The mechanisms by which ANP32 proteins support the influenza polymerase

Influenza A naturally resides asymptomatically in wild aquatic birds. However, cross over into humans can occur and may result in a pandemic if the virus adapts for efficient replication and transmission between immunologically naïve human hosts. Fortunately, pandemics occur infrequently due to the existence of host range barriers. Poor replication of the influenza genome in the human cell nucleus is one of the main barriers that restricts avian origin influenza virus. The most common mutation which can overcome this block is the E627K mutation in the PB2 subunit of the trimeric influenza polymerase complex. Both the absence of a compatible positive factor or the presence of an inhibitory factor in human hosts have been proposed to explain why a polymerase bearing a glutamic acid at position 627 of PB2 is restricted in human cells. Avian ANP32A has been identified as a host factor which can support the activity of an avian origin viral polymerase. However, this polymerase is not compatible with the shorter human orthologues of this protein, human ANP32A and ANP32B. Since these human proteins have been implicated in supporting the activity of a human adapted influenza polymerase, it is hypothesised that the PB2 E627K mutation is an adaptation towards utilising huANP32A and -B. In this study we investigate how the ANP32A proteins are able to support polymerase activity and why species differences in this protein determine its compatibility with polymerases bearing different mutations. This was primarily examined by assessing differences in the interactions between specific ANP32A proteins and viral polymerases. We found that the interactions with trimeric polymerase complex were dependent on the species of ANP32A. These interactions were stabilised at inactive ribonucleoproteins, but dissociated as replication of the viral genome occurred. However, using multiple methods, we concluded that the ability of viral polymerase to co-opt ANP32A was determined by more than differences in interactions alone. As ANP32A can act as an adapter protein in some cases, we also investigated whether the interactome of the polymerase differed in the presence or absence of avian ANP32A. We further explored the impact of two human proteins, SRPK1 and importin alpha7, which differed in their interaction with avian origin polymerase when chicken ANP32A was co-expressed. Finally, we visualised the viral polymerase by overexpression of PA-GFP in human cells and observed differences between an avian origin polymerase and that with humanising mutations. Specifically, the avian origin polymerase appeared to form speckles in the nucleus of human cells. We explored the requirements for formation of these speckles as well as their relationship with ANP32A. This study has allowed us to gain insight into the mechanism by which ANP32A can support influenza virus polymerase. This was explored via several avenues, which led to the identification of other host factors which may affect polymerase activity. Overall, these data have enhanced our understanding of host restriction of influenza virus polymerase and the interplay between host and viral factors.Open Acces

Species difference in ANP32A underlies influenza A virus polymerase host restriction.

Influenza pandemics occur unpredictably when zoonotic influenza viruses with novel antigenicity acquire the ability to transmit amongst humans. Host range breaches are limited by incompatibilities between avian virus components and the human host. Barriers include receptor preference, virion stability and poor activity of the avian virus RNA-dependent RNA polymerase in human cells. Mutants of the heterotrimeric viral polymerase components, particularly PB2 protein, are selected during mammalian adaptation, but their mode of action is unknown. We show that a species-specific difference in host protein ANP32A accounts for the suboptimal function of avian virus polymerase in mammalian cells. Avian ANP32A possesses an additional 33 amino acids between the leucine-rich repeats and carboxy-terminal low-complexity acidic region domains. In mammalian cells, avian ANP32A rescued the suboptimal function of avian virus polymerase to levels similar to mammalian-adapted polymerase. Deletion of the avian-specific sequence from chicken ANP32A abrogated this activity, whereas its insertion into human ANP32A, or closely related ANP32B, supported avian virus polymerase function. Substitutions, such as PB2(E627K), were rapidly selected upon infection of humans with avian H5N1 or H7N9 influenza viruses, adapting the viral polymerase for the shorter mammalian ANP32A. Thus ANP32A represents an essential host partner co-opted to support influenza virus replication and is a candidate host target for novel antivirals

Efficacy and safety of two neutralising monoclonal antibody therapies, sotrovimab and BRII-196 plus BRII-198, for adults hospitalised with COVID-19 (TICO): a randomised controlled trial

We aimed to assess the efficacy and safety of two neutralising monoclonal antibody therapies (sotrovimab [Vir Biotechnology and GlaxoSmithKline] and BRII-196 plus BRII-198 [Brii Biosciences]) for adults admitted to hospital for COVID-19 (hereafter referred to as hospitalised) with COVID-19. In this multinational, double-blind, randomised, placebo-controlled, clinical trial (Therapeutics for Inpatients with COVID-19 [TICO]), adults (aged ≥18 years) hospitalised with COVID-19 at 43 hospitals in the USA, Denmark, Switzerland, and Poland were recruited. Patients were eligible if they had laboratory-confirmed SARS-CoV-2 infection and COVID-19 symptoms for up to 12 days. Using a web-based application, participants were randomly assigned (2:1:2:1), stratified by trial site pharmacy, to sotrovimab 500 mg, matching placebo for sotrovimab, BRII-196 1000 mg plus BRII-198 1000 mg, or matching placebo for BRII-196 plus BRII-198, in addition to standard of care. Each study product was administered as a single dose given intravenously over 60 min. The concurrent placebo groups were pooled for analyses. The primary outcome was time to sustained clinical recovery, defined as discharge from the hospital to home and remaining at home for 14 consecutive days, up to day 90 after randomisation. Interim futility analyses were based on two seven-category ordinal outcome scales on day 5 that measured pulmonary status and extrapulmonary complications of COVID-19. The safety outcome was a composite of death, serious adverse events, incident organ failure, and serious coinfection up to day 90 after randomisation. Efficacy and safety outcomes were assessed in the modified intention-to-treat population, defined as all patients randomly assigned to treatment who started the study infusion. This study is registered with ClinicalTrials.gov, NCT04501978. Between Dec 16, 2020, and March 1, 2021, 546 patients were enrolled and randomly assigned to sotrovimab (n=184), BRII-196 plus BRII-198 (n=183), or placebo (n=179), of whom 536 received part or all of their assigned study drug (sotrovimab n=182, BRII-196 plus BRII-198 n=176, or placebo n=178; median age of 60 years [IQR 50–72], 228 [43%] patients were female and 308 [57%] were male). At this point, enrolment was halted on the basis of the interim futility analysis. At day 5, neither the sotrovimab group nor the BRII-196 plus BRII-198 group had significantly higher odds of more favourable outcomes than the placebo group on either the pulmonary scale (adjusted odds ratio sotrovimab 1·07 [95% CI 0·74–1·56]; BRII-196 plus BRII-198 0·98 [95% CI 0·67–1·43]) or the pulmonary-plus complications scale (sotrovimab 1·08 [0·74–1·58]; BRII-196 plus BRII-198 1·00 [0·68–1·46]). By day 90, sustained clinical recovery was seen in 151 (85%) patients in the placebo group compared with 160 (88%) in the sotrovimab group (adjusted rate ratio 1·12 [95% CI 0·91–1·37]) and 155 (88%) in the BRII-196 plus BRII-198 group (1·08 [0·88–1·32]). The composite safety outcome up to day 90 was met by 48 (27%) patients in the placebo group, 42 (23%) in the sotrovimab group, and 45 (26%) in the BRII-196 plus BRII-198 group. 13 (7%) patients in the placebo group, 14 (8%) in the sotrovimab group, and 15 (9%) in the BRII-196 plus BRII-198 group died up to day 90. Neither sotrovimab nor BRII-196 plus BRII-198 showed efficacy for improving clinical outcomes among adults hospitalised with COVID-19. US National Institutes of Health and Operation Warp Spee