Viral growth kinetics of silent HA mutants

Main text contribution

[FROM ANETH MANUSCRIPT START] In order to further investigate potential differences in the fitness effects of silent mutations, we performed infection kinetics experiments on 4 silent substitutions, \(L5L^{CTC}\), \(L6L^{CTC}\), \(L6L^{TTG}\) and \(L8L^{CTA}\). Of note, three of these mutants exhibited growth defects in bulk competition (\(L6L^{CTC}\),\(L6L^{TTG}\) and \(L8L^{CTA}\)),while \(L5L^{CTC}\) did not exhibit a large growth effect in the bulk competition (Figure 2A). [FROM ANETH MANUSCRIPT OVER]

We used phenomenological model of viral kinetics (Paradis 2015) to fit our experimental data in order to quantitatively characterize infection kinetics for the mutants (see Methods/Supplement for details). Sensitivity analysis of the model (see Supplement) showed that we can reliably estimate only 2 model parameters (out of 5): \(\beta\) rate of infection of target cells and \(\tau_{E}\) average duration of an eclipse phase (time infected cells spend before they start yielding virus), the results are presented in a Table \ref{table:kinetics}. According to the model fit, \(L6L^{TTG}\) demonstrates \(WT\)-like kinetics of viral yield, while other 3 mutants \(L5L^{CTC}\), \(L6L^{CTC}\), and \(L8L^{CTA}\) demonstrate delayed eclipse phase along with improved infection rate, while being virtually indistinguishable from each other.

[FROM ANETH MANUSCRIPT START] The discrepancy between the viral kinetic assay and bulk competitions for the \(L5L^{CTC}\) mutation could be due to inherent challenges in measuring fitness effects with precision using these techniques. This is consistent with our analyses of the reproducibility of estimates of fitness effects that indicate that the precision of these estimates can distinguish overall trends, but are not precise enough to discern the fitness effect of a precise mutation with strong confidence. For this reason, we have taken multiple approaches to investigate fitness effects. The slowed infection kinetics of this panel of four individual mutations lends additional evidence that silent mutations in the signal sequence can cause strong fitness defects. [FROM ANETH MANUSCRIPT OVER]

\label{table:kinetics} Viral kinetics parameters are estimated for the wild-type WSN/33 and four silent mutants using MCMC procedure. Median of the estimated parameter is reported along with the 95% bootstrap confidence intervals (BCI). One mutant demonstrating essentially WT-like kinetics, while 3 others are characterized by the prolonged eclipse phase and slightly elevated infection rates.
\(\log_{10}\beta\,\left[95\%\,BCI\right]\) \(\tau_{E}\,\left[95\%\,BCI\right]\)
WT -6.28, [ -6.50 -6.05] 6.61 [5.82 7.64]
L6L(TTG) -6.16, [ -6.40 -5.87] 8.82 [7.52 10.9]
L5L(CTC) -4.97, [ -5.26 -4.69] 20.8 [19.0 22.3]
L6L(CTC) -4.85, [ -5.10 -4.58] 18.6 [17.1 20.0]
L8L(CTA) -5.11, [ -5.30 -4.92] 23.1 [22.0 24.0]