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Functionality of IAV packaging signals depends on site-specific charges within the viral nucleoprotein.

ABSTRACT: The coordinated packaging of the segmented genome of the influenza A virus (IAV) into virions is an essential step of the viral life cycle. This process is controlled by the interaction of packaging signals present in all eight viral RNA (vRNA) segments and the viral nucleoprotein (NP), which binds vRNA via a positively charged binding groove. However, mechanistic models of how the packaging signals and NP work together to coordinate genome packaging are missing. Here, we studied genome packaging in influenza A/SC35M virus mutants that carry mutated packaging signals as well as specific amino acid substitutions at the highly conserved lysine (K) residues 184 and 229 in the RNA-binding groove of NP. Because these lysines are acetylated and thus neutrally charged in infected host cells, we replaced them with glutamine to mimic the acetylated, neutrally charged state or arginine to mimic the non-acetylated, positively charged state. Our analysis shows that the coordinated packaging of eight vRNAs is influenced by (i) the charge state of the replacing amino acid and (ii) its location within the RNA-binding groove. Accordingly, we propose that lysine acetylation induces different charge states within the RNA-binding groove of NP, thereby supporting the activity of specific packaging signals during coordinated genome packaging.

Importance

Influenza A viruses (IAVs) have a segmented viral RNA (vRNA) genome encapsidated by multiple copies of the viral nucleoprotein (NP) and organized into eight distinct viral ribonucleoprotein complexes. Although genome segmentation contributes significantly to viral evolution and adaptation, it requires a highly sophisticated genome-packaging mechanism. How eight distinct genome complexes are incorporated into the virion is poorly understood, but previous research suggests an essential role for both vRNA packaging signals and highly conserved NP amino acids. By demonstrating that the packaging process is controlled by charge-dependent interactions of highly conserved lysine residues in NP and vRNA packaging signals, our study provides new insights into the sophisticated packaging mechanism of IAVs.

SUBMITTER: Ciminski K

PROVIDER: S-EPMC11019843 | biostudies-literature | 2024 Apr

REPOSITORIES: biostudies-literature

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Functionality of IAV packaging signals depends on site-specific charges within the viral nucleoprotein.

Ciminski Kevin K Flore Viktoria V Jakob Celia C Mues Helen H Smedegaard Frederiksen Anne A Schwemmle Martin M Bolte Hardin H Giese Sebastian S

Journal of virology 20240312 4

The coordinated packaging of the segmented genome of the influenza A virus (IAV) into virions is an essential step of the viral life cycle. This process is controlled by the interaction of packaging signals present in all eight viral RNA (vRNA) segments and the viral nucleoprotein (NP), which binds vRNA via a positively charged binding groove. However, mechanistic models of how the packaging signals and NP work together to coordinate genome packaging are missing. Here, we studied genome packagin ...[more]

PMID: 38470155

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Project description:The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV ψ.

Dataset Information

Functionality of IAV packaging signals depends on site-specific charges within the viral nucleoprotein.

Importance

Publications

Functionality of IAV packaging signals depends on site-specific charges within the viral nucleoprotein.

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