| Background:As a member of the paramyxovirus family, human parainfluenza virus type 3 (HPIV3) is an enveloped virus harboring a nonsegmented, negative-sense, single-stranded RNA genome. HPIV3 can cause serious respiratory illnesses including pediatric croup, bronchiolitis, and pneumonia in children aged younger than 5 years all over the world. At present, there are still virtually no licensed vaccines or antiviral agents are available for HPIV3. The main hurdle for developing safe and effective vaccines is that the mechanism of HPIV3 infecting host cells is unclear. Therefore, understanding this mechanism is required first.Fusion of the viral envelope with a host cell membrane is an essential step for its entry into the cytoplasm of the target cells to cause virus spread and the cell-cell fusion, and the cell-cell fusion is also the pathological characteristics of infected cells. Two types of virion-associated surface glycoproteins, the hemagglutinin-neuraminidase (HN) protein and the fusion (F) protein, cooperate to facilitate this process. The HPIV3 fusion (F) glycoprotein, belonging to the class I virus membrane fusion protein, is synthesized as an inactive precursor (F0) that is cleaved by a host cell proteolytic enzyme in the trans Golgi apparatus into a biologically active form consisting of a larger carboxy-terminal subunit (F1) and a smaller amino-terminal subunit (F2).Several structural features of this protein are known to be important for its fusion activity. Fusion peptide is responsible for inserting into the host cell membrane to initiate the fusion process. Two heptad repeat domains (HRA and HRB), which have a strong affinity, could form a conserved six-helical bundle (6HB) that pulls the viral and target cell membranes together. Between the two heptad repeat domains there are about 250 amino acid residues that comprise three domains (DⅠ-DⅢ) and two linkers (the DⅠ-DⅡ linker and the HRB linker). The DⅠ-DⅡ linker (369aa-374aa) connects domains DⅠ and DⅡ. It is noteworthy that only the X-ray crystallographic structure of the HPIV3 F protein in the postfusion conformation is currently available and HRA, HRB, domains DⅠ-DⅢ and the two linkers have been found in this structure. Up to now, although several F protein regions have been considered to have important effects on regulating the membrane fusion activity, whether the DⅠ-DⅡ linker of the HPIV3 F protein has an effect on fusogenicity is as yet unclear.Objectives:To explore the role of the DⅠ-DⅡ linker of the HPIV3 F protein in fusion activity, to illustrate the mechanisms of this domain affecting membrane fusion, to provide clues for the developing of antiviral vaccines and therapeutic drugs to prevent and cure HPIV3 infection.Methods:1. The homology structural model of the HPIV3 F protein in the prefusion conformation based on the PIV5 (parainfluenza virus 5, PIV5) prefusion F-trimer structure (PDB ID 4GIP) was initially generated to relocate the new position of this linker in it.2. Site-directed mutagenesis was employed to substitute each amino acid residue in this domain. A transient expression system using the vaccinia virus-T7 RNA polymerase was used to express the wild-type or mutated F proteins3. Three different types of membrane fusion assays (Giemsa stain assay, the report gene assay and the dye transfer assay) were employed to analyze the fusogenic activity.4. Fluorescence-activated cell sorting (FACS) analysis was executed to examine the cell surface expression level.5. A co-immunoprecipitation assay was conducted to probe the HN-F interaction at the cell surface.6. Statistical analysis was performed by SPSS 17.0 software. All results were expressed as the mean ± SD from at least three separate experiments. Statistical analysis was conducted using Student’s t-test, P< 0.05 was considered statistically significant.Results:1. The homology model was successfully generated and the DI-DII linker (residues369-374) of the HPIV3 F protein in the postfusion conformation still acted as the DI-DII linker in its prefusion form by the amino acid sequence alignment.2. Nine mutants (K369A, K369E, S370A, D371A, I372A, V373A, V373T, P374A and P374T) in this domain was successfully generated.3. All mutants exhibited diminished fusion activity in all steps of the fusion process when coexpressed with their homologous HN proteins. The syncytia formed by these mutants were not only smaller but also fewer in comparison with those produced by wt F protein, the K369E, S370A, V373A, V373T and P374T mutations almost abrogated their abilities to form syncytia, with extents less than 5.0% of the wt F and HN level, and resulted in debilitated content mixing, which retained only 18.0%, 17.3%,16.4%,18.9% and 15.4% of the wt F level, respectively, their hemifusion activity also sharply diminished, both the rate and extent of the initial fusion were less than 15% of those of the levels of wt F and HN. The remainder of the mutants in this domain (K369A, D371A, I372A and P374A) were capable of forming syncytia, though at lower levels than those seen in the wt F protein, the K369A and D371A mutants directed approximately two-thirds of the levels of content mixing of the wild-type protein, with the extents and rates of hemifusion about three quarters (74.5%,75.2%, respectively) and four fifths (82.2%,82.0%, respectively) of those for the wt F and HN; the I372A and P374A mutants reduced the levels of content mixing to 57.6% and 54.5% of that of wt F, respectively, decreased the initial rate to 58.5% and 51.8%, respectively, and diminished the initial extent to 62.6% and 60.6%, respectively.4. Substitutions in the DI-DII linker had no effect on the cell surface expression of F. Each of the mutated proteins gave a mean fluorescent intensity that was similar to that of the wt F protein, ranging from 93.1% to 105.9% of the wt F level.5. Mutations in the DI-DII linker exhibited defects in the F-HN interaction. The K369E, V373A, V373T and P374T mutated F proteins in the presence of HPIV3 HN coexpression, each of which fused less than 19% of the wt level, almost lost their abilities to co-IP with HN protein, corresponding to percentages of less than 10.0%; The S370A mutant co-immunoprecipitated at approximately one fifth of the wt F level. K369A, D371A, I372A and P374A mutated F proteins co-immunoprecipitated at 41.4%,66.0%,26.9% and 15.2%, respectively.Conclusions:The HPIV3 F protein DI-DII linker has an important effect on the fusogenic activity of F, and not all residues were of equal effects on F-induced cell-to-cell fusion. The reduction in the ability of each mutated F protein to interact with their homologous HN protein at the cell surface may be responsible for the loss of fusion activity.Background:The penetration of HPIV3 into host cells occurs via the merging of viral envelopes with susceptible cell membranes, which results in the delivery of the viral genome (a single-stranded, nonsegmented, negative-sense RNA) into the cytoplasm of the target cell. Two types of virion-associated surface glycoproteins, the hemagglutinin-neuraminidase (HN) protein and the fusion (F) protein, cooperate to facilitate this process. The HN glycoprotein is responsible for initial binding between the virus and the target cells, whereas the F glycoprotein is involved in disrupting the host-cell plasma membrane and directly inducing membrane fusion.There is a large intervening region between HRA and HRB, which comprise three domains or domains DⅠ-DⅡ, they were connected through the linker regions. The DⅠ-DⅡ linker (369aa-374aa) connects domains DⅠ and DⅡ; The HRB linker connects domains DⅡ and HRB; Domains DI and DⅢ connects directly, there is no amino acid sequence to connect the two domains, so we defined it as "false linker region", and sequence analysis indicates that it contains a leucine zipper-like motif.Leucine zippers are a special class of heptad repeats that contain leucine or isoleucine at every seventh amino acid residue, containing nonpolar residues in all "a" positions and in most "d" positions when displayed on an α-helical wheel. The leucine zipper motif was first described in several DNA-binding proteins including transcriptional regulators (e.g., yeast GCN4 and mammalian C/EBP) and nuclear transforming proteins such as Jun, Fos, and Myc. Previous studies of this motif in three different paramyxovirus systems have suggested that this region is important for fusion activity. However, whether the leucine zipper-like motif at the DI-DIII linker of the HPIV3 F protein plays an important role in fusion activity has not yet been determined.Objectives:To investigate the effect of the leucine zipper-like motif at the DI-DIII linker of the HPFV3 F protein on fusion activity, and to seek for the mechanisms of this domain affecting membrane fusion.Methods:Point mutations were introduced by using site-directed mutagenesis to substitute the heptadic residues at amino acids 257,264,271,278,285,292, and 299 in this motif with an alanine residue. Additionally, three highly conserved middle heptadic leucine residues (L271, L278, and L285) were replaced with alanine in various combinations. A transient expression system using the vaccinia virus-T7 RNA polymerase was used to express the wild-type or mutated F proteins. Three different types of membrane fusion assays i.e., syncytium formation assay, content mixing assay and lipid mixing assay, were performed. Flow cytometry was performed to assay the cell surface expression level. A co-immunoprecipitation assay was conducted to probe the abilities of HPIV3 wt or mutated F proteins to interact with their homologous HN proteins at the cell surface. Statistical analysis was performed by SPSS 17.0 software. All results were expressed as the mean ± SD from at least three separate experiments. Statistical analysis was performed using Student’s t-test, P < 0.05 was considered statistically significant.Results:1. Mutations were introduced both individually and in combination. Seven single mutants (L257A, V264A, L271 A, L278A, L285A, Q292A and I299A), three double mutants (L271A-L278A, L271A-L285A and L278A-L285A) and one triple mutant (L271A-L278A-L285A) in this domain was successfully generated.2. All mutants reduced or even abrogated fusion activity in all fusion steps when coexpressed with the HPIV3 HN protein. The syncytia formed in monolayer cells coexpressing mutated F and HN proteins were not only smaller but also fewer in contrast to syncytia observed in cells coexpressing the HPIV3 wt F and HN proteins, Three single mutants (L257A, L271A, and