitation showed that hCKα was recruited onto the viral RC presumably through its binding to NS5A domain 1 (D1). hCKα silencing or treatment with CK37, an hCKα activity inhibitor, abolished HCV-induced MW formation. In addition, hCKα depletion hindered NS5A localization on the ER, interfered with NS5A and NS5B colocalization, and mitigated NS5A-NS5B interactions but had no apparent effect on NS5A-NS4B and NS4B-NS5B interactions. Nevertheless, hCKα activity was not essential for the binding of NS5A to hCKα or NS5B. These findings demonstrate that hCKα forms a complex with NS5A and that hCKα activity enhances the targeting of the complex to the ER, where hCKα protein, not activity, mediates NS5A binding to NS5B, thereby promoting functional membranous viral RC assembly and viral RNA replication. IMPORTANCE: HCV infection reorganizes the cellular membrane to create an active viral replication site named the membranous web (MW). Here, we report that human choline kinase-α (hCKα) acts as an essential host factor for HCV RNA replication. A fraction of hCKα colocalizes with the viral replication complex (RC) on the endoplasmic reticulum (ER) in HCV-infected cells. NS3-NS5B expression increases ER localization of wild-type, but not D288A mutant, hCKα, and hCKα activity facilitates the transport of itself and NS5A to the ER. Silencing or inactivation of hCKα abrogates MW formation. Moreover, hCKα is recruited by NS5A independent of hCKα activity, presumably through binding to NS5A D1. hCKα activity then mediates the ER targeting of the hCKα-NS5A complex. On the ER membrane, hCKα protein, per se, induces NS5A binding to NS5B, thereby promoting membranous RC formation and viral RNA replication. Our study may benefit the development of hCKα-targeted anti-HCV therapeutics.

Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication.

The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein.

Emerging roles of interferon-stimulated genes in the innate immune response to hepatitis C virus infection.

Infection with hepatitis C virus (HCV), a major viral cause of chronic liver disease, frequently progresses to steatosis and cirrhosis, which can lead to hepatocellular carcinoma. HCV infection strongly induces host responses, such as the activation of the unfolded protein response, autophagy and the innate immune response. Upon HCV infection, the host induces the interferon (IFN)-mediated frontline defense to limit virus replication. Conversely, HCV employs diverse strategies to escape host innate immune surveillance. Type I IFN elicits its antiviral actions by inducing a wide array of IFN-stimulated genes (ISGs). Nevertheless, the mechanisms by which these ISGs participate in IFN-mediated anti-HCV actions remain largely unknown. In this review, we first outline the signaling pathways known to be involved in the production of type I IFN and ISGs and the tactics that HCV uses to subvert innate immunity. Then, we summarize the effector mechanisms of scaffold ISGs known to modulate IFN function in HCV replication. We also highlight the potential functions of emerging ISGs, which were identified from genome-wide siRNA screens, in HCV replication. Finally, we discuss the functions of several cellular determinants critical for regulating host immunity in HCV replication. This review will provide a basis for understanding the complexity and functionality of the pleiotropic IFN system in HCV infection. Elucidation of the specificity and the mode of action of these emerging ISGs will also help to identify novel cellular targets against which effective HCV therapeutics can be developed.

Autophagy in hepatitis C virus-host interactions: potential roles and therapeutic targets for liver-associated diseases.

Autophagy is a lysosome-associated, degradative process that catabolizes cytosolic components to recycle nutrients for further use and maintain cell homeostasis. Hepatitis C virus (HCV) is a major cause of chronic hepatitis, which often leads to end-stage liver-associated diseases and is a significant burden on worldwide public health. Emerging lines of evidence indicate that autophagy plays an important role in promoting the HCV life cycle in host cells. Moreover, the diverse impacts of autophagy on a variety of signaling pathways in HCV-infected cells suggest that the autophagic process is required for balancing HCV-host cell interactions and involved in the pathogenesis of HCV-related liver diseases. However, the detailed molecular mechanism underlying how HCV activates autophagy to benefit viral growth is still enigmatic. Additionally, how the autophagic response contributes to disease progression in HCV-infected cells remains largely unknown. Hence, in this review, we overview the interplay between autophagy and the HCV life cycle and propose possible mechanisms by which autophagy may promote the pathogenesis of HCV-associated chronic liver diseases. Moreover, we outline the related studies on how autophagy interplays with HCV replication and discuss the possible implications of autophagy and viral replication in the progression of HCV-induced liver diseases, e.g., steatosis and hepatocellular carcinoma. Finally, we explore the potential therapeutics that target autophagy to cure HCV infection and its related liver diseases.

