Critical role of the fusion protein cytoplasmic tail sequence in parainfluenza virus assembly

Abstract

Interactions between viral glycoproteins, matrix protein and nucleocapsid sustain assembly of parainfluenza viruses at the plasma membrane. Although the protein interactions required for virion formation are considered to be highly specific, virions lacking envelope glycoprotein(s) can be produced, thus the molecular interactions driving viral assembly and production are still unclear. Sendai virus (SeV) and human parainfluenza virus type 1 (hPIV1) are highly similar in structure, however, the cytoplasmic tail sequences of the envelope glycoproteins (HN and F) are relatively less conserved. To unveil the specific role of the envelope glycoproteins in viral assembly, we created chimeric SeVs whose HN (rSeVhHN) or HN and F (rSeVh(HN+F)) were replaced with those of hPIV1. rSeVhHN grew as efficiently as wt SeV or hPIV1, suggesting that the sequence difference in HN does not have a significant impact on SeV replication and virion production. In sharp contrast, the growth of rSeVh(HN+F) was significantly impaired compared to rSeVhHN. rSeVh(HN+Fstail) which expresses a chimeric hPIV1 F with the SeV cytoplasmic tail sequence grew similar to wt SeV or rSeVhHN. Further analysis indicated that the F cytoplasmic tail plays a critical role in cell surface expression/accumulation of HN and F, as well as NP and M association at the plasma membrane. Trafficking of nucelocapsids in infected cells was not significantly affected by the origin of F, suggesting that F cytoplasmic tail is not involved in intracellular movement. These results demonstrate the role of the F cytoplasmic tail in accumulation of structural components at the plasma membrane assembly sites.

Figure 1. Rescue and characterization of rSeVhHN, rSeVh(HN+F) and rSeVh(HN+Fstail).

(A) Aligned amino acid sequences of the F and HN cytoplasmic tails of hPIV1 and SeV. Conserved amino acids are highlighted in red. (B) Schematic diagram of rSeV genomes compared with wt SeV and hPIV1. With rSeVhHN, the SeV HN gene was replaced with that of hPIV1. In rSeVh(HN+F), both HN and F genes of SeV were replaced with those of hPIV1. For rSeVh(HN+Fstail), the entire cytoplasmic tail portion of hPIV1 F shown in Figure 1A was replaced with that of SeV F. (C) Expression of HN and F from rSeVs. IF analysis of A549 cells infected with wt SeV, hPIV1 or rSeV. Origins of HN or F were confirmed using cross reactive (cr) or specific (sp) mAb for NP (cr M52), HN (SeV-sp S16, hPIV1-sp P24), F (SeV-sp M38, cr P38) or M (hPIV1-sp P3) , , , , .

Figure 2. Virus growth kinetics and plaque formation of rSeVs.

(A) Multi-step growth curve of the viruses in LLC-MK₂ cells. Cells were infected with wt or chimeric viruses at MOI 0.01 and incubated at 34°C. Aliquots of infected cell supernatants were collected at indicated times after infection and viral titers of supernatants were determined in LLC-MK₂ cells. (B) Plaque formation of the wt and rSeVs. LLC-MK₂ cells were infected with SeV, rSeVhHN, rSeVh(HN+F), rSeVh(HN+Fstail) or hPIV1 and cultured at 34°C with medium containing agarose. Plaques were identified using crystal violet staining.

Figure 3. Virus production from infected cells.

(A) Virion production from LLC-MK₂ cells infected with SeV (lane 1), rSeVhHN (lane 2), rSeVh(HN+F) (lane 3), or rSeVh(HN+Fstail) (lane 4). Cells were infected at a MOI of 1 and labeled with [³⁵S] Met/Cys for 16 h. Labeled progeny virions released from the cells were purified and analyzed by SDS-PAGE. (B) Viral proteins produced in infected cells. HN, F and NP proteins in [³⁵S]-labeled cell lysates as described in (A) were immunoprecipitated using anti-SeV or hPIV1 HN, F or NP antibodies. (C) Amounts of NP in released virions (A) were quantified and shown as the average of three independent experiments with standard deviations.

Figure 4. Surface and subcellular localization of HN and F proteins.

A549 cells were infected with SeV, hPIV1 or rSeVs and incubated for 16 h at 34°C. Cells were then fixed and treated with mAbs against SeV or hPIV1 F (A) or HN (B) without (upper panels) or with (lower panels) permeabilization. Anti-mouse IgG-Texas Red was used as secondary.

Figure 5. M and NP localization below the plasma membrane of infected cells.

A549 cells were infected with the indicated viruses for 16 h at 34°C. Cells were processed for IF using anti-SeV NP mAb or M rabbit serum. Top 3 panels represent localization of M and NP. Merged images are deconvoluted z-stack images of the xy-plane created using an Olympus FV1000 confocal microcope. Bottom 3 panels show plasma membrane, M and NP and are z-stack reconstructions of the ROIs indicated in the whole cell images by white rectangles in the merged images.

Figure 6. Accumulation of vRNP in rSeVh(HN+F)LeGFP-infected cells.

A549 infected with rSeVLeGFP or rSeVh(HN+F)LeGFP were fixed at the indicated hours after infection, and processed for IF to detect M protein (red).