I299A) and three double mutants were severely debilitated for syncytium formation, less than 6.0% of that of the wt F and HN proteins, had markedly diminished content mixing, corresponding to a percentage of less than 16.0% of the wt F and HN level, and gave only a very small number of fusion events, both the rate and extent of hemifusion were less than 9.5% of the wt F and HN level; whereas the V264A, L285A and Q292A mutants displayed a slightly decreased extent of syncytium formation,64.7%,69.1%,68.5% of the wt F and HN level, respectively, retained about 85% of wild-type level in content mixing, the initial rates of fusion for them were 88.8%,87.6%, and 85.0%, respectively, and the extents of dequenching at 5 min were 85.5%,87.2% and 83.1%, respectively. In addition, while the L278A mutant resulted in a decrease in syncytium formation, to 38.1% of the wt F and HN level, reduced fusion activity to approximately 50% of the wt F level, and caused approximately half of the dye transfer of wt F, with the initial rate and extent of hemifusion reduced to 52.8% and 60.6% of the wt level; the triple mutant (L271A-L278A-L285A) lost its ability to form syncytia, only a background level of fusion activity (2.2% of wt F) was detected, no dye transfer events was observed from it, both the rate and extent of hemifusion were less than 9.5% of the wt F and HN level.3. Substitutions in this motif cause no reductions in the cell surface expression of F. All of the mutated F proteins were expressed at the cell surface at levels quite comparable to that of the wt F protein.4. All mutants interfered with the formation of HN-F complexes at the cell surface. Three single mutant proteins (L257A, L271A, and I299A) were co-immunoprecipitated with the anti-HN MAbs less than 30% of the wt F amount. Three double mutant proteins co-IPs less than half of the wt F protein. The V264A, L285A and Q292A mutant F proteins can be co-immunoprecipitated at 63.3%,64.3% and 66.7% of the wt level, respectively. In addition, the L278A mutant co-immunoprecipitated at 65.1% of the wt F level, and there is no detectable co-immunoprecipitation of the F protein for the triple mutant.Conclusions:This leucine zipper-like motif at the DⅠ-DⅢ linker of the HPIV3 F protein has an important influence on fusion activity; the mutations in combitions have the most influence on membrane fusion; and its integrality is indispensable for membrane fusion.Background:The HPIV3 fusion (F) glycoprotein, belonging to the class Ⅰ virus membrane fusion protein, is synthesized as a precursor designated as FO and is cleaved during transport through the Golgi membranes into two disulfide-linked subunits, F1 and F2.Previous studies have suggested that several structural features, such as FP, HRA and HRB, of this protein are known to be important for its fusion activity. It is noteworthy that Yin et al. discovered that in the prefusion conformation of the PIV5 F protein, there is a region that shows weak electron density, defined as the HRB linker. Mutational analysis of the 454 amino acid of the NDV F protein HRB linker indicated that this amino acid is important for membrane fusion. Russell et al mutated the SV5 F protein at conserved residues L447 and 1449, which are proximal to HRB, discoveres that the two amio acids are important for the 6HB structure and fusion activity, suggesting this region is involved in a conformational switch. Up to now, whether the HRB linker of the HPIV3 F protein has an effect on fusogenicity is as yet unclear.Objectives:To explore the effect of the HRB linker of HPIV3 F protein on fusion activity, and to probe the mechanisms of this domain affecting membrane fusion.Methods:Site-directed mutagenesis was employed to substitute T429, E432,1443 and N446 in this domain. A transient expression system using the vaccinia virus-T7 RNA polymerase was used to express the wild-type or mutated F proteins. Three different types of membrane fusion assays (syncytium formation assay, content mixing assay and lipid mixing assay) were executed to analyze the fusogenic activity. Statistical analysis was performed by SPSS 17.0 software. All results were expressed as the mean ± SD from at least three separate experiments. Statistical analysis was conducted using Student’s t-test, P< 0.05 was considered statistically significant.Results:1. Six mutants (T429A, T429L, E432A, I443A and N446S) in this domain was successfully generated.2. When coexpressed with their homologous HN proteins, these mutants exhibited two types of membrane fusion.The syncytia formed by T429A, T429L and N446A were not only smaller but also fewer in comparison with those produced by wt F protein, the levels of content mixing diminished, which retained only 36.6%,42.3%, 15.2% of the wt F level, respectively, and their hemifusion activity decreased to 34.8% 42.7% and 12.4%, respectively; the fusion activity of E432A,1443A and N446S is mildly higher than wt F in whole, corresponding to 109.4%,113.2% and 118.6% of the wt F level, respectively, but their hemifusion was comparable to that of the wt F.Conclusions:The HRB linker of HPIV3 F protein has an important effect on the fusogenic activity of F, and the N446 was the key amino acid. |
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