EFF-1 promotes muscle fusion, paralysis and retargets infection by AFF-1-coated viruses in C. elegans

  1. Anna Meledin
  2. Xiaohui Li
  3. Elena Matveev
  4. Boaz Gildor
  5. Ofer Katzir
  6. Benjamin Podbilewicz

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

A hallmark of muscle development is that myoblasts fuse to form myofibers. However, smooth muscles and cardiomyocytes do not generally fuse. In C. elegans , the body wall muscles (BWMs), the physiological equivalents of skeletal muscles, are mononuclear. Here, to determine what would be the consequences of fusing BWMs, we express the cell-cell fusogen EFF-1 in these cells. We find that EFF-1 induces paralysis and dumpy phenotypes. To determine whether EFF-1-induced muscle fusion results in these pathologies we injected viruses pseudotyped with AFF-1, a paralog of EFF-1, into the pseudocoelom of C. elegans . When these engineered viruses encounter cells expressing EFF-1 or AFF-1 they are able to infect them as revealed by GFP expression from the viral genome. We find that AFF-1 viruses can fuse to EFF-1-expressing muscles revealing multinucleated fibers that cause paralysis and abnormal muscle morphogenesis. Thus, aberrant fusion of otherwise non-syncytial muscle cells may lead to pathological conditions.

Significance statement

Most cells are individual units that do not mix their cytoplasms. However, some cells fuse to become multinucleated in placenta, bones and muscles. In most animals, muscles are formed by myofibers that originate by cell-cell fusion. In contrast, in C. elegans the body wall muscles are mononucleated cells that mediate worm-like movement. EFF-1 and AFF-1 fusogens mediate physiological cell fusion in C. elegans . By ectopically expressing EFF-1 in body wall muscles we induce their fusion resulting in behavioral and morphological deleterious effects, revealing possible causes of congenital myopathies in humans. Using AFF-1-coated pseudoviruses we infect EFF-1-expressing muscle cells retargeting viral infection into these cells. We suggest that virus retargeting can be utilized to study myogenesis, neuronal regeneration, gamete fusion and screens for new fusogens in different organisms. In addition, our virus retargeting system can be used in gene-therapy, viral-based oncolysis and to study viral-host interactions.

Article activity feed

  1. eLife

    ###Reviewer #3:

    The manuscript "EFF-1 promotes muscle fusion, paralysis and retargets infection by AFF-1-coated viruses in C. elegans" describes the ability to VSV virus coated with AFF-1 fusogen can be targeted to specific cells in vivo using C. elegans. Using this technique, the authors elegantly show that AFF-1 viruses show tissue/cellular tropism in vivo that largely match known AFF-1 or EFF-1 receptor expression, which they verify through genetic mutation and ectopic expression. Overall, I would like to commend the authors on a fascinating and scientifically thorough manuscript that would be of interest to a broad range of scientists, from C. elegans researchers to viral engineers. However, while there are several lines of evidence that suggest cell-to-cell fusion in the muscle upon EFF-1 ectopic expression, they are all circumstantial. So I suggest the authors tone down the strong language used throughout the manuscript that outright state EFF-1 induces muscle fusion, including in the title, unless they use EM or photoconvertible fluorescent markers that show actual shared cytoplasm between cells.

    Major issues:

    1. The authors have not clearly shown that EFF-1 and VSV-EFF-1 cause muscle cell fusion. Nuclei count is not evidence of cell-cell fusion (Fig. 4I) and it is not clear from the images how the authors can distinguish the plasma membrane of muscle cells in order to count nuclei per cell in Fig 4I and Fig 7O-P. Furthermore, the authors claim muscle cell fusion in the myo-3p::eff-1 strain based on indistinguishable membranes expressing membrane-bound YFP and even distribution of mCherry (Fig 5). But loss of membrane bound YFP and distribution of mCherry are not clear evidence of cell fusion, especially when qualified and not quantified. Definitive evidence of cell-cell fusion in the muscle can be shown with EM or using a photoconvertible fluorescent protein which could show actual sharing of cytoplasm between cells. So claims like the following (and many others including the title) are too strong given the data in the manuscript:

    a) "EFF-1 expression in BWMs induces their fusion" (Line 331)

    b) "evenly distributed cytoplasmic myo-3p::mCherry indicating fusion and content mixing between these cells during development" (lines 297-299)

    c) EFF-1 expression in fused BWMs enables VSV∆G-AFF-1 and VSV∆G-G spreading (line 349)

