Fig 2: Virion-independent genome transfer leads to productive infection by an actin-dependent mechanism.[A] Experimental workflow for MDCK, A549 WT or A549 Rab11a KO infection and co-culture with MDCK WSN HA cells [generated via BioRender.com]. Plotted is the infectious virus yield from co-culture of MDCK WSN HA cells with MDCK cellsinfected with NL09 ?HA Venus WT and NL09 ?HAScarlet VAR viruseseither treated with Cytochalasin D [B] or Nocodazole [D]. [C] Infectious virus yield from MDCK cells infected with NL09 viruses and treated with Cytochalasin D. [E] Infectious virus yield from co-culture of MDCK WSN HA cells with A549 WT or Rab11a KO cells infected with either NL09 ?HA Venus WT or NL09 ?HAScarlet VAR viruses. Significance of differences between the control and Cytochalasin D or Nocodazole treated cells or between WT and KO cells was tested using 2-way ANOVA with Bonferroni’s correction for multiple comparisons [* P-value <0.1; ** P-value <0.01; *** P-value <0.001; **** P-value <0.0001; ns = not significant]. Error bars represent the SEM of two biological replicates,each comprising three replicate infections[black circles]. The dotted line represents the limit of detection of the plaque assay.

Fig 3: Working model for Rab11a mediated vRNP transport across TNTs.vRNP complexes synthesized within the nucleus are exported out and form Rab11a-vRNP complexes. Two potential fates of these complexes are shown- the classical assembly and egress pathway for production of progeny virions and transport of these complexes via TNTs to an uninfected cell. A new infection is initiated in the recipient cell, resulting in progeny virion production. Generated via BioRender.com.

Fig 4: Rab11a does not modulate TNT formation.A549 WT and Rab11a KO cells were mock infected or infected with NL09 viruses and the total number of TNTs formed between 50 cell pairs were counted manually. Significance of differences in the number of TNTs between mock and NL09 infected groups was tested using 1-way ANOVA [** P-value <0.01]. Error bars represent the SEM of two biological replicates.

Fig 5: TNTs serve as conduits for genome mixing and reassortment in a Rab11a dependent manner.Genotypes of clonal viral isolates collected from the culture medium of NL09 ?HA Venus WT and NL09 ?HA Scarlet VAR virus infected A549 WTcells [A]or A549 Rab11a KOcells [B] co-cultured with MDCK WSN HA cells. Three technicalreplicate co-cultures per condition were sampled serially at the time points indicated and 21 plaque isolates were analyzed per sample. The origin of the gene segments, represented by the columns in each table, is denoted by the colored boxes [blue = WT and red = VAR]. The segments are in order PB2, PB1, PA, NP, NA, M and NS moving from left to right. The white panels indicate samples where no plaques were detected [ND = not detected].