# Polyclonal LC3B Antibodies Generate Non-Specific Staining in the Nucleus of Herpes Simplex Virus Type 1-Infected Cells: Caution in the Interpretation of LC3 Staining in the Immunofluorescence Analysis of Viral Infections

## TL;DR

This study warns that polyclonal LC3B antibodies can produce misleading nuclear staining in cells infected with herpes simplex virus, potentially leading to incorrect conclusions about autophagy.

## Contribution

The study reveals that polyclonal LC3B antibodies cause non-specific nuclear staining during HSV-1 infection, which was previously unrecognized.

## Key findings

- Polyclonal LC3B antibodies show nuclear staining in HSV-1-infected cells, but monoclonal antibodies and GFP-LC3 do not.
- LC3B is not detected in the nuclear fraction of infected cells by Western blot, even with polyclonal antibodies.
- Nuclear staining persists in LC3B knockout cells, indicating non-specific antibody binding.

## Abstract

The most common marker used to monitor autophagy is the microtubule-associated protein light chain 3 (LC3). Upon induction of autophagy, LC3 is conjugated to phosphatidylethanolamine and targeted to autophagic membranes, which can be easily detected by immunofluorescence. However, this technique has some limitations. During the early stages of HSV-1 infection, strong LC3B nuclear staining is observed within the viral replication compartments. This staining is only detected when using polyclonal antibodies. It is noteworthy that monoclonal antibodies or the GFP-LC3 plasmid do not reveal any nuclear LC3 staining. Interestingly, LC3B is not detected in the nuclear fraction of infected cells by Western blotting, even when polyclonal antibodies are used. In infected LC3B knockout cells, nuclear staining is still observed when using polyclonal LC3B antibodies. This suggests that polyclonal LC3B antibodies generate non-specific nuclear staining in infected cells, which could result in misinterpretation and erroneous conclusions. These findings raise questions about the reliability of LC3-immunofluorescence assays in herpesvirus infections. It is imperative that the methodology employed for monitoring autophagy by immunofluorescence in viral infections be reviewed and updated, and that the specificity of anti-LC3B antibodies be tested before use. To ensure the accuracy of the results, it is essential to validate this technique with additional assays, such as by immunoblot analysis or via the use of autophagy-deficient cell lines.