Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family of nonsegmented, negative-sense, single-stranded RNA genome viruses, is a leading cause of lower respiratory tract infections in infants, young children, and the elderly or immunocompromised. There are many open questions regarding the processes that regulate human RSV (hRSV) assembly and budding. Here, using cryo-electron tomography, we identified virus particles that were spherical, filamentous, and asymmetric in structure, all within the same virus preparation. The three particle morphologies maintained a similar organization of the surface glycoproteins, matrix protein (M), M2-1, and the ribonucleoprotein (RNP). RNP filaments were traced in three dimensions (3D), and their total length was calculated. The measurements revealed the inclusion of multiple full-length genome copies per particle. RNP was associated with the membrane whenever the M layer was present. The amount of M coverage ranged from 24% to 86% in the different morphologies. Using fluorescence light microscopy (fLM), direct stochastic optical reconstruction microscopy (dSTORM), and a proximity ligation assay (PLA), we provide evidence illustrating that M2-1 is located between RNP and M in isolated viral particles. In addition, regular spacing of the M2-1 densities was resolved when hRSV viruses were imaged using Zernike phase contrast (ZPC) cryo-electron tomography. Our studies provide a more complete characterization of the hRSV virion structure and substantiation that M and M2-1 regulate virus organization. IMPORTANCEhRSV is a leading cause of lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. We used cryo-electron tomography and Zernike phase contrast cryo-electron tomography to visualize populations of purified hRSV in 3D. We observed the three distinct morphologies, spherical, filamentous, and asymmetric, which maintained comparable organizational profiles. Depending on the virus morphology examined, the amount of M ranged from 24% to 86%. We complemented the cryo-imaging studies with fluorescence microscopy, dSTORM, and a proximity ligation assay to provide additional evidence that M2-1 is incorporated into viral particles and is positioned between M and RNP. The results highlight the impact of M and M2-1 on the regulation of hRSV organization.

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Vibrio vulnificus is an opportunistic human pathogen [1]. Its virulence is regulated by cellular appendages (flagellum and pili) as well as other structural components, such as the capsular polysaccharide [2]. Recently, V. vulnificus was found to produce outer membrane vesicles (OMVs) that can induce host cell death [3] and may contain a variety of virulence factors [4]. Our goal was to study the formation V. vulnificus OMVs using cryo-electron microscopy.V. vulnificus clinical isolates MO6-24/O and CMCP6 were grown in Luria-Bertani medium supplemented with 2.0% (w/v) NaCl. Cultures were incubated overnight (250 rpm at 28°C) and flash frozen onto glow-discharged Quantifoil carbon grids in liquid ethane with an FEI Vitrobot. Cryo-EM data was collected with a JEOL 2200FS 200 kV, field emission TEM with an incolumn energy filter, on a Gatan 4kx4k high resolution CCD camera.