Peer review of: The RNA quality control pathway nonsense-mediated mRNA decay targets cellular and viral (2020)

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

The major claims of this well-written manuscript are 1. previous splicing events have been missed by 
previous reports, 2. repression of NMD genes enhances lytic reactivation and virion production, 3. 
some important viral transcripts are targets of NMD. These claims will be interesting to the 
expanding NMD field and virologists. This report also brings to light another example of interactions 
with NMD factors and noncoding RNAs. The work is convincing with genomics-based sequencing 
methods followed by validation assays that included the important controls. It is important to 
distinguish between effect lytic reactivation and abortive reactivation by showing a new round of 
infection with virus from the NMD repression assays. They showed this striking result in Figure 2. 
These are some of the highest amounts of lytic reactivation that this reviewer has witnessed. A 
significant contribution in the many splicing events that have been mapped in this report. This 
challenges an incorrect assumption that many viral genes are not spliced. They have validated some 
of the new introns by cloning and sequencing the splice junctions. The statistical methods and 
details of the methods were fine. 

 

 

Below are some specific comments that would improve this strong and thought-provoking 
manuscript. 

 

1. What is the significance of NMD in the lytic cycle in the context of host shutoff? 

2. Representative flow cytometry histogram plots should be included to complement the column 
graphs of RFP+ or PAN+ cells. 

3. The GEO accession number appears to be from a previous manuscript. It is unknown if the current 
sequencing data have been deposited. 

4. How can the authors determine whether repression of UPF1 and UPF3X is affecting lytic 
reactivation by NMD-independent mechanisms? 

5. How was the antibody immunoprecipitation of p-UPF1 validated before fRIP-seq? 

6. Lines 241-242: Have these “noncoding” RNAs been found to be associated with ribosomes in 
other reports? 

7. Figure 5F: Measuring additional negative control regions would be helpful in Fig. 5 to show viral 
messages that were not found in fRIP-seq have low levels of detection by qPCR. 

8. The spliced form of XBP1 in activated in some developmental stages of B cells, which are relevant 
to KSHV infections. This could be explored in the Discussion section. 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

In this paper, Zhao et al. explore the effects of nonsense-mediated RNA decay on KSHV lytic 
reactivation. The paper makes three important claims. First, and most importantly, they claim that 
NMD “poses a significant barrier” to KSHV lytic reactivation. Second, they use transcriptomics to 
identify host and viral NMD targets in infected cells and to identify novel introns in viral genes. Third, 
they link NMD with the unfolded protein response and expression of the KSHV transcription factor 
RTA. The strength of the paper is the exciting and novel observation that NMD restricts viral lytic 
reactivation. The work supporting that conclusion is strong and rigorous. The weakness in the paper 
(discussed more below) is that the paper is full of potentially important hints at what the role and/or 
mechanisms of NMD in viral lytic reactivation, none of which are are fully fleshed out to provide a 
solid conclusion. The paper would be significantly improved if they would focus on one potential 
driving factor (e.g. RTA as NMD substrate) and investigate it thoroughly. As it stands now, the paper 
conflates multiple loosely connected observations that do not support any single firm conclusion 
about any of the role(s) of NMD in KSHV reactivation. 

 

Major concerns: 

 

1. The role of NMD in KSHV lytic reactivation is the main idea of the paper. However, the paper 
starts with an in depth splicing analysis that is never tied into the rest of the paper in any concrete 
fashion. They imply that the 3´ UTR splice sites may be driving NMD on viral transcripts but no 
substantiation of this is found anywhere in the paper. Even with RTA, which had previously 
described 3´ UTR introns, no validation that splicing was linked to NMD was presented. For example, 
If they knockdown UPF1, do they see accumulation of spliced 3´ UTR isoforms? Does removal of the 
3´ UTR intron abolish NMD, or is it driven by long 3´ UTRs? Given its central role in KSHV lytic 
reactivation, it would be compelling to know whether expression of an NMD-insensitive RTA mRNA 
complements most of the NMD phenotypes. Currently, Figure 1 is a descriptive dataset only 
tangentially related to the rest of the paper. 

