Peer review of: Reverse engineering synthetic antiviral amyloids (2023)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
In the present work, Michiels and co-workers investigate whether amyloid-virus interactions are 
driven by APR-APR interactions. The results of the study are well supported by the experimental 
data, and this work is a significant contribution to the field. They have performed a multi-faceted 
analysis incorporating both molecular and biophysical methods. 
 
Minor revisions: 
 
1. The authors should reread their manuscript in order to correct some grammatical and 
syntactical errors. A few sentences are quite long, making it difficult to understand their meaning. 
2. The authors are advised to write the numbers of the amino acids corresponding to the ends of 
the peptides, wherever this is possible. For example, VGGVVI could be written as 36VGGVVI41. 
3. Additionally, the numbers of the amino acids should not be in the sequence. For example, 
L381IQLIVS387 should be modified to 381LIQLIVS387. 
4. In lines 991 and 1051, “ONE-way ANOVA” should be replaced with “one-way ANOVA”. 
5. Figure 2 is blurred. If this is not due to the pdf conversion process, consider increasing the size 
of the image. 
6. All decimal numbers should be separated by a dot, not a comma. 
7. There are references that have different font from the main text. 
8. The authors mention that they have conducted local alignment utilizing BLAST. Have they tried 
an alignment with a more qualified program, like CLUSTAL Omega? And are the results the same? 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
Previous studies have identified the amyloid protein, associated with Alzheimer’s Disease (AD), can 
inhibit virus and bacteria. The authors used bioinformatics to design very short aggregation 
peptide region from the viral proteins (of influenza and Zika virus) which are homologous to 
human amyloid, and confirm that these short peptides have anti-flu and anti-zika-viral activity. 
The idea is not new. But they are first group to make such short peptides for antiviral action and 
they further improve them by flanking them with hydrophilic amino acids so as to improve 
solubility and availability. They demonstrated that 12B binds to PB2 with broad-spectrum anti-
influenza A activity, and showed that the antiviral activity of this peptide can tolerate a single 
conservative substitution. 
 
 
 
Major concerns: 
 
1. The antiviral activity of the peptides shown in this study was very modest. Only plaque size 
reduction in cell culture assay can be achieved. And only a 2-3 fold viral load reduction in mice, 
not at the magnitude of log reduction. There is no survival data to prove that the antiviral peptide 
can salvage challenged mice. 
 
2. The findings of the Cryo-EM used in this study is just like the TEM, and there was no structural 
binding analysis provided. 
 
3. Lots of data are given but the general quality of the data are low, especially for those given in 
supplementary information. 
 
4. The text is very difficult to follow and need extensive rewriting. 

 
 
 
Individual comments: 
 
1, Figure 1. mainly confirmed the findings of previous studies but using short peptides. 
 
 
 
2, Supplementary Figure 2 AB and Fig S7-8, there are no error bars. All these experimental studies 
must be done by at least 3 independent experiments to correct for random errors. 
 
 
 
3, There are no peptide sequences for Supplementary Figure 2 AB. We ask this question is because 
authors claimed in line 128,129 in results (page 6) that they supercharged the APR (including 
peptide 12 and therefore variants A to H) with basic arginine residues, but when we look at 
supplementary table 2, we found that the peptide 12 variants including 12 A,B, C and D are 
endowed with negative charges. This finding is completely contradictory to their claim. 
 
 
 
4, The author claims that 12B targeted PB2. Then the authors should not select NP inhibitor and 
NA inhibitor as the positive control. They should select PB2 inhibitor, VX-787, as the positive 
control in Fig. 2C. 
 
It is difficult to understand why did authors use the plaque size to calculate the IC50, but not used 
the plaque reduction assay to test IC50. The gold standard for antiviral activity is the Plaque 
reduction assay, the reduction of virus titre in culture supernatant of multicycle infection by cell 
culture and the protection against cytopathic effects. These findings MUST be provided in any 
antiviral studies. 
 
