Peer review of: Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functiona (2020)

Reviewer #1 (Remarks to the Author): 

Arragain et al have used X-ray crystallography and cryo-EM to obtain a high-resolution structure of 
the La Crosse virus L protein. This approach enabled them to build a full-length structure of the 
polymerase core and the flexible cap binding and endonuclease domains. In addition, the authors 
were able to obtain a structure of the L protein bound to RNA in an elongation complex. The work 
also advances our understanding of this enzyme complex by showing new orientations of the 
flexible domains, suggesting that these move around during cap snatching. The identification of 
the flexible zinc binding domain is also very intriguing. Overall the work is of high quality, the 
interpretation of the structures very sensible and I believe that this work significantly advances our 
understanding of RNA virus RNA polymerases and, in particular, our understanding of bunyavirus 
RNA synthesis. I highly recommend this manuscript’s publication and just have a couple of minor 
comments that the authors could discuss. 

1. The authors speculate that the zinc binding domain is a host factor binding platform and make a 
comparison with the 627-domain of the influenza virus RNA polymerase, which is involved in host 
adaptation and viral replication. The NCBI lists at least 84 La Crosse virus L protein sequences. Did 
the authors check if mutations are present in the zinc binding domain that could reveal more about 
its function? 

2. The authors say that the active site is slightly more open than normal (line 210). What post-
incorporation complex are they comparing the LACV polymerase too? I presume the influenza A 
virus one, but it would be good to have this specified and perhaps even quantified since this 
structure is such an interesting intermediate between incorporation and translocation. In addition, 
do the authors assume that some of the energy that will power translocation is stored in this open 
conformation? 



Reviewer #2 (Remarks to the Author): 

Arragain et al. reports the X-ray crystallographic cryo-EM structures of the polymerase of La 
Crosse Virus (LACV). Only a single existing structure of a Bunyavirales polymerase exists and is 
missing crucial information for the C-terminal portion of the enzyme which contains the cap-
binding domain (CBD) and a zinc-binding domain (ZBD). Bunyavirales members utilize a cap-
snatching mechanism to grab a cellular mRNA and utilize an N-terminal endonuclease domain to 
cleave the mRNA, leaving a capped template that serves as a primer for transcription and 
replication. The length of the truncated RNA differs based on virus species and it’s unknown 
whether L interacts with cellular factors like RNA polymerase II to grab the mRNA as has been 
shown in influenza. Here the authors present both the x-ray crystallographic and cryo-EM 
structures of full length LACV-L protein in both an apo state and bound to a duplexed RNA 
template that mimics the template and product strand. Overall this is an important structure that 
reveals the full structure of an L protein from this order of viruses. However, the manuscript could 
use some editing in order to polish the text to make it easier for the reader to understand. 

1) on line 51 it should read RNA polymerase II instead of just polymerase II. 
2) Interestingly the conformation of the endonuclease domain differs from the previously published 
structure of a C-terminally truncated (1-1750) LACV-L construct by ~180 degrees. This was 
somewhat hard to visualize in Supplemental Figure 4 and the authors might consider adding labels 
to helices to better visualize this difference. 
3) Why this difference in position of the endonuclease is not addressed by the authors. Might it be 
due to the presence of the full length construct forming additional interactions between 
endonuclease and the CBD? 
4) In general I found many figures to be very cluttered and difficult to focus on. The previously 
missing C-terminal portion of L projects away from the body of the polymerase with the CBD 
interacting with the endonuclease domain. The CBD is flexible as shown in Figure 3C but the 
authors might consider using different colors so as not to be confusing with the N to C terminal 
coloring in Figure 3D & E. 
5) This flexibility is compared to existing structures of the influenza virus polymerase but the 
range of motion identified here (~35 degrees) doesn’t encompass the states seen in influenza that 
correlate with bringing the cellular RNA first to the endonuclease for cleavage and then to the 
template entry site. 
6) The reference on line 158 to Supplemental Figure 10 needs to be corrected. 
7) The authors claim that alpha helix 91 folds back into the hydrophobic pocket of the ZBD. It 
suggests a stable interaction although the electron density for that helix is largely missing at a 
standard threshold, indicating it is highly flexible and not very stable. 
8) Surprisingly the authors were able to identify a subset of particles with duplexed RNA in the 
active site cavity which the authors suggest it represents an elongation state. It is consistent with 
the path of the RNA from the 5’ RNA promoter bound in the 5’ stem-loop pocket. The authors were 
able to identify differences in the position of the priming loop and a portion of the mid-domain that 
it interacts with in the elongation state compared to the pre-initiation state. On lines 248-251 a 
reference is made to residue Y1696 forcing separation of the two strands but I don’t see any 
labeling of that residue in the referenced figure, 4b. Might the authors have meant 4c? 




