Over the past 20 years, several coronaviruses have crossed the species barrier into humans, causing outbreaks of severe, and often fatal, respiratory illness. Since SARS-CoV was first identified in ...animal markets, global viromics projects have discovered thousands of coronavirus sequences in diverse animals and geographic regions. Unfortunately, there are few tools available to functionally test these viruses for their ability to infect humans, which has severely hampered efforts to predict the next zoonotic viral outbreak. Here, we developed an approach to rapidly screen lineage B betacoronaviruses, such as SARS-CoV and the recent SARS-CoV-2, for receptor usage and their ability to infect cell types from different species. We show that host protease processing during viral entry is a significant barrier for several lineage B viruses and that bypassing this barrier allows several lineage B viruses to enter human cells through an unknown receptor. We also demonstrate how different lineage B viruses can recombine to gain entry into human cells, and confirm that human ACE2 is the receptor for the recently emerging SARS-CoV-2.
Spillover of sarbecoviruses from animals to humans has resulted in outbreaks of severe acute respiratory syndrome SARS-CoVs and the ongoing COVID-19 pandemic. Efforts to identify the origins of ...SARS-CoV-1 and -2 has resulted in the discovery of numerous animal sarbecoviruses–the majority of which are only distantly related to known human pathogens and do not infect human cells. The receptor binding domain (RBD) on sarbecoviruses engages receptor molecules on the host cell and mediates cell invasion. Here, we tested the receptor tropism and serological cross reactivity for RBDs from two sarbecoviruses found in Russian horseshoe bats. While these two viruses are in a viral lineage distinct from SARS-CoV-1 and -2, the RBD from one virus, Khosta 2, was capable of using human ACE2 to facilitate cell entry. Viral pseudotypes with a recombinant, SARS-CoV-2 spike encoding for the Khosta 2 RBD were resistant to both SARS-CoV-2 monoclonal antibodies and serum from individuals vaccinated for SARS-CoV-2. Our findings further demonstrate that sarbecoviruses circulating in wildlife outside of Asia also pose a threat to global health and ongoing vaccine campaigns against SARS-CoV-2
Human APOBEC3H (A3H) is a cytidine deaminase that inhibits HIV-1 replication. To evade this restriction, the HIV-1 Vif protein binds A3H and mediates its proteasomal degradation. To date, little ...information on the Vif-A3H interface has been available. To decipher how both proteins interact, we first mapped the Vif-binding site on A3H by functionally testing a large set of A3H mutants in single-cycle infectivity and replication assays. Our data show that the two A3H α-helixes α3 and α4 represent the Vif-binding site of A3H. We next used viral adaptation and a set of Vif mutants to identify novel, reciprocal Vif variants that rescued viral infectivity in the presence of two Vif-resistant A3H mutants. These A3H-Vif interaction points were used to generate the first A3H-Vif structure model, which revealed that the A3H helixes α3 and α4 interact with the Vif β-sheet (β2-β5). This model is in good agreement with previously reported Vif and A3H amino acids important for interaction. Based on the predicted A3H-Vif interface, we tested additional points of contact, which validated our model. Moreover, these experiments showed that the A3H and A3G binding sites on HIV-1 Vif are largely distinct, with both host proteins interacting with Vif β-strand 2. Taken together, this virus-host interface model explains previously reported data and will help to identify novel drug targets to combat HIV-1 infection.
HIV-1 needs to overcome several intracellular restriction factors in order to replicate efficiently. The human APOBEC3 locus encodes seven proteins, of which A3D, A3F, A3G, and A3H restrict HIV-1. HIV encodes the Vif protein, which binds to the APOBEC3 proteins and leads to their proteasomal degradation. No HIV-1 Vif-APOBEC3 costructure exists to date despite extensive research. We and others previously generated HIV-1 Vif costructure models with A3G and A3F by mapping specific contact points between both proteins. Here, we applied a similar approach to HIV-1 Vif and A3H and successfully generated a Vif-A3H interaction model. Importantly, we find that the HIV-1 Vif-A3H interface is distinct from the Vif-A3G and Vif-A3F interfaces, with a small Vif region being important for recognition of both A3G and A3H. Our Vif-A3H structure model informs on how both proteins interact and could guide toward approaches to block the Vif-A3H interface to target HIV replication.
Middle East Respiratory Syndrome Coronavirus (MERS-CoV) likely originated in bats and passed to humans through dromedary camels; however, the genetic mechanisms underlying cross-species adaptation ...remain poorly understood. Variation in the host receptor, dipeptidyl peptidase 4 (DPP4), can block the interaction with the MERS-CoV spike protein and form a species barrier to infection. To better understand the species adaptability of MERS-CoV, we identified a suboptimal species-derived variant of DPP4 to study viral adaption. Passaging virus on cells expressing this DPP4 variant led to accumulation of mutations in the viral spike which increased replication. Parallel passages revealed distinct paths of viral adaptation to the same DPP4 variant. Structural analysis and functional assays showed that these mutations enhanced viral entry with suboptimal DPP4 by altering the surface charge of spike. These findings demonstrate that MERS-CoV spike can utilize multiple paths to rapidly adapt to novel species variation in DPP4.
