Incomplete influenza A virus genomes are abundant but readily complemented during spatially structured viral spread

Abstract

the frequency of co-infection and consequently reassortment are therefore likely to play an 53 important role in viral evolution. 54While the ability of a virus particle to enter a cell depends only on the proteins that line 55 the virion surface, subsequent production of viral progeny requires successful expression and 56 replication of the genome. A virion that does not contain, or fails to deliver, a complete genome 57 could therefore infect a cell but fail to produce progeny. IAV particles outnumber plaque-forming 58 units (PFUs) by approximately 10-100 fold 17 , meaning that only a minority of particles establish 59 productive infection at limiting dilution. Recent data suggest that IAV infection is not a binary 60 state, however. Efforts to detect viral proteins and mRNAs at the single cell level have revealed 61 significant heterogeneity in viral gene expression [18][19][20] . These data furthermore suggest that a 62 subset of gene segments is often missing entirely from cells infected at low MOI. Thus, many non-63 plaque-forming particles appear to be semi-infectious, giving rise to incomplete viral genomes 64 (IVGs) within the infected cell 21 . 65Replication and expression of only a subset of the genome may be explained by two 66 potential mechanisms: either the majority of particles lack one or more genome segments, or 67 segments are readily lost in the process of infection before they can be replicated. Electron 68 microscopy has shown that most particles contain eight distinct RNA segments 22 , and FiSH-based 69 detection of viral RNAs indicated that a virion tends to contain one copy of each segment 23 , 70suggesting that most particles contain full genomes. Regardless of the molecular mechanisms 71 that lead to the phenomenon of incomplete IAV genomes, their frequent occurrence suggests 72 that complementation by co-infection at the cellular level is an underappreciated aspect of the 73 viral life cycle. The observation of appreciable levels of reassortment following co-infection at 74Interactions between vRNP segments are thought to play an important role in the 126 assembly of new virions 10,26-29 . To determine whether similar interactions exist that could 127 mediate the co-delivery of segments to the cell, the patterns of segment co-occurrence were 128 analyzed. In performing this analysis, it was again important to take into account the known 129 probability of multiple infection in our single cell assay. As shown in Supplementary Figure 1, cells 130 containing more segments were likely to have been infected with multiple virions. Because such 131 cells are less informative for this analysis, we applied a weighting factor to ensure that results 132 relied more strongly on data from cells with fewer WT segments. Namely, we determined the 133 probability that a given cell acquired its gene constellation by infection with a single virion and 134 weighted data according to this probability to calculate the pairwise correlation between 135 segments. While some significant interactions were obser...

Single-Cell Virus Sequencing of Influenza Infections That Trigger Innate Immunity

“…S4 for frequencies of individual genes). This measured frequency of infected cells expressing all eight genes is slightly higher than in our own prior work using the WSN strain (12) and slightly to substantially higher than that reported in studies by others using different viral strains or methodologies (15, 43, 61, 62).…”

“…Despite the fact that we used a low-passage-number viral stock generated from plasmids, most infected cells did not express unmutated copies of all viral genes. Although our study is certainly not the first to note that influenza virus has a high mutation rate (30–34) and sometimes fails to express genes (12, 15, 43, 61, 62), it is the first to directly observe the full spectrum of these defects across single cells. Visual inspection of Fig.…”

A method for the unbiased quantification of reassortment in segmented viruses

“…In co-infections between highly divergent parents, an inability to package any of the three segments results in attenuation, which has the potential to limit emergence of reassortant viruses. Conversely, only ∼1% of eight-segmented influenza A viruses replicate a full complement of segments within the infected cell, so co-infection is often necessary for successful completion of the viral life cycle 41 . As co-infected cells are the only ones capable of yielding reassortant progeny, a requirement for co-infection increases the likelihood that reassortment will occur.…”

Replication competent, 10-segmented influenza viruses as antiviral therapeutics

“…Finally, the fact that 9S and 10S viruses can interfere with WT viral propagation strongly supports the notion that cellular co-infection is a common occurrence in vivo. Despite the historical notion that most viral particles are fully infectious, likely due to the fact that IAV particles package all eight genomic segments the majority of the time [59,60], recent work has suggested that co-infection may actually be not only a frequent occurrence that allows viral reassortment [61], but also a critical aspect of normal viral spread across infected tissues [62]. Since our 9S or 10S interfering effects are dependent on coinfection with WT viruses, we not only favor this model, but propose that even distinct viral infections that begin at different times are also subject to this co-infection phenomenon.…”