Cell-cell and virus-cell fusion assay-based analyses of alanine insertion mutants in the distal α9 portion of the JRFL gp41 subunit from HIV-1.

Membrane fusion is the first essential step in HIV-1 replication. The gp41 subunit of HIV-1 envelope protein (Env), a class I fusion protein, achieves membrane fusion by forming a structure called a six-helix bundle composed of N- and C-terminal heptad repeats. We have recently shown that the distal portion of the α9 helix in the C-terminal heptad repeat of X4-tropic HXB2 Env plays a critical role in the late-stage membrane fusion and viral infection. Here, we used R5-tropic JRFL Env and constructed six alanine insertion mutants, 641+A to 646+A, in the further distal portion of α9 where several glutamine residues are conserved (the number corresponds to the position of the inserted alanine in JRFL Env). 644+A showed the most severe defect in syncytia formation. Decreased fusion pore formation activity, revealed by a dual split protein assay, was observed in all mutants except 641+A. Sequence analysis and substitution of inserted 644A with Gln revealed that the glutamine residue at position 644 that forms complex hydrogen-bond networks with other polar residues on the surface of the six-helix bundle is critical for cell-cell fusion. We also developed a split NanoLuc® (Nluc) reporter-based assay specific to the virus-cell membrane fusion step to analyze several of the mutants. Interestingly syncytia-competent mutants failed to display Nluc activities. In addition to defective fusion activity, a reduction of Env incorporation into virions may further contribute to differences in cell-cell and virus-cell fusions.

Six-helix bundle completion in the distal C-terminal heptad repeat region of gp41 is required for efficient human immunodeficiency virus type 1 infection.

BACKGROUND: The native pre-fusion structure of gp120/gp41 complex of human immunodeficiency virus type 1 was recently revealed. In the model, the helices of gp41 (α6, α7, α8, and α9) form a four-helix collar underneath trimeric gp120. Gp41 is a class I fusion protein and mediates membrane fusion by forming a post-fusion structure called the six-helix bundle (6HB). The comparison of the pre- and post-fusion structures revealed the large conformational changes in gp41 during the antiparallel packing of the N- and C-terminal heptad repeats (NHRs and CHRs) in membrane fusion. Several mutagenesis studies of gp41 performed in the past were interpreted based on 6HB, the only available structure at that time. To obtain an insight about the current pre-fusion structural model and conformational changes during membrane fusion, alanine insertion mutagenesis of the NHR, CHR and connecting loop regions of HXB2 gp41 was performed. The effects of mutations on biosynthesis and membrane fusion were analyzed by immunoblotting and fusion assays, respectively. The extent of membrane fusion was evaluated by split luciferase-based pore formation and syncytia formation assays, respectively. RESULTS: Consistent with the current structural model, drastic negative effects of mutations on biosynthesis and membrane fusion were observed for NHR, loop, and proximal regions of CHR (up to amino acid position 643). The insertions in α9 after it leaves the four-helix collar were tolerable for biosynthesis. These CHR mutants showed varying effects on membrane fusion. Insertion at position 644 or 645 resulted in poor pore and syncytia formation. Efficient pore and syncytia formation almost similar to that of the wild type was observed for insertion at position 647, 648 or 649. However, recovery of virus infectivity was only observed for the insertions beyond position 648. CONCLUSIONS: The mutagenesis data for HXB2 gp41 is in agreement with the recent pre-fusion structure model. The virus infection data suggested that fusion pores sufficiently large enough for the release of the virus genome complex are formed after the completion of 6HB beyond position 648.

Identification and Functional Analysis of Apoptotic Protease Activating Factor-1 (Apaf-1) from Spodoptera litura.

