Three-dimensional analysis of ribonucleoprotein complexes in influenza A virus

Abstract

The influenza A virus genome consists of eight single-stranded negative-sense RNA (vRNA) segments. Although genome segmentation provides advantages such as genetic reassortment, which contributes to the emergence of novel strains with pandemic potential, it complicates the genome packaging of progeny virions. Here we elucidate, using electron tomography, the three-dimensional structure of ribonucleoprotein complexes (RNPs) within progeny virions. Each virion is packed with eight well-organized RNPs that possess rod-like structures of different lengths. Multiple interactions are found among the RNPs. The position of the eight RNPs is not consistent among virions, but a pattern suggests the existence of a specific mechanism for assembly of these RNPs. Analyses of budding progeny virions suggest two independent roles for the viral spike proteins: RNP association on the plasma membrane and the subsequent formation of the virion shell. Our data provide further insights into the mechanisms responsible for segmented-genome packaging into virions.

Herpes Simplex Virus 1 UL47 Interacts with Viral Nuclear Egress Factors UL31, UL34, and Us3 and Regulates Viral Nuclear Egress

“…Vero cells infected with wild-type HSV-1(F), YK545 (⌬UL47), or YK546 (⌬UL47-repair) at a multiplicity of infection (MOI) of 5 for 18 h were examined by ultrathin-section electron microscopy as described previously (31). Immunoelectron microscopy was performed as described previously (33,34). Briefly, Vero cells infected with wild-type HSV-1(F) or YK536 (MEF-UL47) at an MOI of 5 for 18 h were fixed with 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) on ice for 1 h. After a wash with the same buffer, the cells were postfixed with 2% osmium tetroxide on ice for 1 h, washed with distilled water, dehydrated with an ethanol gradient series, incubated in propylene oxide, and embedded in an Epon 812 resin mixture.…”

Packaging of influenza virus genome: Robustness of selection

“…Importantly, no progeny virus particles contain more than eight RNPs (11). Recent 3D analyses using electron tomography (ET) showed that the eight RNPs arranged in the aforementioned "seven plus one" configuration differ in length (12,13) (Fig. 1C).…”

“…(B) EM analysis shows that the eight RNPs are arranged in a distinct pattern when packaged in a virus particle (11). (C) A 3D model of the eight RNPs within a virus particle reconstructed by using ET (13). The RNPs differ in length and are labeled with different colors.…”

“…1C). They are composed of three long RNPs and five shorter RNPs of significantly different lengths, suggesting that the eight RNPs contain at least six different kinds of viral RNA segments (13,14). Interestingly, ET also suggests that the eight RNPs within a virus particle are connected to each other, forming a supramacromolecular complex (12,13).…”

“…They are composed of three long RNPs and five shorter RNPs of significantly different lengths, suggesting that the eight RNPs contain at least six different kinds of viral RNA segments (13,14). Interestingly, ET also suggests that the eight RNPs within a virus particle are connected to each other, forming a supramacromolecular complex (12,13). This observation is consistent with reverse genetics studies demonstrating that mutations in a packaging signal of a viral RNA segment affect the packaging efficiency of the other viral RNA segments in the progeny virus particles (15).…”