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•MERS-CoV infected cells expressing DPP4 from 16 bat species•MERS-CoV spike rapidly adapted to species variation in DPP4•Viral adaptations modified the surface charge of viral spike•Different routes of spike adaptation enhanced entry with the same DPP4 variant
MERS-CoV is a zoonotic pathogen capable of infecting numerous species. However, our understanding of how this virus adapts to new species remains unclear. Letko et al. experimentally observe several different routes in the stepwise, adaptive evolution of MERS-CoV to a unique host-species variant of the viral receptor.
Following emergence in late 2019, SARS-CoV-2 rapidly became pandemic and is presently responsible for millions of infections and hundreds of thousands of deaths worldwide. There is currently no ...approved vaccine to halt the spread of SARS-CoV-2 and only very few treatment options are available to manage COVID-19 patients. For development of preclinical countermeasures, reliable and well-characterized small animal disease models will be of paramount importance. Here we show that intranasal inoculation of SARS-CoV-2 into Syrian hamsters consistently caused moderate broncho-interstitial pneumonia, with high viral lung loads and extensive virus shedding, but animals only displayed transient mild disease. We determined the infectious dose 50 to be only five infectious particles, making the Syrian hamster a highly susceptible model for SARS-CoV-2 infection. Neither hamster age nor sex had any impact on the severity of disease or course of infection. Finally, prolonged viral persistence in interleukin 2 receptor gamma chain knockout hamsters revealed susceptibility of SARS-CoV-2 to adaptive immune control. In conclusion, the Syrian hamster is highly susceptible to SARS-CoV-2 making it a very suitable infection model for COVID-19 countermeasure development.
Human cells express natural antiviral proteins, such as APOBEC3G (A3G), that potently restrict HIV replication. As a counter-defense, HIV encodes the accessory protein Vif, which binds A3G and ...mediates its proteasomal degradation. Our structural knowledge on how Vif and A3G interact is limited, because a co-structure is not available. We identified specific points of contact between Vif and A3G by using functional assays with full-length A3G, patient-derived Vif variants, and HIV forced evolution. These anchor points were used to model and validate the Vif-A3G interface. The resultant co-structure model shows that the negatively charged β4-α4 A3G loop, which contains primate-specific variation, is the core Vif binding site and forms extensive interactions with a positively charged pocket in HIV Vif. Our data present a functional map of this viral-host interface and open avenues for targeted approaches to block HIV replication by obstructing the Vif-A3G interaction.
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•Three specific HIV Vif-A3G contact points were identified using functional assays•Protein docking using Vif-A3G anchor points reveals the Vif-A3G interface•The A3G binding site on Vif consists of a defined, positively charged pocket•The negatively charged species-specific β4-α4 A3G loop fits within the Vif pocket
The HIV Vif protein binds and degrades the human restriction factor APOBEC3G (A3G), but our knowledge on how these two proteins interact is limited. Letko et al. identify and use specific interaction points between Vif and A3G to generate a functional Vif-A3G interface model.
The sarbecovirus subgenus of betacoronaviruses is widely distributed throughout bats and other mammals globally and includes human pathogens, SARS-CoV and SARS-CoV-2. The most studied sarbecoviruses ...use the host protein, ACE2, to infect cells. Curiously, the majority of sarbecoviruses identified to date do not use ACE2 and cannot readily acquire ACE2 binding through point mutations. We previously screened a broad panel of sarbecovirus spikes for cell entry and observed bat-derived viruses that could infect human cells, independent of ACE2. Here we further investigate the sequence determinants of cell entry for ACE2-independent bat sarbecoviruses.
We employed a network science-based approach to visualize sequence and entry phenotype similarities across the diversity of sarbecovirus spike protein sequences. We then verified these computational results and mapped determinants of viral entry into human cells using recombinant chimeric spike proteins within an established viral pseudotype assay.
We show ACE2-independent viruses that can infect human and bat cells in culture have a similar putative receptor binding motif, which can impart human cell entry into other bat sarbecovirus spikes that cannot otherwise infect human cells. These sequence determinants of human cell entry map to a surface-exposed protrusion from the predicted bat sarbecovirus spike receptor binding domain structure.
Our findings provide further evidence of a group of bat-derived sarbecoviruses with zoonotic potential and demonstrate the utility in applying network science to phenotypic mapping and prediction.
This work was supported by Washington State University and the Paul G. Allen School for Global Health.