Apoptotic protease activating factor-1 (Apaf-1) is an adaptor molecule, essential for activating initiator caspase and downstream effector caspases, which directly cause apoptosis. In fruit flies, nematodes, and mammals, Apaf-1 has been extensively studied. However, the structure and function of Apaf-1 in Lepidoptera remain unclear. This study identified a novel Apaf-1 from Spodoptera litura, named Sl-Apaf-1. Sl-Apaf-1 contains three domains: a CARD domain, as well as NOD and WD motifs, and is very similar to mammalian Apaf-1. Interference of Sl-apaf-1 expression in SL-1 cells blocked apoptosis induced by actinomycin D. Overexpression of Sl-apaf-1 significantly enhances apoptosis induced by actinomycin D in Sf9/SL-1/U2OS cells, suggesting that the function of Sl-Apaf-1 is evolutionarily conserved. Furthermore, Sl-Apaf-1 could interact with Sl-caspase-5 (a homologue of mammalian caspase-9) and yielded a binding affinity of 1.37 × 106 M-1 according isothermal titration calorimetry assay. Initiator caspase (procaspase-5) of S. litura could be activated by Sl-Apaf-1 (without WD motif) in vitro, and the activated Sl-caspase-5 could cleave Sl-procaspase-1 (a homologue of caspase-3 in mammals), which directly caused apoptosis. This study demonstrates the key role of Sl-Apaf-1 in the apoptosis pathway, suggesting that the apoptosis pathway in Lepidopteran insects and mammals is conserved.

The Antimalarial Compound Atovaquone Inhibits Zika and Dengue Virus Infection by Blocking E Protein-Mediated Membrane Fusion.

Flaviviruses bear class II fusion proteins as their envelope (E) proteins. Here, we describe the development of an in vitro quantitative mosquito-cell-based membrane-fusion assay for the E protein using dual split proteins (DSPs). The assay does not involve the use of live viruses and allows the analysis of a membrane-fusion step independent of other events in the viral lifecycle, such as endocytosis. The progress of membrane fusion can be monitored continuously by measuring the activities of Renilla luciferase derived from the reassociation of DSPs during cell fusion. We optimized the assay to screen an FDA-approved drug library for a potential membrane fusion inhibitor using the E protein of Zika virus. Screening results identified atovaquone, which was previously described as an antimalarial agent. Atovaquone potently blocked the in vitro Zika virus infection of mammalian cells with an IC90 of 2.1 µM. Furthermore, four distinct serotypes of dengue virus were also inhibited by atovaquone with IC90 values of 1.6-2.5 µM, which is a range below the average blood concentration of atovaquone after its oral administration in humans. These findings make atovaquone a likely candidate drug to treat illnesses caused by Zika as well as dengue viruses. Additionally, the DSP assay is useful to study the mechanism of membrane fusion in Flaviviruses.

The V1 region of gp120 is preferentially selected during SIV/HIV transmission and is indispensable for envelope function and virus infection.

A transmission bottleneck occurs during each human immunodeficiency virus (HIV) transmission event, which allows only a few viruses to establish new infection. However, the genetic characteristics of the transmitted viruses that are preferentially selected have not been fully elucidated. Here, we analyzed amino acids changes in the envelope protein during simian immunodeficiency virus (SIV)/HIV deep transmission history and current HIV evolution within the last 15-20 years. Our results confirmed that the V1V2 region of gp120 protein, particularly V1, was preferentially selected. A shorter V1 region was preferred during transmission history, while during epidemic, HIV may evolve to an expanded V1 region gradually and thus escape immune recognition. We then constructed different HIV-1 V1 mutants using different HIV-1 subtypes to elucidate the role of the V1 region in envelope function. We found that the V1 region, although highly variable, was indispensable for virus entry and infection, probably because V1 deletion mutants exhibited impaired processing of gp160 into mature gp120 and gp41. Additionally, the V1 region affected Env incorporation. These results indicated that the V1 region played a critical role in HIV transmission and infection.

Comparison of the effect of increased hepatitis B vaccine dosage on immunogenicity in healthy children and adults.

Hepatitis B (HepB) infection remains a global public health problem, particularly in China. Vaccination for children and adult who are unvaccinated is an effective method for preventing the disease. In this study, we aimed to compare the effects of increased dosage of HepB vaccine on immunogenicity in healthy children and adults. A phase III, controlled, double-blinded clinical trial was performed. The subjects were assigned into groups I (age 5-14 y), II (age 15-24 y), and III (age ≥ 25 y). Subjects were randomly administered either 10 or 5 µg recombinant HepB vaccine; blood samples were collected before and after vaccination to estimate immunogenicity. The results showed that the seroconversion rate and geometric mean concentration of antibody to hepB surface antigen (anti-HBs) did not differ significantly between the dosages in each age group. Anti-HBs levels were reduced with age, and this effect was more obvious in adults administered 5 µg HepB vaccine. In conclusion, both vaccine dosages could be used to immunize children, and the 10 µg vaccine could be used for adults ages 15-24 y, whereas a higher dosage of the HepB vaccine may be required for adults ages 25 y and older.