    1. Figure 3 does not convincingly show key data to fit with their hypothesis that VSV-AFF-1 infection would increase upon EFF-1 expression in a dose-dependent manner. Based off of Figure 3, the authors conclude that "hypodermal infection by VSV∆G-AFF-1 increases with conditional induction of eff-1." (Lines 229-230). But they use an assay counting GFP-positive nuclei. So the result showing a decrease in GFP+ nuclei as eff-1 levels decrease is likely due to a loss of natural syncytium formation in the hypodermis rather than due decreased infection by VSV-AFF-1. As they stated in lines 199-200, GFP+ nuclei in the hypodermis are localized closer to the injection region of the head in eff-1 mutants. So higher eff-1 expression would lead to both a larger hypodermal target for viral infection and more posterior nuclei within that target for the virus to spread towards, showing GFP expression when the syncytium becomes infected. To control for this, the authors could infect the eff-1-ts mutant with VSV-G and show no dose dependent effect.
  2. eLife

    ###Reviewer #2:

    The manuscript by Meledin et al have used the C. elegans model to investigate two interesting aspects: (1) The consequence of ectopically fusing the normally mononuclear body wall muscle cells by expressing the eff-1 fusogen (2) using VSV∆G virus particles coated with the AFF-1 fusogen to change the tropism of the virus and preferentially infect muscle cells. This manuscript describes a novel and truly innovative approach in the C. elegans model to develop methods for cell-specific viral targeting by modifying the host genome. I find the data showing preferential and efficient infection of EFF-1 expressing cells by VSV∆G-AFF-1 spectacular, as there are many applications that could be developed using this approach. In addition, showing that fused body wall muscles do not function normally is a significant finding, even though the exact causes of the strong defects that were observed are not investigated in detail. Here, the manuscript could be strengthened, for example by including an ultrastructural (EM) analysis of the fused muscle cells.

    Overall, the manuscript is very well written and based on solid data. Some figures are a bit difficult to interpret (e.g. fig. 6 showing the fused muscle cells).

  3. eLife

    ###Reviewer #1:

    In their manuscript, the authors examine Vesicular Stomatitis Virus (VSV) coated with fusogen infection in C. elegans based on previously developed pseudotyped virus VSVG-AFF-1. They show VSVG-AFF-1 can efficiently infect C. elegans multiple tissues through microinjection, and the infection requires the function of bilateral fusogen (AFF-1 or EFF-1) on the target cells. Furthermore, using the genetic and living imaging techniques, they observed that overexpression of EFF-1 in muscle leads to paralysis, dumpy, and uncoordinated phenotype. AFF-1 coated pseudovirus can thus infect BWMs with ectopically express EFF-1, and significantly enhance the uncoordinated behavior, which may be due to the merge of BWMs or formation of non-functional syncytial muscle fibers. This is an interesting, well-written, and thoughtful study to show that C. elegans can be infected by a virus with the bilateral fusogen and represents a significant advance in identifying important players mediating virus infection in C. elegans.

    Major Comments:

    1. myo-3 encodes a myosin heavy chain, and its promoter is very strong for the gene expression. Overexpression of myo-3p::GFP/mCherry with high concentration extrachromosome array frequently results in uncoordinated, dumpy, or paralysis phenotype, which due to inconsistent expression, chimeric expression, leak expression and varies copies expression that inhibits the endogenous promoter. The authors show that extrachromosome array of muscle expression of EFF-1 causes uncoordinated, dumpy, larval arrest, and paralysis phenotypes, which may be due to both myo-3 promoter or EFF-1 expression in the muscle. It is very difficult to draw any solid conclusion here. As most of the data were based on the extrachromsome muscle expression of EFF-1, it is important to generate a single-copy insertion of myo-3p::EFF-1 to mimic the endogenous expression levels and test whether ectopic expression of EFF-1 is required for VSVG-AFF-1 infection and others.

    2. Is it possible to examine/observe AFF-1 and EFF-1 interaction after VSVG-AFF-1 infection and in the fused BMWs in vivo?

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript.

    ###Summary:

    All reviewers thought this is an interesting study and most of the experiments are convincingly performed. However, they also raised a number of concerns.

  5. Version published to 10.1101/2020.05.17.099622 on bioRxiv