 

2. Similarly, the host targets (Figure 4) and XBP1 data (Figure 6) do not lead to any solid conclusions. 
There’s nothing wrong with the data in Figure 6, but they do not convincingly demonstrate that 
NMD regulation of XBP1 has any effect on the virus. The data say that XBP1 might be an important 

NMD target related to KSHV reactivation. The data also show that upon lytic reactivation, there is a 
less than 2-fold difference in ORF50 RNA levels in the siUPF1 treatment plus/minus KIRA6. While this 
may indeed be biologically relevant, it’s difficult to say if/how this change contributes to the robust 
effects of NMD on KSHV reactivation. Perhaps there is a link, but Figure 6 data seems more 
appropriate for a “Figure 1” of a new paper rather than a mechanistic conclusion (and model) for the 
current paper. 

 

3. The fRIP studies are interpreted too strongly. In many cases, this is the only experimental support 
that the authors use to classify something as a “NMD target”. It is well established that 
formaldehyde RIP approaches often has high background, so fRIP should be interpreted with 
considerable caution. Conversely, they are also seeing false negatives because it appears that 
approximately one-third of the 89 “NMD Targets” have greater signal in the IgG than the anti-UPF1p 
pulldowns (i.e. log2FC<0)(Fig 4b/c). Moreover, rigorous validation needs to be done. For example, 
how many of these genes are up-regulated upon NMD inhibition? Admittedly, this is difficult (or 
even impossible) to do with viral lytic RNAs because of the confounding factor of increased 
reactivation upon NMD inhibition. However, this should not be a confounding issue with host RNAs 
or viral latent genes. The analysis is also confusing. Most CLIP-type experiments employ peak-calling 
algorithms, but it doesn’t seem to be the case here. By eye, it appears that they find nearly 50% of 
their transcripts have more signal in anti-UPF1 than IgG (Fig 4b/4c) and they suggest thousands of 
genes are NMD targets. What are their cutoffs and how are they justified? This seems like 
significantly more than expected, but no comparisons to similar experiments be other groups are 
discussed so it’s difficult assess. Moreover, there’s no correction for RNA levels. For example, for the 
ORF45 traces show what they interpret to be a 3´ UTR peak (Fig 5). However, this could simply arise 
due to high background because the ORF45 transcript is expressed at higher levels than ORF46-48. 

 

Other: 

1. The fRIP-PCR assays require a control of a viral nonspliced (Fig 1) or non-NMD target mRNA in 
addition to vtRNA1-1. 

2. In Figure 1, they “preference for a G nucleotide in the 3´ of the splice acceptor site. The 
identification of consensus splice site sequences…” The 3´ splice sites shown do not show a strong 
preference for AG. This is therefore clearly not representative of a consensus splice site. 

3. More browser shots in supplemental of KSHV genes would help readers assess the fRIP peaks, 
particularly if compared to RNA-seq from the same time point. 

Minor: 

1. The GSE number is incorrect. 

2. In Figure 1d, ORF45, the slash is at wrong place. 

3. In figures such as 3b and 3d which are so similar, it could be helpful to give headers or color code 
according to if they are BCBL vs iSLK2.19 

4. Figure 2m is UPF3X, called UPF1 in text. 

 

 

 

 

 

Reviewer #3 (Remarks to the Author): 

 

The impact of NMD on DNA virus infections is largely unexplored. In this study, the authors use 
global analyses to greatly expand (~10X) the number of splice junctions in the KSHV transcriptome, 
many of which were found in the 3’ UTR and result in the deposition of the EJC complex as inferred 
by RIP experiments using eIF4A3 – hence leading them to examine a role for NMD. UPF1 KD resulted 
in a minor increase in lytic viral growth but interestingly a major increase in latent reactivation. The 
authors go onto to examine NMD targets globally and focus on the ORF50 transcript as a regulated 
example. Finally, the authors identify the UPR-associated ORF50 transcription factor sXBP1as 
another NMD-regulated factor during infection. 

Overall the manuscript is well-written, the data are extremely convincing, and conclusions are well-
supported. The study does a great job at establishing NMD as a factor that influences the regulation 
of a DNA virus infection and will be of very high interest and impact in the field. 

 

I have only one very minor suggestion to polish the manuscript: The EJC label in the model figure 
(6K) is compacted/compressed and should be redrawn to enhance readability.