Empowering village doctors and enhancing rural healthcare using cloud computing in a rural area of mainland China.

BACKGROUND: China's healthcare system often struggles to meet the needs of its 900 million people living in rural areas due to major challenges in preventive medicine and management of chronic diseases. Here we address some of these challenges by equipping village doctors (ViDs) with Health Information Technology and developing an electronic health record (EHR) system which collects individual patient information electronically to aid with implementation of chronic disease management programs. METHODS: An EHR system based on a cloud-computing architecture was developed and deployed in Xilingol county of Inner Mongolia using various computing resources (hardware and software) to deliver services over the health network using Internet when available. The system supports the work at all levels of the healthcare system, including the work of ViDs in rural areas. An analysis done on 291,087 EHRs created from November 2008 to June 2011 evaluated the impact the EHR system has on preventive medicine and chronic disease management programs in rural China. RESULTS: From 2008 to 2011 health records were created for 291,087 (26.25%) from 1,108,951 total Xilingol residents with 10,240 cases of hypertension and 1152 cases of diabetes diagnosed and registered. Furthermore, 2945 hypertensive and 305 diabetic patients enrolled in follow-up. Implementing the EHR system revealed a high rate of cholecystectomies leading to investigations and findings of drinking water contaminated with metals. Measures were taken to inform the population and clean drinking water was supplied. CONCLUSIONS: The cloud-based EHR approach improved the care provision for ViDs in rural China and increased the efficiency of the healthcare system to monitor the health status of the population and to manage preventive care efforts. It also helped discover contaminated water in one of the project areas revealing further benefits if the system is expanded and improved.

Active RNA replication of hepatitis C virus downregulates CD81 expression.

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

Hepatitis C virus and cellular stress response: implications to molecular pathogenesis of liver diseases.

Infection with hepatitis C virus (HCV) is a leading risk factor for chronic liver disease progression, including steatosis, cirrhosis, and hepatocellular carcinoma. With approximately 3% of the human population infected worldwide, HCV infection remains a global public health challenge. The efficacy of current therapy is still limited in many patients infected with HCV, thus a greater understanding of pathogenesis in HCV infection is desperately needed. Emerging lines of evidence indicate that HCV triggers a wide range of cellular stress responses, including cell cycle arrest, apoptosis, endoplasmic reticulum (ER) stress/unfolded protein response (UPR), and autophagy. Also, recent studies suggest that these HCV-induced cellular responses may contribute to chronic liver diseases by modulating cell proliferation, altering lipid metabolism, and potentiating oncogenic pathways. However, the molecular mechanism underlying HCV infection in the pathogenesis of chronic liver diseases still remains to be determined. Here, we review the known stress response activation in HCV infection in vitro and in vivo, and also explore the possible relationship of a variety of cellular responses with the pathogenicity of HCV-associated diseases. Comprehensive knowledge of HCV-mediated disease progression shall shed new insights into the discovery of novel therapeutic targets and the development of new intervention strategy.

Identification of hnRNPH1, NF45, and C14orf166 as novel host interacting partners of the mature hepatitis C virus core protein.

