Crimean–Congo Hemorrhagic Fever Virus (CCHFV) is a tick-borne bunyavirus with widespread and growing geographic distribution that causes severe hemorrhagic fever and death. Vaccine candidates targeting the viral nucleoprotein (NP) have shown efficacy in both mouse and non-human primate models but the mechanism of protection is unclear. Here we employ the closely related Hazara virus (HAZV) to investigate how the intracellular antibody receptor TRIM21 uses anti-NP antibodies to neutralize infection. We show that TRIM21 can detect incoming NP particles within hours of infection and that this results in a potent block to infection. Electroporated-antibody-dependent neutralization assay (EDNA) experiments reveal that TRIM21 inhibits viral transcription, protein expression and genome synthesis and reduces the production of infectious virions. Mutations and domain deletions within TRIM21 reveal that neutralization requires antibody-binding by the PRYSPRY domain but is only partially dependent on the E3 ubiquitin ligase RING domain. The data suggest a dual restriction mechanism in which NP cross-linking by TRIM21 physically interferes with NP function whilst parallel ubiquitination labels the protein for degradation. This dual mechanism is similar to that used by TRIM5 against retroviruses and suggests that antiviral TRIMs may utilize their capacity for self-assembly both for catalytic activation and viral caging.

The HIV-1 capsid, which orchestrates several key post-entry events to facilitate infection, is composed of hexamers and pentamers (capsomers) of the capsid (CA) protein. The capsomers are arranged in a closed, conical structure - the capsid - that protects the viral RNA genome and replicative enzymes reverse transcriptase (RT) and integrase (IN). Each capsomer contains a central pore lined with rings of positively charged amino acid side chains - Arg-18 (R18) and Lys-25 (K25). The R18 and K25 rings drive capsid assembly by binding the host polyanion inositol hexakisphosphate (IP6) and are proposed to mediate the import of dNTPs into the capsid to facilitate reverse transcription. Here we demonstrate that the R18 ring can be functionally replaced by the N21K substitution that establishes a new electropositive ring within the central pore. In contrast with previous studies in which R18 mutants were unable to adapt in culture, the N21K substitution facilitated the acquisition of second-site compensatory mutations that restored near-WT fitness to viral mutants lacking the R18 ring. Comparative analysis of several central pore mutants lacking the R18 ring revealed that particle infectivity was not correlated with IP6 binding or capsid assembly but rather with capsid stability and key post-entry events including reverse transcription and nuclear entry. Our results indicate that the central pore plays critical roles in both the assembly of capsids and their sustained stability post-entry.