The spike protein on sarbecovirus virions contains two external, protruding domains: an N-terminal domain (NTD) with unclear function and a C-terminal domain (CTD) that binds the host receptor, ...allowing for viral entry and infection. While the CTD is well studied for therapeutic interventions, the role of the NTD is far less well understood for many coronaviruses. Here, we demonstrate that the spike NTD from SARS-CoV-2 and other sarbecoviruses binds to unidentified glycans in vitro similarly to other members of the Coronaviridae family. We also show that these spike NTD (S-NTD) proteins adhere to Calu3 cells, a human lung cell line, although the biological relevance of this is unclear. In contrast to what has been shown for Middle East respiratory syndrome coronavirus (MERS-CoV), which attaches sialic acids during cell entry, sialic acids present on Calu3 cells inhibited sarbecovirus infection. Therefore, while sarbecoviruses can interact with cell surface glycans similarly to other coronaviruses, their reliance on glycans for entry is different from that of other respiratory coronaviruses, suggesting sarbecoviruses and MERS-CoV have adapted to different cell types, tissues, or hosts during their divergent evolution. Our findings provide important clues for further exploring the biological functions of sarbecovirus glycan binding and adds to our growing understanding of the complex forces that shape coronavirus spike evolution. IMPORTANCE Spike N-terminal domains (S-NTD) of sarbecoviruses are highly diverse; however, their function remains largely understudied compared with the receptor-binding domains (RBD). Here, we show that sarbecovirus S-NTD can be phylogenetically clustered into five clades and exhibit various levels of glycan binding in vitro. We also show that, unlike some coronaviruses, including MERS-CoV, sialic acids present on the surface of Calu3, a human lung cell culture, inhibit SARS-CoV-2 and other sarbecoviruses. These results suggest that while glycan binding might be an ancestral trait conserved across different coronavirus families, the functional outcome during infection can vary, reflecting divergent viral evolution. Our results expand our knowledge on the biological functions of the S-NTD across diverse sarbecoviruses and provide insight on the evolutionary history of coronavirus spike.
Significance Understanding emerging disease origins is important to gauge future human infection risks. This is particularly true for the various forms of the AIDS virus, HIV-1, which were ...transmitted to humans on four independent occasions. Previous studies identified chimpanzees in southern Cameroon as the source of the pandemic M group, as well as the geographically more restricted N group. Here, we show that the remaining two groups also emerged in southern Cameroon but had their origins in western lowland gorillas. Although group P has only been detected in two individuals, group O has spread extensively throughout west central Africa. Thus, both chimpanzees and gorillas harbor viruses that are capable of crossing the species barrier to humans and causing major disease outbreaks.
HIV-1, the cause of AIDS, is composed of four phylogenetic lineages, groups M, N, O, and P, each of which resulted from an independent cross-species transmission event of simian immunodeficiency viruses (SIVs) infecting African apes. Although groups M and N have been traced to geographically distinct chimpanzee communities in southern Cameroon, the reservoirs of groups O and P remain unknown. Here, we screened fecal samples from western lowland ( n = 2,611), eastern lowland ( n = 103), and mountain ( n = 218) gorillas for gorilla SIV (SIVgor) antibodies and nucleic acids. Despite testing wild troops throughout southern Cameroon ( n = 14), northern Gabon ( n = 16), the Democratic Republic of Congo ( n = 2), and Uganda ( n = 1), SIVgor was identified at only four sites in southern Cameroon, with prevalences ranging from 0.8–22%. Amplification of partial and full-length SIVgor sequences revealed extensive genetic diversity, but all SIVgor strains were derived from a single lineage within the chimpanzee SIV (SIVcpz) radiation. Two fully sequenced gorilla viruses from southwestern Cameroon were very closely related to, and likely represent the source population of, HIV-1 group P. Most of the genome of a third SIVgor strain, from central Cameroon, was very closely related to HIV-1 group O, again pointing to gorillas as the immediate source. Functional analyses identified the cytidine deaminase APOBEC3G as a barrier for chimpanzee-to-gorilla, but not gorilla-to-human, virus transmission. These data indicate that HIV-1 group O, which spreads epidemically in west central Africa and is estimated to have infected around 100,000 people, originated by cross-species transmission from western lowland gorillas.
Some human APOBEC3 cytidine deaminases have antiviral activity against HIV-1 and other retroviruses. The single deaminase domain APOBEC3H (A3H) enzyme is highly polymorphic and multiple A3H ...haplotypes have been identified. A3H haplotype II (A3H-hapII) possesses the strongest activity against HIV-1. There remains, however, uncertainty regarding the extent to which A3H-hapII is sensitive to HIV-1 Vif mediated degradation. We tested, therefore, the two different reference Vif proteins widely used in previous studies. We show that A3H-hapII is resistant to NL4-3 Vif while it is efficiently degraded by LAI Vif. Co-immunoprecipitation assays demonstrate that LAI Vif, but not NL4-3 Vif associates with A3H-hapII. Chimeras between NL4-3 and LAI Vif identify the amino acid responsible for the differential degradation activity: A histidine at position 48 in Vif confers activity against A3H-hapII, while an asparagine abolishes its anti-A3H activity. Furthermore, the amino acid identity at position 48 only affects the degradation of A3H-hapII, whereas recognition of and activity against human A3D, A3F and A3G are only minimally affected. NL4-3 encoding 48H replicates better than NL4-3 WT (48N) in T cell-lines stably expressing A3H hapII, whereas there is no fitness difference in the absence of APOBEC3. These studies provide an explanation for the conflicting reports regarding A3H resistance to Vif mediated degradation.
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