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. 2005 Jan;79(2):1262-70.
doi: 10.1128/JVI.79.2.1262-1270.2005.

Influenza a viruses with mutations in the m1 helix six domain display a wide variety of morphological phenotypes

Laura M Burleigh  1 , Lesley J Calder, John J Skehel, David A Steinhauer
Affiliations

Influenza a viruses with mutations in the m1 helix six domain display a wide variety of morphological phenotypes

Laura M Burleigh et al. J Virol. 2005 Jan.

Abstract

Several functions required for the replication of influenza A viruses have been attributed to the viral matrix protein (M1), and a number of studies have focused on a region of the M1 protein designated "helix six." This region contains an exposed positively charged stretch of amino acids, including the motif 101-RKLKR-105, which has been identified as a nuclear localization signal, but several studies suggest that this domain is also involved in functions such as binding to the ribonucleoprotein genome segments (RNPs), membrane association, interaction with the viral nuclear export protein, and virus assembly. In order to define M1 functions in more detail, a series of mutants containing alanine substitutions in the helix six region were generated in A/WSN/33 virus. These were analyzed for RNP-binding function, their capacity to incorporate into infectious viruses by using reverse genetics, the replication properties of rescued viruses, and the morphological phenotypes of the mutant virus particles. The most notable effect that was identified concerned single amino acid substitution mutants that caused significant alterations to the morphology of budded viruses. Whereas A/WSN/33 virus generally forms particles that are predominantly spherical, observations made by negative stain electron microscopy showed that several of the mutant virions, such as K95A, K98A, R101A, and K102A, display a wide range of shapes and sizes that varied in a temperature-dependent manner. The K102A mutant is particularly interesting in that it can form extended filamentous particles. These results support the proposition that the helix six domain is involved in the process of virus assembly.

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Figures

FIG. 1.
FIG. 1.
(A) Ribbon diagram of the M1 protein structure. The right panel is rotated by 90° relative to the left panel, such that the top of helix six in the left panel orients toward the reader in the right panel. The residues indicated in yellow are (bottom to top, left panel; back to front, right panel) N91, K95, K98, R101, K102, and R105. (B) Schematic diagram of mutations introduced into amino acids 75 to 110 of the M1 gene and results of virus rescue. Blue boxes indicate residues mutated to alanine.
FIG. 2.
FIG. 2.
(A) Autoradiograph of in vitro-transcribed and translated M1 proteins. (B) SDS-PAGE analysis and Coomassie blue staining of RNP purification. Lane 1, untreated virus; lane 2, RNP purified by low-pH and detergent treatment. (C) Binding of mutant and wild-type 35S-labeled M1 to RNP. A total of 100,000 cpm of M1 was incubated with 10 μg of RNP. RNPs were pelleted through 20% glycerol, and the M1 associated with RNP was quantitated. Experiments were carried out three times.
FIG. 3.
FIG. 3.
Plaque morphology of mutant and wild-type viruses. MDCK cells were infected at equal multiplicities of infection. After incubation for 3 days at 37°C, the cells were stained with crystal violet. No difference in plaque size was observed when cells were incubated at 33 and 38.5°C.
FIG. 4.
FIG. 4.
Negative stain electron microscopy of wild-type viruses and mutant viruses with wild type-like morphology at 33°C. Morphology did not vary at 37°C (data not shown). The scale bars represent 100 nm. Viruses were stained with 1% sodium silicotungstate and examined under low-dose, high-resolution conditions.
FIG. 5.
FIG. 5.
Negative stain electron microscopy of mutant viruses with an altered morphology at 33 and 37°C. A/Udorn/72 virus is shown as an example of filamentous morphology. Black arrows indicate areas of membrane disruption; white arrows indicate shed HA. The scale bars represent 100 nm. Viruses were stained with 1% sodium silicotungstate and examined under low-dose, high-resolution conditions.
FIG. 6.
FIG. 6.
(A) M1 arrangement in filamentous K102A mutants and in A/Udorn/72 virions. Arrows indicate areas where the M1 arrangement is visible. (B) Reduced magnification of K102A mutant and Udorn, showing filaments >1 μm in length. Scale bars, 100 nm. Viruses were stained with 1% sodium silicotungstate and examined under low-dose, high-resolution conditions.
FIG. 7.
FIG. 7.
Protein composition of wild-type and K102A virions. Virions were analyzed on a 12% polyacrylamide gel. The band density was determined by using Quantity One (Bio-Rad) software.
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