The hepatitis C virus core protein (HCVc) forms the viral nucleocapsid and is involved in viral persistence and pathogenesis, possibly by interacting with host factors to modulate viral replication and cellular functions. Here, we identified 36 cellular protein candidates by one-dimensional SDS-PAGE and LC-MS/MS-based proteomics after affinity purification with HCVc174, a matured form of HCVc from HCV-1b genotype, tagged with biotin and calmodulin-binding peptide/protein A at N- and C-termini, respectively. By pull-down and confocal imaging techniques, we confirmed that heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1), nuclear factor 45 (NF45), and C14orf166 are novel HCVc174-interacting host proteins, known to participate in mRNA metabolism, gene regulation, and microtubule organization, respectively. Unlike the other 2 proteins, NF45 interacted with HCVc174 in an RNA-dependent manner. These 3 proteins colocalized with ectopic HCVc-1b in both the cytoplasm and nucleus, which demonstrated their spatial interaction with naturally translocated HCVc174 after HCVc biogenesis. Such colocalization, however, shifted to the cytoplasm in cells with replicating virus of 1b or 2a genotype, indicating that active viral replication confined these interacting proteins in the cytoplasm. Collectively, our findings suggest that spatial interactions of hnRNPH1, NF45, and C14orf166 with HCVc174 likely modulate HCV or cellular functions during acute and chronic HCV infection.

An efficient production and characterization of HIV-1 gp41 ectodomain with fusion peptide in Escherichia coli system.

We demonstrated a high level expression and purification of recombinant human immunodeficiency virus type 1 gp41 ectodomain (gp41e-FP) using glass bead approach with a final yield of 12±2mg/L bacterial culture. The proper folding of gp41e-FP encompassing the fusion peptide (FP) was ascertained by circular dichroism (CD) measurement and recognition by NC-1 antibody. The latter assay revealed stabilization of the gp41 coiled coil structure in the presence of liposome dispersion. The differential affinity of gp41e-FP and gp41e (devoid of FP) by NC-1 suggested an aggregated state for gp41e-FP and/or possible proximity of the fusion peptide domain to the coiled coil structure of gp41 ectodomain. Perfluorooctanoate (PFO)-PAGE electrophoresis experiment revealed the trimeric propensity of the recombinant gp41e-FP. In comparison to gp41e, the lipid mixing activity of gp41e-FP was two-fold higher suggesting a role of FP in promoting membrane fusion. The present approach to efficiently and quantitatively preparing the functional full-length recombinant gp41 ectodomain protein can be employed for structural and biomedical investigations and the extraction of other inclusion body-embedded recombinant proteins.

Autophagy: a novel guardian of HCV against innate immune response.

Autophagy is an evolutionarily conserved process that catabolizes intracellular components and maintains cellular homeostasis. Autophagy involves the sequestration of cytoplasmic content within a double-membraned autophagosome, and the fusion of the autophagosome with a lysosome to form an autolysosome for subsequent degradation (Fig. 1A). Autophagy plays a pivotal role in various aspects of cellular responses to stresses, such as nutrient deprivation, damaged organelles, aggregated proteins, exposure to endoplasmic reticulum (ER) stress and pathogen infections. Virus infection often leads to ER stress and induction of the unfolded protein response (UPR). Recent studies reveal that virus-induced UPR may activate autophagy to support the virus life cycle. However, the exact roles of the UPR and autophagy in host cell-virus interactions are still enigmatic.

Activation of the unfolded protein response and autophagy after hepatitis C virus infection suppresses innate antiviral immunity in vitro.

Autophagy, a process for catabolizing cytoplasmic components, has been implicated in the modulation of interactions between RNA viruses and their host. However, the mechanism underlying the functional role of autophagy in the viral life cycle still remains unclear. Hepatitis C virus (HCV) is a single-stranded, positive-sense, membrane-enveloped RNA virus that can cause chronic liver disease. Here we report that HCV induces the unfolded protein response (UPR), which in turn activates the autophagic pathway to promote HCV RNA replication in human hepatoma cells. Further analysis revealed that the entire autophagic process through to complete autolysosome maturation was required to promote HCV RNA replication and that it did so by suppressing innate antiviral immunity. Gene silencing or activation of the UPR-autophagy pathway activated or repressed, respectively, IFN-ß activation mediated by an HCV-derived pathogen-associated molecular pattern (PAMP). Similar results were achieved with a PAMP derived from Dengue virus (DEV), indicating that HCV and DEV may both exploit the UPR-autophagy pathway to escape the innate immune response. Taken together, these results not only define the physiological significance of HCV-induced autophagy, but also shed light on the knowledge of host cellular responses upon HCV infection as well as on exploration of therapeutic targets for controlling HCV infection.

The cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 harbors lipid raft association determinants.

The molecular basis for localization of the human immunodeficiency virus type 1 envelope glycoprotein (Env) in detergent-resistant membranes (DRMs), also called lipid rafts, still remains unclear. The C-terminal cytoplasmic tail of gp41 contains three membrane-interacting, amphipathic alpha-helical sequences, termed lentivirus lytic peptide 2 (LLP-2), LLP-3, and LLP-1, in that order. Here we identify determinants in the cytoplasmic tail which are crucial for Env's association with Triton X-100-resistant rafts. Truncations of LLP-1 greatly reduced Env localization in lipid rafts, and the property of Gag-independent gp41 localization in rafts was conserved among different strains. Analyses of mutants containing single deletions or substitutions in LLP-1 showed that the alpha-helical structure of the LLP-1 hydrophobic face has a more-critical role in Env-raft associations than that of the hydrophilic face. With the exception of a Pro substitution for Val-833, all Pro substitution and charge-inverting mutants showed wild-type virus-like one-cycle viral infectivity, replication kinetics, and Env incorporation into the virus. The intracellular localization and cell surface expression of mutants not localized in lipid rafts, such as the TM844, TM813, 829P, and 843P mutants, were apparently normal compared to those of wild-type Env. Cytoplasmic subdomain targeting analyses revealed that the sequence spanning LLP-3 and LLP-1 could target a cytoplasmic reporter protein to DRMs. Mutations of LLP-1 that affected Env association with lipid rafts also disrupted the DRM-targeting ability of the LLP-3/LLP-1 sequence. Our results clearly demonstrate that LLP motifs located in the C-terminal cytoplasmic tail of gp41 harbor Triton X-100-resistant raft association determinants.

Mutagenesis of the fusion peptide-like domain of hepatitis C virus E1 glycoprotein: involvement in cell fusion and virus entry.

BACKGROUND: Envelope (E) glycoprotein E2 of the hepatitis C virus (HCV) mediates binding of the virus to target cell receptors. Nevertheless, the precise role of E1 in viral entry remains elusive. METHODS: To understand the involvement of the fusion peptide-like domain positioned at residues 264 to 290 within envelope glycoprotein E1 in HCV infection, mutants with Ala and Asn substitutions for residues conserved between HCV and E proteins of flaviviruses or the fusion proteins of paramyxoviruses were constructed by site-directed mutagenesis and their effects on membrane fusion and viral infectivity were examined. RESULTS: None of these mutations affected the synthesis or cell surface expression of envelope proteins, nor did they alter the formation of a non-covalent E1-E2 heterodimer or E2 binding to the large extracellular loop of CD81. The Cys residues located at positions 272 and 281 were unlikely involved in intra- or intermolecular disulfide bond formation. With the exception of the G267A mutant, which showed increased cell fusion, other mutants displayed reduced or marginally inhibited cell fusion capacities compared to the wild-type (WT) E1E2. The G267A mutant was also an exception in human immunodeficiency virus type 1 (HIV-1)/HCV E1E2 pseudotyping analyses, in that it showed higher one-cycle infectivity; all other mutants exhibited greatly or partially reduced viral entry versus the WT pseudotype. All but the G278A and D279N mutants showed a WT-like profile of E1E2 incorporation into HIV-1 particles. Since C272A, C281A, G282A, and G288A pseudotypes bound to Huh7 cells as effectively as did the WT pseudotype, the reduced infectivity of these pseudotypes was due to their ability to inhibit cell fusion. CONCLUSION: Our results indicate that specific residues, but not the structure, of this fusion peptide-like domain are required for mediating cell fusion and viral entry.

Characterization of hepatitis C virus core protein multimerization and membrane envelopment: revelation of a cascade of core-membrane interactions.

