Entry receptors — the gateway to alphavirus infection

Zimmerman, Ofer; Holmes, Autumn C.; Kafai, Natasha M.; Adams, Lucas J.; Diamond, Michael S.

doi:10.1172/jci165307

Cited by 22 publications

(22 citation statements)

References 175 publications

(232 reference statements)

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“…Thus, we infected various cell lines with rVSV-dG-RABV-GFP which showed the wide tropism of rVSV-dG-RABV-G-GFP, exemplified in its ability to replicate in the wide spectrum of tested cell lines. However, a distinct alteration in cell tropism was observed among the VSV-GFP WT and the rVSV-dG-RABV-G-GFP infection on HaCaT cells which revealed resistance to the recombinant virus displaying the RABV-G protein, in contrast to their permissiveness to the rVSV -GFP-WT, suggesting the suitability of HaCaT cells to act as a cellular model for studying RABV receptor preference (27). In our study, we Interestingly, the higher levels of released virus progeny demonstrated on HaCaT cells co expressing the ITGB1 with nAChR receptors is supported by findings from previous study which demonstrated the interaction of both receptors through CO-IP assay (1).…”

Section: Discussionmentioning

confidence: 98%

“…Thus, we infected various cell lines with rVSV-dG-RABV-GFP which showed the wide tropism of rVSV-dG-RABV-G-GFP, exemplified in its ability to replicate in the wide spectrum of tested cell lines. However, a distinct alteration in cell tropism was observed among the VSV-GFP WT and the rVSV-dG-RABV-G-GFP infection on HaCaT cells which revealed resistance to the recombinant virus displaying the RABV-G protein, in contrast to their permissiveness to the rVSV -GFP-WT, suggesting the suitability of HaCaT cells to act as a cellular model for studying RABV receptor preference (27). In our study, we ectopically expressed the P.alecto receptors on HaCaT cells (Human cell line) to establish a heterologous system in which other factors that could affect the cellular receptors interactions are eliminated Additionally, to alleviate the cellular stress induced by transfection, we incorporated an empty vector control throughout the entire study as a means of normalization.…”

Section: Discussionmentioning

confidence: 99%

“…Hence the ectopic expression of RABV receptors on HaCaT cells showed enhanced virus binding and virus release but could not render them susceptible to infection (no GFP was observed). This could be explained by the fact that accumulation or high binding between the surface glycoprotein and the receptor does not necessarily trigger post-binding events that allow viral entry, due to the possible presence of restriction factors or lack of other genes expression required for viral infection (27,28). Another probable reason for the absence of CPE in HaCaT cells infected with rVSV-dG-RABV-GFP might be due to the conservation of HaCaT cell integrity as previously observed in HaCaT cells infected with rhinovirus (29).…”

Section: Discussionmentioning

confidence: 99%

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Structural and Mechanistic Usage and Preference of Human, Dog, and Bat Receptors by Rabies Virus

