Architecture of ribonucleoprotein complexes in influenza A virus particles

Abstract

In viruses, as in eukaryotes, elaborate mechanisms have evolved to protect the genome and to ensure its timely replication and reliable transmission to progeny. Influenza A viruses are enveloped, spherical or filamentous structures, ranging from 80 to 120 nm in diameter. Inside each envelope is a viral genome consisting of eight single-stranded negative-sense RNA segments of 890 to 2,341 nucleotides each. These segments are associated with nucleoprotein and three polymerase subunits, designated PA, PB1 and PB2; the resultant ribonucleoprotein complexes (RNPs) resemble a twisted rod (10-15 nm in width and 30-120 nm in length) that is folded back and coiled on itself. Late in viral infection, newly synthesized RNPs are transported from the nucleus to the plasma membrane, where they are incorporated into progeny virions capable of infecting other cells. Here we show, by transmission electron microscopy of serially sectioned virions, that the RNPs of influenza A virus are organized in a distinct pattern (seven segments of different lengths surrounding a central segment). The individual RNPs are suspended from the interior of the viral envelope at the distal end of the budding virion and are oriented perpendicular to the budding tip. This finding argues against random incorporation of RNPs into virions, supporting instead a model in which each segment contains specific incorporation signals that enable the RNPs to be recruited and packaged as a complete set. A selective mechanism of RNP incorporation into virions and the unique organization of the eight RNP segments may be crucial to maintaining the integrity of the viral genome during repeated cycles of replication.

Influenza virus pleiomorphy characterized by cryoelectron tomography

“…RNPs. EM studies of stained plastic sections of budding influenza virions have presented evidence for a (7 ϩ 1) configuration of aligned RNPs (17). Our observations of complete ice-embedded elongated virions (class II) are consistent with this model.…”

“…During assembly, a dilemma common to all viruses with segmented genomes must be resolved: how to assign the various segments appropriately to nascent virions? In transverse thinsection electron micrographs of budding influenza virions, a commonly observed motif is a ring with seven features, each thought to be an RNP, surrounding a central such feature (16,17). Their total number, eight, matches the number of distinct RNA segments in the influenza virus genome.…”

Stabilization of protein–protein interactions by small molecules

“…Influenza nucleoprotein (NP) is an essential viral factor, necessary for replication and virion assembly [80]. Hence, inhibitors of NP function could be of interest as therapeutic agents.…”

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.…”

“…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. Ultrathin sections were prepared on nickel grids as described previously (34) and incubated with a saturated sodium periodate solution (33), followed by 0.2 M glycine in phosphate-buffered saline (PBS) buffer. After a PBS wash, the sections were incubated with 1% bovine serum albumin in PBS and then with anti-Myc mouse monoclonal antibody.…”