The molecular basis underlying hepatitis C virus (HCV) core protein maturation and morphogenesis remains elusive. We characterized the concerted events associated with core protein multimerization and interaction with membranes. Analyses of core proteins expressed from a subgenomic system showed that the signal sequence located between the core and envelope glycoprotein E1 is critical for core association with endoplasmic reticula (ER)/late endosomes and the core's envelopment by membranes, which was judged by the core's acquisition of resistance to proteinase K digestion. Despite exerting an inhibitory effect on the core's association with membranes, (Z-LL)(2)-ketone, a specific inhibitor of signal peptide peptidase (SPP), did not affect core multimeric complex formation, suggesting that oligomeric core complex formation proceeds prior to or upon core attachment to membranes. Protease-resistant core complexes that contained both innate and processed proteins were detected in the presence of (Z-LL)(2)-ketone, implying that core envelopment occurs after intramembrane cleavage. Mutations of the core that prevent signal peptide cleavage or coexpression with an SPP loss-of-function D219A mutant decreased the core's envelopment, demonstrating that SPP-mediated cleavage is required for core envelopment. Analyses of core mutants with a deletion in domain I revealed that this domain contains sequences crucial for core envelopment. The core proteins expressed by infectious JFH1 and Jc1 RNAs in Huh7 cells also assembled into a multimeric complex, associated with ER/late-endosomal membranes, and were enveloped by membranes. Treatment with (Z-LL)(2)-ketone or coexpression with D219A mutant SPP interfered with both core envelopment and infectious HCV production, indicating a critical role of core envelopment in HCV morphogenesis. The results provide mechanistic insights into the sequential and coordinated processes during the association of the HCV core protein with membranes in the early phase of virus maturation and morphogenesis.

Identification of the LWYIK motif located in the human immunodeficiency virus type 1 transmembrane gp41 protein as a distinct determinant for viral infection.

The highly conserved LWYIK motif located immediately proximal to the membrane-spanning domain of the gp41 transmembrane protein of human immunodeficiency virus type 1 has been proposed as being important for the surface envelope (Env) glycoprotein's association with lipid rafts and gp41-mediated membrane fusion. Here we employed substitution and deletion mutagenesis to understand the role of this motif in the virus life cycle. None of the mutants examined affected the synthesis, precursor processing, CD4 binding, oligomerization, or cell surface expression of the Env, nor did they alter Env incorporation into the virus. All of the mutants, particularly the DeltaYI, DeltaIK, and DeltaLWYIK mutants, in which the indicated residues were deleted, exhibited greatly reduced one-cycle viral replication and the Env trans-complementation ability. All of these deletion mutant proteins were still localized in the lipid rafts. With the exception of the Trp-to-Ala (WA) mutant, which exhibited reduced viral infectivity albeit with normal membrane fusion, all mutants displayed loss of some or almost all of the membrane fusion ability. Although these deletion mutants partially inhibited in trans wild-type (WT) Env-mediated fusion, they were more effective in dominantly interfering with WT Env-mediated viral entry when coexpressed with the WT Env, implying a role of this motif in postfusion events as well. Both T20 and L43L peptides derived from the two gp41 extracellular C- and N-terminal alpha-helical heptad repeats, respectively, inhibited WT and DeltaLWYIK Env-mediated viral entry with comparable efficacies. Biotin-tagged T20 effectively captured both the fusion-active, prehairpin intermediates of WT and mutant gp41 upon CD4 activation. Env without the deletion of the LWYIK motif still effectively mediated lipid mixing but inhibited content mixing. Our study demonstrates that the immediate membrane-proximal LWYIK motif acts as a unique and distinct determinant located in the gp41 C-terminal ectodomain by promoting enlargement of fusion pores and postfusion activities.

The dominant-negative action of a fusion protein containing the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 in virus replication.

