TRIM21 Neutralizes Hazara Virus Using a Dual Mechanism of Nucleoprotein Caging and Ubiquitination

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper by Jahun, Vamos et al. is well written, the Figures are almost perfect. The presented hypothesis, how TRIM21 restricts the Hazara virus is supported by solid experiments. I am happy to recommend this paper for publication and I have only very few question below.

 

Major comments

1. Why the Hazara virus (HAZV) was used instead of the Crimean-Congo Hemorrhagic Fever Virus and how closely it is related to CCHFV should be better explain in the Introduction (it is somewhat in the Discussion section). Although I suppose the reason is no access to a BSL-4 facility by the authors?

 

2. I do not suggest this experiment because it would require too much time. But do the authors think that nnAb against other viral proteins such as the RNA polymerase would also confer protection?

 

Minor comments

1. Shoud the EDNA be rather called electroporated-antibody-dependent neutralization assay instead of electroporated antibody neutralization assay (in Abstract) as it is in the text?

 

2. Some shortcuts could be explained when first used e.g. Fc_gama_Rs.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Jahun et al present work on the impact of non-neutralizing antibodies on Hazara virus (HAZV), a model used to mirror CCHFV infection, and how these antibodies work with TRIM21 to impact infection. Anti-nucleoprotein antibodies have shown promise in preventing disease but the mechanims are not well understood.

Non-neutralizing antibodies can be produced at high levels and their effects can complement those of neutralizing antibodies, or perhaps be exploited separately. Most results presented here are based on a simple but elegant assay dubbed EDNA, in which antibodies are electroporated into cells which are then infected with HAZV tagged with an epitope that matches the electroporated antibody (HA tag - structure-guided location). Results are sometimes further supported by intracellular co-localization of TRIM21, the antibody used, and the epitope. In this way, the authors provide solid evidence that TRIM21 works together with the antibody to block infection and replication of HAZV, in a RING domain -dependent and -independent manner. The data suggest that the RING-independent manner works via 'caging' of NP protein, likely making the NP protein inaccessible. The RING-dependent manner appears to involve TRIM21 mediated ubiquitination, but the impact of proteasome inhibitors on infection did not allow direct demonstration of the impact of proteasome-dependent NP degradation on infection. In the discussion, the authors also point out a major gap in understanding of how antibodies that function intracellularly actually get inside cells. There is no data trying to fill this gap in understanding. Nevertheless, using epitope-tagged NP protein and viruses expressing such protein, the authors provide strong evidence that non-neutralizing anti-NP antibodies inhibit HAZV infection (and likely by extension CCHFV) by more than one mechanism. The paper is interesting and well written. Only minor concerns were noted:

 

Minor comments:

Line 347. Explain briefly in the text how the results were quantified.

Lines 383-389. In addition to giving a conclusion, explain how the authors arrived at this conclusion.

Fig 3i: explain why anti-HA antibody is absent from the western blot in lane 2.

Line 499. Please explain/speculate how/why anti-NP antibodies would arise much earlier than other antibodies.

Author Response

Please see the attachment

Author Response File: Author Response.pdf