Reviewer #3 (Remarks to the Author): 

La Crosse virus (LACV) is a significant pathogen of Bunyavirales; however, no effective treatment 
and vaccine are available. The viral RNA-dependent RNA polymerase of LACV is responsible for 
both replication and transcription of the viral genome. Arragain et al. described both the crystal 
and cryo-EM structures of full-length LACV polymerase in two different functionally states, pre-
initiation and elongation. In particular, compared to the previously reported structure of LACV-L(1-
1750) that lacks C-terminal domains (CTD), Arragain et al. reported the structure of the full-length 
of LACV-L, including the model of the C-terminal domains and revealed the changes between two 
captured functional states. Arragain et al. also demonstrated that the priming loop, lid domain, 
and C-terminal region are involved during RNA synthesis. 

Overall, it is a nice paper with robust experimental data. Arragain et. al. used a combination of x-
ray crystallography and cryo-EM to determine the structures of the full-length LACV polymerase 
with two functional states. The Arragain et al. revealed the difference between the pre-initiation 
and elongation states, highlighted the structural features of the cap-binding domain (CBD, 
residues 1842-1977) and zinc-binding domain (ZBD, residues 2026-2263) within the CTD. 

However, there are some issues that need to be addressed: 

1. Due to a large amount of information, in the data statistics table, the author should list the 
components for each structure, including the sequence of 3' template, 3' 1-16, 5' hook, ENDO 
CORE, and the range of protein residues. 

2. Page 5 “adjusted version of the protocol described previously”. Please briefly highlight and 
describe the difference compared to the protocol by Gerlach et al. (2013). 

3. It is interesting to see not only the pre-initiation state of complex but also an elongation state 
with a bound product-template duplex. The 5ʹ promoter 1-10 and 3ʹ promoter 7-16 is not a perfect 
match (with one UG mismatch), but very close. The captured state is without the incoming NTP 
and longer template at the 5' end. So it is curious to see what are the distances between the 
active site residues and the U16? Is this close enough to catalyze the reaction? Also, since it is 
unexpected, could this be an inhibition state rather than an elongation state? 

4. Please make the residue numbering consistent throughout the text. For example, on pages 5 
and 6, LACV-L 1-1750 vs and the polymerase core (residues 186-1751). It is not apparent that the 
C-terminal region (residues 1752-2263) includes the mid-domain (residues 1752-1841 and 1978-
2025), C-terminal domain (CBD, residues 1842-1977), and zinc-binding domain (ZBD, residues 
2026-2263), and the β-hairpin strut (residues 2084-2102) is part of ZBD. Please rearrange the 
text and make it easy to appreciate the relationships among domains. 

5. Page 7: Fig.2d and Fig.2e are missing? Should be Fig.3d and Fig.3e? Not clear about the 
extreme position 1 and 2. The color is confusing. The color of the ribbons in Fig. 3a is also gold 
and orange. Do the gold and red in Fig.3c and Fig.3d represent the entire CBD or part of the CBD? 
What regions are they comparing to? Please show the comparison with one region (such as 
endonuclease domain) is fixed. 

6. “Its two sub-domains of equivalent size surround a metal ion that is coordinated by four 
residues highly conserved amongst peribunyaviruses: C2064, H2169, D2178 and H2182, 
suggesting that it is a zinc ionThe overall topology of the ZBD has not been previously observed 
according to a DALI search." It is interesting as a ZBD; it doesn’t have a structural homology. 
Does any other related polymerase have a similar ZBD? Or is this unique in LACV. 

7. Figure 1. Too many colors in Figure 1 make it hard to recognize the details of the structures. 
Also, please do not switch the color for the same component. For example, the template 5’vRNA, 
and the product 3’vRNA should keep with consistent color within the same figure. The name of the 
sequences in the figure should be labeled clearly and consistent with the main text, including 3’ 
vRNA, 5’ vRNA, template, and product. The figure legends should contain more details to illustrate 
the figures clearly, such as the color and residue numbers in Figure 1a. 

8. The sequence alignments should include the protein ID (such as the UniProt #) in Figure 3b and 
Supplementary Figure 10. 

9. Supplementary figure 9: the comparison between LACV and influenza polymerase priming loop 
at elongation. It seems that the priming loop in LACV is not as flexible as influenza polymerase. 
Because the priming loop is supposed to be critical in the initiation stage, but not in the elongation 
stage, does this mean the priming loop in LACV is in a state between initiation and elongation? 

10. There are two polymerases in the asymmetric unit of the crystal structure. Are they identical? 
Please also compare them with the cryo-EM structure at the pre-initiation state. 

11. Page 16. “Before freezing and storing at -80°C, LACV-L bound to 3ʹ (1-16) and 5ʹ (9-16) vRNA 
were mixed with 5ʹ (1-10) vRNA hook in a 1:2 molar ratio.” Why not incubate them at the same 
time? Also, why choose 1:2 ratio? In addition, “The 5ʹ (1-10) vRNA end was later soaked into 
crystals.” Using the same ratio?