Specific Residues of the Influenza A Virus Hemagglutinin Viral RNA Are Important for Efficient Packaging into Budding Virions

Abstract

The influenza A virus genome is composed of eight negativesense RNA segments (22, 26), which can encode up to 11 viral proteins (3, 23). Upon infection, the viral RNA (vRNA) is transported to the nucleus, the site of vRNA transcription and replication. At late stages of the infectious cycle, the viral ribonucleoprotein complex, composed of the three influenza polymerase proteins, the nucleoprotein, and vRNA, is exported from the nucleus in association with the influenza virus matrix (M1) and nuclear export (NEP) proteins (5). The final step of the virus replication cycle involves the assembly of the viral structural proteins and the packaging of the viral genome. For a virus particle to be fully infectious, it must contain a full complement of the eight vRNA segments. The process by which this packaging occurs is not well understood. However, it is known that vRNA is specifically packaged in preference to cellular RNAs and that the different vRNAs are present in an equimolar ratio within a population of virus particles (21).Two models have been proposed for the packaging of vRNA into budding virions: the random incorporation model and the selective incorporation model (23). The random incorporation model assumes that a common structural feature is present on all vRNAs which enables them to be randomly incorporated into budding virions. In other words, the virus randomly incorporates vRNA into budding virions and does not differentiate among the different segments (4). This means that the likelihood of a virion obtaining a full complement of the eight vRNAs is determined entirely by chance. This model is supported by evidence that infectious viruses may possess more than eight vRNAs (8). Mathematical modeling suggests that if eight vRNAs are packaged randomly, 0.24% of released virus particles would contain a full complement of vRNAs and be infectious (1,8). If 12 vRNAs were packaged randomly, the mathematical models suggest that infectivity increases to approximately 10%, which is comparable with the experimental data (6). As only 1 to 2% of the weight of the influenza virus particle is vRNA, it is difficult to accurately quantify the exact number of vRNA segments packaged. The earliest evidence suggesting that influenza vRNAs have packaging signals was from Luytjes et al. (14). Utilizing both the 3Ј and 5Ј terminal untranslated regions of the NS gene, it was possible to package a chloramphenicol acetyltransferase gene into influenza virus particles. This foreign gene was packaged into infectious particles and passaged several times, suggesting that the terminal 22 5Ј and 26 3Ј nucleotides are sufficient to provide the signals for RNA transcription and replication as well as for the packaging of RNA into influenza virus particles.The selective incorporation model suggests that each vRNA segment contains a unique "packaging signal" allowing it to act independently, with each vRNA segment being packaged selectively. Evidence supporting this model comes from several reports demonstrating that defective interfering ...

Genome packaging in influenza A virus

“…4c), and in every case, the terminal coding regions, comprising sequences unique for each segment, have been found to promote more efficient packaging than the UTRs alone (Dos Santos Afonso et al, 2005;Fujii et al, 2003;Fujii et al, 2005;Gog et al, 2007;Liang et al, 2005Liang et al, , 2008Marsh et al, 2007;Muramoto et al, 2006;Ozawa et al, 2007Ozawa et al, , 2009Watanabe et al, 2003). The observation that virus formation is inefficient in the absence of the terminal regions of all eight vRNAs has also been corroborated and extended by examining the effects of removing other segments (de Wit et al, 2006;Fujii et al, 2009;Gao et al, 2008;Gao & Palese, 2009;Marsh et al, 2007).…”

“…The finding that alteration of a small number of nucleotides (even as few as one) can have a profound effect on segment incorporation is perhaps surprising, given the apparently large size of the signals. However, in many cases, other mutations within areas predicted to be part of the packaging signal had no apparent effect on its function (Fujii et al, 2005;Gog et al, 2007;Hutchinson et al, 2008Hutchinson et al, , 2009Liang et al, 2008;Marsh et al, 2007Marsh et al, , 2008. This may, in part, reflect the discontinuous nature of the signals, as suggested by analysis of sequence conservation (Gog et al, 2007).…”

“…A major advantage of this approach is that it can be applied to otherwise wild-type virus (Fujii et al, 2005;Hutchinson et al, 2008Hutchinson et al, , 2009Liang et al, 2008;Marsh et al, 2007Marsh et al, , 2008Ozawa et al, 2009) as well as to constructs containing reporter genes (Fujii et al, 2005;Fujii et al, 2009;Gog et al, 2007;Liang et al, 2008). For all segments, point mutagenesis has identified nucleotides, individually or in small clusters, whose alteration causes significant reductions in packaging of the altered segment as well as (where examined) reductions in the titre of replicated virus (Fujii et al, 2005;Fujii et al, 2009;Gog et al, 2007;Hutchinson et al, 2008Hutchinson et al, , 2009Liang et al, 2008;Marsh et al, 2007Marsh et al, , 2008 (Fig. 4e).…”

“…(e) Point mutagenesis. Red lines indicate regions where clusters of mutations or small deletions have substantially reduced segment packaging (Fujii et al, 2005;Gog et al, 2007;Hutchinson et al, 2008Hutchinson et al, , 2009Liang et al, 2008;Marsh et al, 2007Marsh et al, , 2008.…”

“…(c) Packaging of reporter genes. The shortest sequences shown to cause efficient packaging of reporter genes are indicated by green bars Fujii et al, 2005;Liang et al, 2005;Marsh et al, 2007;Ozawa et al, 2007Ozawa et al, , 2009). The region indicated for segment 7 promotes packaging relatively poorly, and may not contain the complete packaging signal (Muramoto et al, 2006;Ozawa et al, 2009).…”