 
 
5, The authors should choose an antiviral such as VX-787 that can inhibit viral replication in Fig 
2G. The authors used oseltamivir which did not show any antiviral effect in the mice model. Note 
that oseltamivir by oral gavage should give obvious reduction in lung virus titer of treated mice 
after challenge. This is again contradictory and throws lots of doubt on the creditability of this 
study. Moreover, there was only 2-3 fold viral load reduction in lung tissue after treatment of 
infected mice by the 12B peptide. No survival data was given to prove the efficacy of the 12B 
peptide given by intravenous injection. 
 
 
 
6, In Fig S5F, the TEM pictures indicated that the PB2 is not visualized as single particle which 
suggested that there are purification problem. How was PB2 protein being purified? 
 
 
 
8, In Fig 4I, at virus inoculum of 1MOI, immunofluorescence microscopy done at 16h post-
infection, why was there no PB2 visualized in 12Bpro2 treated cell and the buffer treated cells? 
 
 
 
9, In Fig 5A and Line 286, authors claimed that sequence-specific process is tolerant to single 

conservative substitutions which suggests the potential of a novel broad-spectrum antiviral. 
However, even though for the 100% matched LIQLIVS, the antiviral activities against the four 
viruses are very different with inhibition potency varies from 20% to 80% at the 10uM peptide 
concentration. Thus it is unlikely that when you have single substitutions, such poor antiviral 
potency can be maintained for all 4 different viruses. 
 
 
 
10, Line 299, Authors claimed that ‘R50 outperforming 7-DMA for anti-Zikavirus’, but the IC50 and 
TC50 of 7-DMA are better than those of R50. This is again contradictory. 
 
 
 
 
Recommendation: There are many serious shortcomings in the work that must be addressed 
before the paper can be considered for publication. 
 
 
Reviewer #3 (Remarks to the Author): 
 
 
 
In their paper Michiels et al. searched for sequence homologies in aggregation-prone regions in 
amyloidogenic proteins and viral proteins speculating that these APR regions may interact 
physically thereby affecting viral replication. They identified such shared APR and engineered a 
designer peptide derived from the Influenza A PB2 peptide. This peptide inhibited Influenza A 
replication in cell culture and in vivo in mice. Using the same approach, they also created a similar 
peptide that was shown to inhibit ZIKV infection. 
 
Although the hypothesis is thrilling I have severe doubts regarding several parts of the 
manuscript: 
 
1. Authors searched for APR in amyloids and viral proteins and found homologies assuming that 
possible interactions of respective regions occur also in vivo. At least in part, the some 
assumptions are wrong, because e.g. full length PAP (Fig 1E and S Table 1) does not interact with 
HIV-1 particles (only fibrils derived from PAP85-120 and PAP248-286) and the fibrisl do not bind 
the viral glycoprotein but the membrane of virions. So, there is no viral counterpart. Whether 
alpha synuclein indeed interacts with specific viral proteins has to my knowledge also yet proven. 
As previously published by the authors and other, APR can be found in almost all proteins and 
buried inside. So it is not surprising that one finds similar sequences in the human and viral 
proteome if all database entries are screened. Are there ARP in non-amyloidogenic proteins that 
share similarities with virus ARPs? 
 
2. Secondly, the 12B peptide has Influenza inhibitory activity in vitro and in vivo, but the 
mechanism of action is hardly explored (see more details below). As shown, 12B rapidly forms 
amyloid. Has the amyloid form of 12B been tested in vivo or the monomer? Moreover, most of the 
results may simply be explained by competition of the amyloid with endocytotic pathway route of 
Influenza virus (we have unpublished data that many amyloids unspecific ally interfere with 
Influenza virus entry by competition with amyloids and the similar mechanism may also play role 
in the antiviral activity of 12B. It is unclear how the peptide or the amyloid derived thereof is 
internalized and by which mechanism it may prevent Influenza replication. A 12B mutant not 
carrying an ARP might be a good control. Moreover, classical time of addition experiment need to 
be performed to dissect whether 12B inhibits entry or whether it acts indeed intracellularly. It is 
also possible the amyloid sequesters the virus rendering it non-infectious which could be tested by 
pre-exposing the virons to 12B first followed by infection. 