Khalifa,

Atasoy,

Rohaim

et al. 2023

Preprint

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Rabies is a lethal zoonotic viral disease causing approximately 59,000 human deaths annually. Recently, several cellular receptors for rabies virus (RABV) entry and internalization have been identified. However, none of these receptors have been demonstrated to be indispensable for RABV entry. Here we describe the RABV receptor preferencein vivo, utilizing a replication-competent vesicular stomatitis virus (VSV), in which the VSV surface glycoprotein was replaced with rabies virus glycoprotein. To investigate the specific role of RABV receptors in promoting RABV entry in non-permissive cell line, HaCaT cells were used as a cellular model refractory for RABV infection. Employing virus binding and quantification studies, we demonstrated that ITGB1 and mGluR2 are potential receptors for RABV entry and replication. Consequently, knockout (KO) cell lines corresponding to each of the ITGB1 and mGluR2 receptors were generated using CRISPR/Cas9 mediated knockout. Surprisingly, RABV was still able to enter and replicate in the generated KO cell lines, yet the replication and entry of RABV in KO cells lacking mGluR2 and ITGB1 were significantly reduced; respectively. These findings suggest that RABV utilize these receptors in series rather than sequentially. To test whether RABV utilizes similar receptor preference among human, dog, and bats, the A549, Pa-Br and MDCK cell lines that overexpress receptor orthologs from their respective species were infected with rVSV-dG-RABV-G-GFP and quantified for virus binding and released virus progeny. Our findings revealed that in human cells, ITGB1 increased virus entry, while nAChR enhanced virus replication. In bat cells, ectopic expression of nAChR allowed enhanced virus entry and internalization. While MDCK cells overexpressing ITGB1 enhanced the levels of virus entry and replication. Conclusively, our study, reveals the RABV distinct receptor preference, influenced by the underlying pathways that occur during the interaction between the virus and receptor in different cell lines. Additionally, it emphasizes the significance of host-specific factors in virus entry and replication.Graphical AbstractAuthor SummaryRabies is a fatal neurological disease, characterized by broad host range and tissue tropism. In accordance with the global goal of eliminating dog-mediated human rabies by 2030, studying the underlying mechanism of RABV entry across distinct species would enable adjustment of RABV control strategies. Owing to RABV wide tropism, multiple cellular receptors have been identified for RABV entry into host cells. Previous studies have proposed that some of RABV receptors could serve as promising candidates for development of antiviral drugs (1). From this perspective, we focused on elucidating RABV receptor preference for viral entry in human, dog, and bat cells. In addition to determining whether RABV utilizes these receptors in parallel or in series which would indicate the potential of the identified RABV cellular receptors as targets for antiviral drugs against rabies. Our results demonstrated varying receptor preference of RABV across species. In addition to revealing that none of RABV receptors solely, govern the broad host range of rabies, suggesting that RABV antiviral drugs targeting host cellular factors may not effectively inhibit RABV entry into cells, while antiviral drugs targeting virus glycoprotein may exhibit greater efficacy. Collectively, our study, contribute to providing mechanistic model for RABV entry in different species.

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Section: Discussionmentioning

confidence: 98%

“…Thus, we infected various cell lines with rVSV-dG-RABV-GFP which showed the wide tropism of rVSV-dG-RABV-G-GFP, exemplified in its ability to replicate in the wide spectrum of tested cell lines. However, a distinct alteration in cell tropism was observed among the VSV-GFP WT and the rVSV-dG-RABV-G-GFP infection on HaCaT cells which revealed resistance to the recombinant virus displaying the RABV-G protein, in contrast to their permissiveness to the rVSV -GFP-WT, suggesting the suitability of HaCaT cells to act as a cellular model for studying RABV receptor preference (27). In our study, we ectopically expressed the P.alecto receptors on HaCaT cells (Human cell line) to establish a heterologous system in which other factors that could affect the cellular receptors interactions are eliminated Additionally, to alleviate the cellular stress induced by transfection, we incorporated an empty vector control throughout the entire study as a means of normalization.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Structural and Mechanistic Usage and Preference of Human, Dog, and Bat Receptors by Rabies Virus

Khalifa,

Atasoy,

Rohaim

et al. 2023

Preprint

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“…We then deciphered prominent biological processes through STRING PPI network 44 and discovered protein complexes related to signal peptidases and type I IFN pathway (Extended data 3). Interestingly, signal peptidases are hijacked for polyprotein maturations of several viruses including alphavirus 45,46 , however, the peptidases involved are still unknown.…”

Section: Identification Of Cellular Factors That Interact With Chikv ...mentioning

confidence: 99%

Chikungunya virus glycoproteins transform macrophages into productive viral dissemination vessels

