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. 2012 Apr;86(8):4432-43.
doi: 10.1128/JVI.06744-11. Epub 2012 Feb 8.

The human respiratory syncytial virus matrix protein is required for maturation of viral filaments

Ruchira Mitra  1 , Pradyumna Baviskar, Rebecca R Duncan-Decocq, Darshna Patel, Antonius G P Oomens
Affiliations

The human respiratory syncytial virus matrix protein is required for maturation of viral filaments

Ruchira Mitra et al. J Virol. 2012 Apr.

Abstract

An experimental system was developed to generate infectious human respiratory syncytial virus (HRSV) lacking matrix (M) protein expression (M-null virus) from cDNA. The role of the M protein in virus assembly was then examined by infecting HEp-2 and Vero cells with the M-null virus and assessing the impact on infectious virus production and viral protein trafficking. In the absence of M, the production of infectious progeny was strongly impaired. Immunofluorescence (IF) microscopy analysis using antibodies against the nucleoprotein (N), attachment protein (G), and fusion protein (F) failed to detect the characteristic virus-induced cell surface filaments, which are believed to represent infectious virions. In addition, a large proportion of the N protein was detected in viral replication factories termed inclusion bodies (IBs). High-resolution analysis of the surface of M-null virus-infected cells by field emission scanning electron microscopy (SEM) revealed the presence of large areas with densely packed, uniformly short filaments. Although unusually short, these filaments were otherwise similar to those induced by an M-containing control virus, including the presence of the viral G and F proteins. The abundance of the short, stunted filaments in the absence of M indicates that M is not required for the initial stages of filament formation but plays an important role in the maturation or elongation of these structures. In addition, the absence of mature viral filaments and the simultaneous increase in the level of the N protein within IBs suggest that the M protein is involved in the transport of viral ribonucleoprotein (RNP) complexes from cytoplasmic IBs to sites of budding.

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Figures

Fig 1
Fig 1
Transient and stable expression of the M protein from a codon-optimized ORF. (A) Western blot analysis. Lysates of transfected HEp-2 cells were prepared at 30 hpx, and a Western blot was generated after SDS-PAGE under reducing conditions. Lane 3 represents cells transfected with a plasmid expressing M from a codon-optimized ORF (pc-Mopt). As controls, lysates with equivalent cell numbers of mock-transfected cells (lane 4), wt virus-infected cells (lane 1), and uninfected cells (lane 2) were included. (For lane 5, see panel C.) The positions of molecular weight markers (in thousands) and the M protein are indicated. (B) Transient expression of M determined by IF microscopy. pc-Mopt-transfected cells (panels 2 and 3) or mock-transfected cells (panel 1) on glass coverslips were fixed and permeabilized at 30 hpx and incubated with anti-M peptide serum and Alexa-488-conjugated anti-rabbit antibodies. Samples were examined with a fluorescence microscope and photographed at a ×600 magnification. Panels 2 and 3 represent differential expression patterns observed within the same sample. (C) Analysis of the M-expressing cell line H2-M. Tet-responsive H2-M cells were induced for M expression by the addition of doxycycline (DOX) to the medium (+DOX) (panels 3 and 4) or were left uninduced (−DOX) (panels 1 and 2), and cells were fixed and permeabilized 30 h after induction. M was visualized as described above for panel B (panels 2 and 4), and cells were counterstained with DAPI to visualize nuclei (panels 1 and 3). The samples were examined on a fluorescence microscope and photographed at a ×200 magnification. A lysate of induced H2-M cells at 30 h postinduction was also examined by Western blotting (panel A, lane 5).
Fig 2
Fig 2
Composition of engineered virus genomes and viral protein expression. (A) Genome content of engineered viruses. In the “M-null” virus, the M ORF was replaced with a modified M ORF in which AUGs were mutated to ablate expression, and the SH ORF was replaced with the EGFP ORF for tracking purposes. (A virus with the SH ORF being replaced with that of EGFP was previously shown to replicate in a manner indistinguishable from that of the wt virus [10, 43, 44].) The properties of the M-null virus were compared to those of the “recWT” control virus. The recWT virus also has a replacement of the SH ORF with that of EGFP but contains an unaltered (wt) M ORF. The recWT virus resembles the previously reported RSΔSH virus (43) but is a second-generation virus in which all artificial restriction sites were removed. (B) Viral protein expression determined by cell ELISA. HEp-2 cells were infected with the M-null or recWT virus or left uninfected as a control and were fixed and permeabilized at 26 hpi. Relative protein levels were measured with anti-N, -M, -G, and -F antibodies (Ab) by a cell ELISA as previously described (42, 44). OD490, optical density at 490 nm. A representative result of several independent experiments is shown.
Fig 3
Fig 3
Viral replication in cell culture in the absence of M. (A) Flow cytometry. HEp-2 cells were infected with the M-null or recWT virus by the addition of an inoculum (∼ 0.2 PFU/cell) and centrifugation for 10 min at 3,000 × g. Immediately after infection and at 1-day intervals, progeny virus was harvested by scraping cells into the medium and transferring the samples to −80°C. Frozen samples were thawed, mixed by gentle pipetting, cleared by low-speed centrifugation, and used to infect freshly plated (receiver) HEp-2 cells. At 24 hpi, receiver cells were trypsinized and fixed with 4% paraformaldehyde in suspension, and the percentage of GFP-expressing cells was determined by flow cytometry. The data represent the average values of duplicate samples. (B) HEp-2 cells were similarly infected with the M-null (top) or recWT (bottom) virus. At days 1, 2, 3, and 4 postinfection, GFP expression within the infected cultures was examined with a fluorescence microscope and photographed at a magnification of ×100. A phase-contrast image at day 4 postinfection was also included [Day 4 (phase)].
Fig 4
Fig 4
Subcellular distribution of the N, G, and F proteins in infected cells in the absence of M. Vero cells infected with the recWT (B, D, and F) or M-null (A, C, and E) virus were fixed at 26 hpi and detergent permeabilized. Samples were incubated with anti-N (A and B), anti-G (C and D), or anti-F (E and F) antibodies. Following incubation with Alexa-594-conjugated secondary antibodies, samples were examined with a confocal microscope by scanning sequentially for EGFP expression (green) and N, G, or F expression (red). Images were overlaid. Magnification, approximately ×2,000.
Fig 5
Fig 5
High-resolution analysis of the surface of M-null virus-infected cells. HEp-2 cells were infected, as described in the legend of Fig. 3A, with the recWT (A and B) or M-null (C and D) virus or left uninfected (E and F). At 26 hpi, cells were incubated stepwise with anti-G (L9) and anti-F (Synagis) antibodies and goat anti-mouse or goat anti-human antibodies conjugated to 15-nm and 25-nm colloidal gold, respectively, and processed for field emission SEM analysis as described in Materials and Methods. Samples (approximately 25 fields containing infected cells) were examined by SEM and scanned at magnifications of ×20,000 using the secondary electron (SE) detection mode (A, C, and E) and ×100,000 using both SE and backscattered electron (BSE) detection modes to also visualize gold particles. Photographs of SE and BSE scans were overlaid (B, D, and F). White arrowheads indicate 25-nm gold particles (F protein), and white arrows indicate 15-nm gold particles (G protein).
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