Research Article: Cryotomography of Budding Influenza A Virus Reveals Filaments with Diverse Morphologies that Mostly Do Not Bear a Genome at Their Distal End

Date Published: June 6, 2013

Publisher: Public Library of Science

Author(s): Swetha Vijayakrishnan, Colin Loney, David Jackson, Worawit Suphamungmee, Frazer J. Rixon, David Bhella, Peter Palese.

http://doi.org/10.1371/journal.ppat.1003413

Abstract

Influenza viruses exhibit striking variations in particle morphology between strains. Clinical isolates of influenza A virus have been shown to produce long filamentous particles while laboratory-adapted strains are predominantly spherical. However, the role of the filamentous phenotype in the influenza virus infectious cycle remains undetermined. We used cryo-electron tomography to conduct the first three-dimensional study of filamentous virus ultrastructure in particles budding from infected cells. Filaments were often longer than 10 microns and sometimes had bulbous heads at their leading ends, some of which contained tubules we attribute to M1 while none had recognisable ribonucleoprotein (RNP) and hence genome segments. Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends. Imaging of purified virus also revealed diverse filament morphologies; short rods (bacilliform virions) and longer filaments. Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length. The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process.

Partial Text

Each year influenza A viruses cause seasonal epidemics, in which many millions of people worldwide become infected. Pandemic strains emerge periodically as a consequence of the segmented nature of the influenza virus genome that predisposes these viruses to reassortment. Complementary subsets of genome segments from two parental strains come together to form a novel virus with a new antigenic character and possibly altered virulence or species specificity. Influenza A viruses are enveloped, single-stranded negative-sense RNA viruses within the family Orthomyxoviridae. The viral envelope is derived from the host cell plasma membrane and bears the glycoproteins haemagglutinin (HA) and neuraminidase (NA) as well as the ion channel protein M2, all of which are critical for virus entry and egress. Beneath the viral envelope is a layer of matrix protein (M1), which is important for virion morphogenesis [1]. The virus interior contains the viral genome, which consists of eight separate RNA molecules [2], [3]. These genome segments are encapsidated by the nucleoprotein (NP) forming eight ribonucleoprotein complexes (RNPs, also termed nucleocapsids), each of which is associated with a viral RNA dependent RNA polymerase (RdRp). Segments one, two and three code for the RdRp proteins (PB2, PB1 and PA respectively), segment four for HA, segment five for NP, segment six for NA, segment seven for M1 and M2, and segment eight for the non-structural protein NS1 and the nuclear export protein (NEP) [4]–[6]. Efficient packing of each segment into a budding virion is directed by specific cis-acting RNA sequences. [7], [8].

We have examined influenza A/Udorn/72 filament formation by CET and immunofluorescent confocal microscopy. Confocal microscopy revealed the presence of a variety of filamentous forms: long straight filaments, flexible filaments and Archetti bodies. CET demonstrated that filaments produced in MDCK cells frequently do not contain ordered RNPs at their distal ends and more often terminate with empty ends or Archetti varicosities. These data represent the first structural analysis of influenza virus filaments budding from the host cell under near native conditions. Early metal shadowing TEM studies of filamentous virions produced in eggs described Archetti bodies and proposed that they might bear spherical virions [12], [28]. Our data refute these suggestions and reveal the absence of RNPs or spherical virions in the terminal varicosities of Archetti bodies. It is unclear from our analysis whether these particles bud from the cell surface as bulbous structures or whether the terminal varicosity forms after budding. The presence of tubular assemblies that we attribute to M1 in many Archetti termini suggests that the latter may be the case. However, the envelope measures approximately 12 nm thick in this region, similar to measurements made of the envelope/matrix component of purified particles, budding filaments and empty Archetti bodies. This suggests that the presence of M1 tubes might not be the result of them having detached from the inner surface of the envelope, an occurrence that could result in loss of filamentous form. We frequently saw density within filaments that appears to be a second layer of M1, such as the helical assemblies shown in figure 6 and inclusions within varicosities (Fig S6D). It is possible then, that concentric layers of M1 may be a common feature of influenza A filaments. Owing to the missing wedge we are unable to unambiguously image the entire envelope of the Archetti varicosity however. Thus the origin of the putative M1 tubes cannot be conclusively proven and further experimentation is required to establish the origin of these structures and the morphogenetic pathway of Archetti particles.

 

Source:

http://doi.org/10.1371/journal.ppat.1003413

 

0 0 vote
Article Rating
Subscribe
Notify of
guest
0 Comments
Inline Feedbacks
View all comments