We previously described a novel mode of downregulation of human immunodeficiency virus type 1 (HIV-1) Gag expression by a cytoplasmic domain fusion protein of the envelope (Env) transmembrane protein, beta-galactosidase (beta-gal)/706-856, which contains the cytoplasmic tail of gp41 fused at the C terminus of Escherichia coli beta-gal. In the present study, we showed that this mediator conferred a dose-dependent dominant interference with virus infectivity. In the context of an HIV-1 provirus, this inhibitor downregulated steady-state Env expression. Paradoxically, Env overexpression suppressed beta-gal/706-856-mediatd Gag downregulation. Sucrose gradient ultracentrifugation and confocal microscopy revealed that Gag, Env, and beta-gal/706-856 had stable interactions and formed aggregated complexes in perinuclear regions. Moreover, Env overexpression hindered colocalization of Gag with beta-gal/706-856 in the perinuclear region. Further cytoplasmic domain mapping analyses showed a correlation between the ability of cytoplasmic subdomains to downregulate Gag expression and the ability of these subdomains to stably interact with Gag. These studies show that redirection of Gag from its cytoplasmic synthesis site to a perinuclear compartment is a prerequisite for beta-gal/706-856-mediated Gag downregulation. The results also illustrate that the dynamic interplay among Gag, Env, and beta-gal/706-856 can modulate Gag and Env expression, thus controlling HIV-1 infection.

The immediate early 2 protein of human cytomegalovirus (HCMV) mediates the apoptotic control in HCMV retinitis through up-regulation of the cellular FLICE-inhibitory protein expression.

Human CMV (HCMV) is a widespread human pathogen that causes blindness by inducing retinitis in AIDS patients. Previously, we showed that viral immediate early 2 (IE2) protein may allow HCMV to evade the immune control by killing the Fas receptor-positive T lymphocytes attracted to the infected retina with increased secretion of Fas ligand (FasL). In this study, we further demonstrate that the secreted FasL also kills uninfected Fas-rich bystander retinal cells and that IE2 simultaneously protects the infected cells from undergoing apoptotic death, in part, by activating the expression of cellular FLIP (c-FLIP), an antiapoptotic molecule that blocks the direct downstream executer caspase 8 of the FasL/Fas pathway. c-FLIP induction requires the N-terminal 98 residues of IE2 and the c-FLIP promoter region spanning nucleotides -978 to -696. In vivo association of IE2 to this region, IE2-specific c-FLIP activation, and decrease of FasL-up-regulated activities of caspases 8 and 3 were all demonstrated in HCMV-infected human retinal cells. Moreover, c-FLIP up-regulation by IE2 appeared to involve PI3K and might also render cells resistant to TRAIL-mediated death. Finally, enhanced c-FLIP signals were immunohistochemically detected in IE-positive cells in the HCMV-infected lesions of the human retina. Taken together, these data demonstrate specific activation of c-FLIP by HCMV IE2 and indicate a novel role for c-FLIP in the pathogenesis of HCMV retinitis.

Functional characterization of heptad repeat 1 and 2 mutants of the spike protein of severe acute respiratory syndrome coronavirus.

To understand the roles of heptad repeat 1(HR1) and HR2 of the spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) in virus-cell interactions, the conserved Leu or Ile residues located at positions 913, 927, 941, and 955 in HR1 and 1151, 1165, and 1179 in HR2 were individually replaced with an alpha-helix-breaker Pro residue. The 913P mutant was expressed mainly as a faster-migrating, lower-molecular-weight S(L) form, while the wild type and all other mutants produced similar levels of both the S(L) form and the slower-migrating, higher-molecular-weight S(H) form. The wild-type S(L) form was processed to the S(H) form, whereas the S(L) form of the 913P mutant was inefficiently converted to the S(H) form after biosynthesis. None of these mutations affected cell surface expression or binding to its cognate ACE2 receptor. In a human immunodeficiency virus type 1/SARS S coexpression study, all mutants except the 913P mutant incorporated the S(H) form into the virions as effectively as did the wild-type S(H) form. The mutation at Ile-1151 did not affect membrane fusion or viral entry. The impaired viral entry of the 927P, 941P, 955P, and 1165P mutants was due to their inability to mediate membrane fusion, whereas the defect in viral entry of the 1179P mutant occurred not at the stage of membrane fusion but rather at a postfusion stage. Our study demonstrates the functional importance of HR1 and HR2 of the SARS-CoV spike protein in membrane fusion and viral entry.