Faculty Bibliography

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 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-β 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 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.

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.

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/CONCLUSIONS: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.

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.

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.

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.

The cytoplasmic domain of human immunodeficiency virus type 1 (HIV-1) envelope (Env) transmembrane protein gp41 interacts with the viral matrix MA protein, which facilitates incorporation of the trimeric Env complex into the virus. It is thus feasible to design an anti-HIV strategy targeting this interaction. We herein describe that Gag expression can be downregulated by a cytoplasmic domain fusion protein of the 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. This mediator depleted intracellular Gag molecules in a dose-dependent manner. Sucrose gradient ultracentrifugation and confocal microscopy revealed that Gag and beta-gal/706-856 had stable interactions and formed aggregated complexes in perinuclear, intracellular sites. Pulse-chase and cycloheximide chase analyses demonstrated that this mediator enhanced unmyristylated Gag degradation. The results demonstrate a novel mode of HIV-1 Gag downregulation by directing Gag to an intracellular site via the interaction of Gag with a gp41 cytoplasmic domain fusion protein.

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.