Influenza A virus has an eight-partite RNA genome that during viral assembly forms a complex containing one copy of each RNA. Genome assembly is a selective process driven by RNA-RNA interactions and is hypothesized to lead to discrete punctate structures scattered through the cytosol. Here, we show that contrary to the accepted view, formation of these structures precedes RNA-RNA interactions among distinct viral ribonucleoproteins (vRNPs), as they assemble in cells expressing only one vRNP type. We demonstrate that these viral inclusions display characteristics of liquid organelles, segregating from the cytosol without a delimitating membrane, dynamically exchanging material and adapting fast to environmental changes. We provide evidence that viral inclusions develop close to endoplasmic reticulum (ER) exit sites, depend on continuous ER-Golgi vesicular cycling and do not promote escape to interferon response. We propose that viral inclusions segregate vRNPs from the cytosol and facilitate selected RNA-RNA interactions in a liquid environment.

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics.

Together, these results indicate that CLCb phosphorylation acts as a discriminator for the endocytosis of specific GPCRs.

In view of the data scarcity to guide decision-making, we evaluated how BNT162b2 and mRNA-1273 vaccines impact the immune response of lactating women and the protective profile of breastmilk. Compared to controls, lactating women had higher frequency of circulating RBD-memory B cells and higher anti-RBD antibody titers, but similar neutralizing capacity. We show that upon vaccination, immune transfer to breastmilk occurs through a combination of anti-spike secretory IgA (SIgA) antibodies and spike-reactive T cells. While we found that the concentration of anti-spike IgA in breastmilk might not be sufficient to directly neutralize SARS-CoV-2, our data suggest that cumulative transfer of IgA might provide the infant with effective neutralization capacity. Our findings put forward that breastmilk might convey both immediate, through anti-spike SIgA, as well as long-lived, via spike-reactive T cells, immune protection to the infant. Further studies are needed to address this possibility and to determine spike-T cells functional profile.

Phosphorylation of clathrin light chains (CLCs) regulates GPCR uptake but is dispensable for transferrin internalization. Maib et al. show that CLCb phosphorylation is required for efficient auxilin-mediated clathrin exchange to promote coated pit invagination in a cargo-specific manner.

Influenza A is a rapidly evolving virus that is successful in provoking periodic epidemics and occasional pandemics in humans. Viral assembly is complex as the virus incorporates an eight-partite genome of RNA (in the form of viral ribonucleoproteins, vRNPs), and viral genome assembly - with its implications to public health - is not completely understood. It has previously been reported that vRNPs are transported to the cell surface on Rab11-containing vesicles by using microtubules but, so far, no molecular motor has been assigned to the process. Here, we have identified KIF13A, a member of the kinesin-3 family, as the first molecular motor to efficiently transport vRNP-Rab11 vesicles during infection with influenza A. Depletion of KIF13A resulted in reduced viral titers and less accumulation of vRNPs at the cell surface, without interfering with the levels of other viral proteins at sites of viral assembly. In addition, when overexpressed and following two separate approaches to displace vRNP-Rab11 vesicles, KIF13A increased levels of vRNP at the plasma membrane. Together, our results show that KIF13A plays an important role in the transport of influenza A vRNPs, a crucial step for viral assembly.This article has an associated First Person interview with the first author of the paper.

18Influenza A virus has an eight-partite RNA genome that during viral assembly forms a 19 supramolecular complex containing one copy of each RNA. Genome assembly is a selective 20 process driven by RNA-RNA interactions and is thought to lead to discrete punctate structures 21 scattered through the cytosol. Here, we show that contrary to the accepted view, formation of 22 these structures is not dependent on RNA-RNA interactions among distinct viral 23 ribonucleoproteins (vRNPs), as they assemble in cells expressing only one vRNP type. We 24 demonstrate that these viral inclusions display characteristics of liquid organelles, segregating 25 from the cytosol without a delimitating membrane, dynamically exchanging material, deforming 26 easily and adapting fast to hypotonic shock. We provide evidence that they develop close to the 27 Endoplasmic Reticulum Exit Sites (ERES), being dependent on continuous ER-Golgi vesicular 28cycling. We show that viral inclusions do not promote escape to interferon response, and 29 propose that they facilitate selected RNA-RNA interactions in a liquid environment of 30 concentrated vRNPs. 31 32 33 34 3 MAIN TEXT 35 Influenza A infections are serious threats to human health causing annual epidemics and 36 occasional pandemics 1 . The virus contains an eight-partite RNA genome, and each segment is 37 encapsidated as an individual viral ribonucleoprotein (vRNP) complex. vRNPs are composed of 38 single-stranded negative-sense RNA, with base paired terminal sequences originating a double 39 stranded RNA portion where the trimeric RNA-dependent RNA polymerase (RdRp), composed 40 of PB1, PB2 and PA, binds. The remaining sequence attaches several copies of unevenly-41 bound nucleoprotein (NP) 2 . The advantages of having a segmented genome are evident for 42 viral evolution 3 and for better gene expression control 4 , but increase the complexity of the 43 assembly of fully infectious virions 5-8 . 44 Viral assembly occurs at the plasma membrane and, in 80% of the cases, 8 distinct 45 vRNPs are packaged selectively into a budding membrane 9 . Seminal work established the 46 requirement of cis-acting and intersegment RNA-RNA interactions for the formation of this 47 supra-molecular complex (reviewed in 5-8 ). However, it is under debate if vRNPs reach the 48 plasma membrane already as complete genome bundles. 49Upon exiting the nucleus, where they replicate, vRNPs accumulate around the 50 microtubule organizing centre 10 and, subsequently, distribute throughout the cytoplasm 51 concentrating in discrete puncta that enlarge as infection progresses 10-14 . Each puncta 52 accommodates different vRNP segments with the diversity in vRNPs increasing proportionally to 53 the proximity of the plasma membrane 11,13 . Such observation led to the proposal that genome 54 assembly preceded vRNP packaging in budding virions by a process intimately linked with the 55 formation of the referred vRNP hotspots 6,11,[13][14][15] . Studies on the biogenesis of vRNP hotspots 56 showed that their formation requires...

In view of data scarcity to guide decision-making in breastfeeding women, we evaluated how mRNA vaccines impact immune response of lactating health care workers (HCW) and the effector profile of breast milk transferred immune protection. We show that upon BNT162b2 vaccination, immune transfer via milk to suckling infants occurs through secretory IgA (SIgA) and T cells. Functionally, spike-SIgA was non-neutralizing and its titers were unaffected by vaccine boosting, indicating that spike-SIgA is produced in a T-cell independent manner by mammary gland. Even though their milk was devoid of neutralizing antibodies, we found that lactating women had higher frequencies of RBD-reactive circulating memory B cells and more RBD-IgG antibodies, when compared to controls. Nonetheless, blood neutralization titers in lactating and non-lactating HCW were similar. Further studies are required to determine transferred antibodies and spike-T cells complete functional profile and whether they can mediate protection in the suckling infant.HighlightsMilk and blood responses to BNT162b2 vaccine are initially isotype discordantImmune transfer via milk to suckling infants occurs by spike-reactive SIgA and T cellsSpike-reactive SIgA in the breastmilk is non-neutralizing and T-cell independentLactating vs non-lactating HCW had distinct cellular responses, despite similar NT50