Ubiquitin Ligase ITCH Regulates Life Cycle of SARS-CoV-2 Virus

Reviewer #1 (Public review):

Summary:

The authors investigated the role of an E3 ubiquitin ligase ITCH in regulating the viral life cycle of SARS-CoV-2. The authors showed that ITCH mediates ubiquitination of the membrane (M) and envelope (E) proteins of SARS-CoV-2. Ubiquitination of E and M results in enhanced interactions between the structural proteins and redistribution of the structural proteins into autophagosomes. The authors claim that the enhanced interactions between structural proteins and trafficking of the structural proteins into autophagosomes contribute to SARS-CoV-2 replication and egress, prompting ITCH as a potential antiviral target. ITCH also alters the cellular distribution of host proteases important for spike cleavage which protect and stabilize spike with cleavage. The authors also demonstrated that SARS-CoV-2 replication is augmented by ITCH in which virus replication is significantly impaired in cells lacking ITCH expression.

Strengths:

The authors provided high-quality data with appropriate experimental controls to justify their claims and conclusions. The mechanistic analyses are excellent and presented in a logical manner. The investigation of the role of ubiquitination in coronavirus assembly and egress is novel as most previous studies focused on its role in mediating innate immune responses.

Weaknesses:

Although the authors showed that ITCH ubiquitinates E and M proteins, the claim that such ubiquitination promotes virion assembly and egress is circumstantial. The enhanced interaction between the structural proteins and targeting of ubiquitinated structural proteins into autophagosomes does not necessarily result in increased virion production and release as suggested by the authors. There is a disconnect between the ubiquitination of structural proteins and the role of ITCH in augmenting virus replication as shown in Fig. 6A and B. In addition, the authors showed that the catalytic activity of ITCH is important for the localization and maturation of host proteases. However, the mechanism behind is unknown. Also, it is unclear how protection of spike from cleavage conferred by ITCH explains its role in promoting replication as a lack of spike cleavage would inevitably compromise entry. The major weakness of the manuscript is the lack of experimental data that explains the molecular role of ITCH in relation to its phenotype observed during SARS-CoV-2 infection.

Reviewer #2 (Public review):

Summary:
In this manuscript Qiwang Xiang et al. investigated the role of the E3 ubiquitin ligase ITCH in the life cycle of SARS-CoV-2. They claim the following:
i) ITCH promotes virion assembly by interacting with E and M proteins and enhancing their K63-linked ubiquitination
ii) ITCH-mediated ubiquitination promotes autophagosome-dependent secretion of viral particles.
iii) ITCH stabilizes the viral spike protein by impairing its processing by furin and catepsin L proteases.
The manuscript provides an interesting exploration of ITCH's role in the SARS-CoV-2 life cycle but requires additional work to strengthen key claims and address potential confounding factors.

Strengths:

The experiments are sufficiently clear in documenting that ITCH activity is critical for efficient SARS-CoV-2 replication and for M and E proteins K63-linked ubiquitination

Weaknesses:

• The manuscript does not convincingly demonstrate how ITCH-mediated ubiquitination of E and M impacts virus assembly and release. Identifying the specific lysine residues in M and E targeted by ITCH, and generating mutant VLPs or recombinant viruses, would strengthen the conclusions.
• Most of the conclusions rely on ITCH overexpression data, which may have off-target effects on Golgi integrity and vesicular trafficking. For instance, figure 4F provides evidence of altered Golgi morphology and TGN46 fragmentation raising concerns that ITCH overexpression could indirectly mislocalize furin, affecting S1/S2 cleavage of the spike protein. In addition, inhibition of furin activity may also lead to off-target effects, given its role in processing numerous host proteins.
• Similarly, ITCH overexpression is likely to indirectly affect cathepsin-L maturation. In addition, the manuscript does not clarify how impaired cathepsin L activity would influence virus assembly or release.
• A major concern is also the lack of quantification and statistical analysis of immunofluorescence images throughout the manuscript, which undermines the reliability of these observations.

Reviewer #3 (Public review):

Summary:

Xiang et al. investigated the role of ubiquitin E3 ligase ITCH in SARS-CoV-2 replication. First, they described the role of ITCH on the structural proteins. Here, the ubiquitination of E and M (but not S) leads to an enhanced interaction and presumably virion assembly. In addition, E and M ubiquitination seems to be necessary for p62-guided sequestration into autophagosomes for secretion. Furthermore, ITCH regulates S proteolytic cleavage by changing furin localization and inhibiting CTSL protease maturation. In addition, SARS-CoV-2 infection upregulates ITCH phosphorylation, whereas knockout of ITCH reduces SARS-CoV-2 replication.

Strengths:

The proposed study is of interest to the virology community because it aims to elucidate the role of ubiquitination by ITCH in SARS-CoV-2 proteins. Understanding these mechanisms will address broadly applicable questions about coronavirus biology and enhance our knowledge of ubiquitination's diverse functions in cell biology.

Weakness:

The involvement of ubiquitin ligases in SARS-CoV-2 replication is not entirely new (see E3 Ubiquitin Ligase RNF5; Yuan et al., 2022; Li et al., 2023). While the data generally support the conclusions, additional work is needed to confirm the role of ITCH in SARS-CoV-2 replication in a biologically relevant context. The vast majority of data is based on transient overexpression experiments of ITCH, which ultimately leads to massive ubiquitination of several viral and host cell factors, including potentially low-affinity substrates not typically recognized under physiological conditions. In addition to that, nearly all experiments were done in cells co-overexpressing ITCH and the viral structural proteins (or cellular proteases) in HEK293T cells. Therefore, a proteomic analysis of protein ubiquitination in a) SARS-CoV-2-infected cells (ideally several cell types) and b) SARS-CoV-2-infected v2T-ITCH-KO cells would verify the ITCH-related ubiquitination of e.g., E and M and would strengthen the whole manuscript. In addition, the few key experiments using SARS-CoV-2 infected cells were performed in VeroE6 cells, which are neither human nor lung-derived. Only in one experiment were lung-derived Calu3 cells included.
Moreover, the manuscript names ITCH as a central regulator of SARS-CoV-2 replication. If ITCH is beneficial for E and M interaction and thereby aids virion assembly, showing its effect on VLP production would be desirable. Clarifications regarding data acquisition and data analysis could strengthen the manuscript and its conclusions.