SARS-CoV-2 spike requires proteolytic processing for viral entry. The presence of a polybasic furin-cleavage site (FCS) in spike, and evolution towards an optimised FCS by dominant variants of concern (VOCs), are linked to enhanced infectivity and transmission. Guanylate binding proteins (GBP) are interferon-inducible restriction factors that target furin-mediated processing of viral envelope proteins and limit infectivity. Here we investigated whether GBPs restrict SARS-CoV-2 infection, and whether VOCs have evolved spikes that escape restriction. We show that GBP2 and 5 interfere with cleavage of the spike proteins of Wuhan-Hu-1, Alpha, Delta and Omicron, consistent with furin inhibition by GBPs. However, while GBP2/5 restrict Wuhan-Hu-1 infectivity, Alpha and Delta escape restriction. GBP exposure in producer cells influences viral entry route into target cells, with a shift towards endosomal entry. We therefore investigated whether GBP-targeting of spike alters sensitivity to endosomal restriction factors, IFITMs. We find IFITM1, but not IFITM 2 or 3, inhibit infection of naturally-permissive epithelial cells by early-lineage SARS-CoV-2, as well as Alpha and Delta, however GBPs did not sensitise to IFITM restriction. Strikingly, we find Omicron is unique amongst VOCs, being sensitive to restriction by GBP2/5, and also IFITM1, 2 and 3. We conclude evolution of Alpha and Delta spikes have conferred resistance to GBP restriction, but this is not solely due to acquisition of an enhanced FCS. Notably, Omicron, which has evolved under different selective pressures, has selected for changes in spike that not only mediate antibody escape, and shift in cell tropism and entry, but also impact the sensitivity of Omicron to innate immunity, potentially contributing to altered pathogenesis.
SARS-CoV-2 spike requires proteolytic processing for viral entry. A polybasic furin-cleavage site (FCS) in spike, and evolution toward an optimized FCS by dominant variants of concern (VOCs), are linked to enhanced infectivity and transmission. Here we show interferon-inducible restriction factors Guanylate-binding proteins (GBP) 2 and 5 interfere with furin-mediated spike cleavage and inhibit the infectivity of early-lineage isolates Wuhan-Hu-1 and VIC. By contrast, VOCs Alpha and Delta escape restriction by GBP2/5 that we map to the spike substitution D614G present in these VOCs. Despite inhibition of spike cleavage, these viruses remained sensitive to plasma membrane IFITM1, but not endosomal IFITM2 and 3, consistent with a preference for TMPRSS2-dependent plasma membrane entry. Strikingly, we find that Omicron is unique among VOCs, being sensitive to restriction factors GBP2/5, and also IFITM1, 2, and 3. Using chimeric spike mutants, we map the Omicron phenotype and show that the S1 domain determines Omicron’s sensitivity to GBP2/5, whereas the S2’ domain determines its sensitivity to endosomal IFITM2/3 and preferential use of TMPRSS2-independent entry. We propose that evolution of SARS-CoV-2 for the D614G substitution has allowed for escape from GBP restriction factors, but the selective pressures on Omicron for spike changes that mediate antibody escape, and altered tropism, have come at the expense of increased sensitivity to innate immune restriction factors that target virus entry.
SERINC5 is a potent lentiviral restriction factor that gets incorporated into nascent virions and inhibits viral fusion and infectivity. The envelope glycoprotein (Env) is a key determinant for SERINC restriction, but many aspects of this relationship remain incompletely understood, and the mechanism of SERINC5 restriction remains unresolved. Here, we have used mutants of HIV-1 and HIV-2 to show that truncation of the Env cytoplasmic tail (ΔCT) confers complete resistance of both viruses to SERINC5 and SERINC3 restriction. Critically, fusion of HIV-1 ΔCT virus was not inhibited by SERINC5 incorporation into virions, providing a mechanism to explain how EnvCT truncation allows escape from restriction. Neutralization and inhibitor assays showed ΔCT viruses have an altered Env conformation and fusion kinetics, suggesting that EnvCT truncation dysregulates the processivity of entry, in turn allowing Env to escape targeting by SERINC5. Furthermore, HIV-1 and HIV-2 ΔCT viruses were also resistant to IFITMs, another entry-targeting family of restriction factors. Notably, while the EnvCT is essential for Env incorporation into HIV-1 virions and spreading infection in T cells, HIV-2 does not require the EnvCT. Here, we reveal a mechanism by which human lentiviruses can evade two potent Env-targeting restriction factors but show key differences in the capacity of HIV-1 and HIV-2 to exploit this. Taken together, this study provides insights into the interplay between HIV and entry-targeting restriction factors, revealing viral plasticity toward mechanisms of escape and a key role for the long lentiviral EnvCT in regulating these processes.
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