Current Peptide and Protein Candidates Challenging HIV Therapy beyond the Vaccine Era

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Adaptor Protein 1A Facilitates Dengue Virus Replication

<div><p>Rearrangement of membrane structure induced by dengue virus (DENV) is essential for replication, and requires host cellular machinery. Adaptor protein complex (AP)-1 is a host component, which can be recruited to components required for membrane rearrangement. Therefore, dysfunction of AP-1 may affect membrane organization, thereby decreasing replication of virus in infected cells. In the present study, AP-1-dependent traffic inhibitor inhibited DENV protein expression and virion production. We further clarified the role of AP-1A in the life cycle of DENV by RNA interference. AP-1A was not involved in DENV entry into cells. However, it facilitated DENV RNA replication. Viral RNA level was reduced significantly in Huh7 cells transfected with AP-1A small interfering RNA (siRNA) compared with control siRNA. Transfection of naked DENV viral RNA into Huh7 cells transfected with AP-1A siRNA resulted in less viral RNA and virion production than transfection into Huh7 cells transfected with control siRNA. Huh7 cells transfected with AP-1A siRNA showed greater modification of membrane structures and fewer vesicular packets compared with cells transfected with control siRNA. Therefore, AP-1A may partly control DENV-induced rearrangement of membrane structures required for viral replication.</p></div

AP-1 was involved in production of four serotypes of DENV.

<p>Inhibitory effect of A5 on virus replication was determined in all four serotypes of DENV. Huh7 cells were infected with DENV-1, -2, -3 and -4 at a MOI of 1 for 2 h. Unbound virus was removed by washing with PBS. DENV-infected cells were incubated with A5 (200 μM) or culture medium (control) for 24 h. Virus titer in culture supernatants was measured by FFU assay. (A) Titer of DENV-1; (B) titer of DENV-2; (C) titer of DENV-3; (D) titer of DENV-4. Statistical significance was analyzed using unpaired <i>t</i> test (*P<0.05). Error bars represent SEM from three independent experiments.</p

AP-1A knockdown affected the DENV replication site.

<p>(A) Ultrastructural analysis of Huh7 cells transfected with control siRNA was observed by TEM at 48 h after second transfection. (B) Cells transfected with control siRNA. (C) Cells transfected with AP-1A siRNA. (D) Cells transfected with AP-2 siRNA were infected with DENV-2 at a MOI of 10 for 24 h. Cells were fixed, processed and analyzed by TEM. Ve, virus-induced vesicles (arrow); Vi, virus particles (arrowhead).</p

Exogenous fatty acid increased DENV production after A5 treatment.

<p>DENV-infected Huh7 cells were incubated with A5 in the presence of oleic acid-BSA or fatty acid free-BSA for 24 h. The culture supernatants were collected for FFU assay. Statistical significance was analyzed using the unpaired <i>t</i> test. *P<0.05. Error bars represent SEM from three independent experiments.</p

Latticed Gold Nanoparticle Conjugation via Monomeric Streptavidin in Lateral Flow Assay for Detection of Autoantibody to Interferon-Gamma

Adult-onset immunodeficiency syndrome (AOID) patients with autoantibodies (autoAbs) against interferon-gamma (IFN-γ) generally suffer from recurrent and recalcitrant disseminated non-tuberculous mycobacterial diseases. Since the early stages of AOID do not present specific symptoms, diagnosis and treatment of the condition are not practical. A simplified diagnostic method for differentiating AOID from other immunodeficiencies, such as HIV infection, was created. Anti-IFN-γ is generally identified using enzyme-linked immunosorbent assay (ELISA), which involves an instrument and a cumbersome process. Recombinant IFN-γ indirectly conjugated to colloidal gold was used in the modified immunochromatographic (IC) strips. The biotinylated-IFN-γ was incorporated with colloidal-gold-labeled 6HIS-maltose binding protein-monomeric streptavidin (6HISMBP-mSA) and absorbed at the conjugate pad. The efficacy of the IC strip upon applying an anti-IFN-γ autoAb cut-off ELISA titer of 2500, the sensitivity and specificity were 84% and 90.24%, respectively. When a cut-off ELISA titer of 500 was applied, the sensitivity and specificity were 73.52% and 100%, respectively

Expression of DENV protein was decreased in Huh7 cells transfected with AP-1A siRNA.

<p>(A) Huh7 cells were transfected with control siRNA and AP-1A siRNA and infected with DENV-2 for 24 h. DENV proteins were examined at 24 h post-infection by western blotting. Band intensity of DENV proteins was quantified using Image J software. (B) Expression of AP-1A, AP-2 or AP-3A in Huh7 cells was examined by real-time RT-PCR at 48 h after second transfection. (C) pRL-SV40 vector, which contains <i>Renilla</i> luciferase gene, was subjected to <i>in vitro</i> transcription. To determine the effect of AP-1A knockdown on translation, Huh7 cells were transfected twice with AP-1A-specific siRNA or control siRNA. After the second round of siRNA transfection, cells were transfected with 2.5 nM reporter RNA followed by replacement with fresh culture medium at 4 h later. Following 8 h after transfection with reporter RNA, cells were harvested and determined for <i>Renilla</i> luciferase expression using Luciferase Reporter Assay System (Promega).</p

AP-1A knockdown affected the DENV replication site.

<p>(A) Ultrastructural analysis of Huh7 cells transfected with control siRNA was observed by TEM at 48 h after second transfection. (B) Cells transfected with control siRNA. (C) Cells transfected with AP-1A siRNA. (D) Cells transfected with AP-2 siRNA were infected with DENV-2 at a MOI of 10 for 24 h. Cells were fixed, processed and analyzed by TEM. Ve, virus-induced vesicles (arrow); Vi, virus particles (arrowhead).</p

AP-1-dependent traffic inhibitor, A5, inhibited DENV production in Huh7 cells.

<p>Huh7 cells were infected with DENV-2 at a MOI of 1 for 2 h. Unbound virus was removed by washing with PBS. Mock- or DENV-infected cells were incubated with A5 at 0, 25, 50, 100 and 200 μM for 24 h. (A) DENV envelope protein in cell lysates was examined at 24 h post-treatment with A5 by western blotting. Band intensity of DENV envelope protein was quantified using Image J software. (B) Virus titer in culture supernatants was measured by FFU assay. (C) Viability of DENV-infected cells was measured using PrestoBlue cell viability reagent. Statistical significance was analyzed using the unpaired <i>t</i> test. *P<0.05. Error bars represent SEM from three independent experiments.</p