Yao

Ramachandran

et al. 2023

Preprint

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Despite their role as innate sentinels, macrophages are cellular reservoirs for chikungunya virus (CHIKV), a highly pathogenic arthropod-borne alphavirus that has caused unprecedented epidemics worldwide. Here, we took interdisciplinary approaches to elucidate the CHIKV determinants that subvert macrophages into virion dissemination vessels. Through comparative infection using chimeric alphaviruses and evolutionary selection analyses, we discovered for the first time that CHIKV glycoproteins E2 and E1 coordinate efficient virion production in macrophages with the domains involved under positive selection. We performed proteomics on CHIKV-infected macrophages to identify cellular proteins interacting with the precursor and/or mature forms of viral glycoproteins. We uncovered two E1-binding proteins, signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 (eIF3k), with novel inhibitory activities against CHIKV production. These results highlight how CHIKV E2 and E1 have been evolutionarily selected for viral dissemination likely through counteracting host restriction factors, making them attractive targets for therapeutic intervention.

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“…Alphaviruses are single-stranded positive-sense RNA viruses responsible for causing arthritic and encephalitic diseases worldwide (1) (2) (3). This genus includes arthropod-borne viruses (arboviruses) such as eastern equine encephalitis virus, chikungunya virus (CHIKV), and Mayaro virus.…”

Section: Introductionmentioning

confidence: 99%

The chikungunya virus E1 glycoprotein fusion loop and hinge alter glycoprotein dynamics leading to cell and host specific changes in infectivity

Thannickal,

Battini,

Spector

et al. 2023

Preprint

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Alphaviruses infect both mammals and insects, yet the distinct mechanisms that alphaviruses use to infect different hosts are not well defined. In this study, we characterize CHIKV E1 variants in the fusion loop (E1-M88L) and hinge region (E1-N20Y)in vitroandin vivoto understand how these regions of the E1 glycoprotein contribute to host-specific infection. Through cell culture assays, we found that CHIKV E1-N20Y enhanced infectivity in mosquito cells while the CHIKV E1-M88L variant enhanced virus binding and infectivity in both BHK-21 and C6/36 cells, and led to changes in the virus cholesterol-dependence in BHK-21 cells. Given thesein vitroresults and that residue E1-M88L is in a defined Mxra8 interacting domain, we hypothesized that this residue may be important for receptor usage. However, while the CHIKV E1-M88L variant increased replication in Mxra8-deficient mice compared to WT CHIKV, it was attenuatedin vitroin mouse fibroblasts, suggesting that residue E1-M88 may function in a cell-type dependent manner to alter entry. Finally, using molecular dynamics to understand how potential changes in the E1 glycoprotein may impact the CHIKV glycoprotein E1-E2 complex, we found that E1-M88L and other E1 domain II variants lead to changes in both E1 and E2 dynamics. Taken together, these studies show that key residues in the CHIKV E1 fusion loop and hinge region function through changes in E1-E2 dynamics to facilitate cell- and host-dependent entry.ImportanceArthropod-borne viruses (arboviruses) are significant global public health threats, and their continued emergence around the world highlights the need to understand how these viruses replicate at the molecular level. The alphavirus class II glycoproteins are critical for virus entry in mosquitoes and mammals, yet how these proteins function is not completely understood. Therefore, to address these gaps in our knowledge, it is critical to dissect how distinct glycoprotein domains functionin vitroandin vivo. Here, we show that changes in the CHIKV E1 fusion loop and hinge contribute to host-specific entry and E1-E2 dynamics, furthering our knowledge of how alphaviruses infect mammals and insects.

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Entry receptors — the gateway to alphavirus infection

Cited by 22 publications

References 175 publications

Structural and Mechanistic Usage and Preference of Human, Dog, and Bat Receptors by Rabies Virus

Structural and Mechanistic Usage and Preference of Human, Dog, and Bat Receptors by Rabies Virus

Chikungunya virus glycoproteins transform macrophages into productive viral dissemination vessels

The chikungunya virus E1 glycoprotein fusion loop and hinge alter glycoprotein dynamics leading to cell and host specific changes in infectivity

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