3. ZIKV inhibitory peptide: Generall, this part is much less elaborated than the Flu part. Several 
controls on the antviral mechanism are missing and also non-specific effects such as toxicity, 
supra structure formation by the peptide, binding of virions etc could account for the observed 
anti-ZIKV activity. Toxicity assays should be performed by incubating cells with peptide at 
concentrations that exceed the IC50 by 100 fold allowing to determine selectivity indexes. Time of 
addition experiments need to be done to test at which stage of the reoication cycle the peptide 
acts (and whether it is active as monomer or amyloid). 
 
More comments: 
 
L 36 PB2 accumulates in influenza-infected tissue in vivo and displays antiviral activity against 
influenza A and its common PB2 polymorphisms: rephrase , 
 
L50 interferors is not the correct wording, e.g. in some instances amyloids clearly enhance 
infection 
 
L51 there are also studies on semenogelins derived fibrils that enhance HIV-1 infection 
 
L42-61: Introduction would benefit from a differentiation in those amyloids that have antiviral and 
those having proviral activity 
 
L79 influenza A we show that the amyloid accumulates: which amyloid is meant? 
 
L90: Semenogelin fragments are missing; 
 
L95 and table 1: Wrong: PAP does not interact with the viral glycoprotein, PAP derived peptides 
248-286 and 85-120 form fibrils that bind the membrane of the virus but not the glycoproteins 
 
L101: scrambeled peptides (same pI and charge) would have been a better control 
 
L111-119: I don’t get the meaning of Fig. 1E. E.g. what is the red mark “HIV-1” in the PAP item 
meaning? Is there a homology in the first PAP residues with those in HIV-1? And if yes, against 
which viral proteins? It would be good to have a control like albumin and hemoglobin and do the 
same analysis for the indicated viruses to show that this kind of assay yields meaningful results. 
 
L124 why not using an APR from the better established Aß- HSV link? 
 
L126 why wasn’t the APR as predicted synthesized but rather a designer peptide (dimer with 
linker) What is the rational? 
 
L138: show hemolytic activity or cytotocity assay of peptide 12, the antiviral effect could be 
caused by toxicity. 
 
L 144: negatively charged, is it just a polyanion if forming fibrils? 
 
Fig. 3F. How has the cytotox assay been performed? How many days were the cells exposed to the 
peptide before toxicity was determined? 
 
Comment on solubility of the peptide? 
 
Typically in vivo experiments are at the end? In which state was 12B applied? As monomer or 
amyloid? 
 
Line 182: the peptide is already present in its amyloid state. Has the amyloid been administered in 
vivo? 

 
Line 188: 3D does not show classical amyloid fibrils it looks like oligomers? What does the inlet 
100 µM mean? 
 
Fig. 3G: It would be much more convincing to titrate the peptides to see whether there is indeed 
activity or not as a general property of the peptides, or just an effect obtained at a certain 
concentration. The antiviral assays are easy to perform. 
 
Line 219: Why is the scrambled peptide binding? 
 
Line 271: same MOI of Influenza A and B viruses used? Mutational analyses of PB2 in Flu A would 
be more convincing 
 
Lien 293: provide sequences as table. 
 
Lines 311-325: many of the mentioned amyloid/virus interaction are only indirect (no physical 
interaction between amyloid and the viral structures); and sometimes data were only derived from 
in vitro experiments when amyloid (eg IAPP) was agitated with virus (e.g. RSV). As outlined 
controls should be included like albumin or hemoglobin subunits as searched for APRs and 
sequence similarities. 
 
The discussion would benefit from a much more detailed explanation of the observed anti-Flu 
effects. How is the peptide internalized? Is it internalized as amyloid or soluble peptide? How does 
the peptide get into contact with PB2 in the cell? What is the mechanism by which it blocks Flu 
replication? How many copies of the peptide per cell are required to block replication? 
Stochiometric or substochiometric amounts required? Is there amyloid stainable inside the cell?