High-Throughput Fitness Profiling of Zika Virus E Protein Reveals Different Roles for Glycosylation during Infection of Mammalian and Mosquito Cells

Gong, Danyang; Zhang, Tian hao; Zhao, Dawei; Du, Yushen; Chapa, Travis J.; Shi, Yuan; Wang, Laurie; Contreras, Deisy; Zeng, Gang; Shi, Pei Yong; Wu, Ting; Arumugaswami, Vaithilingaraja; Sun, Ren

doi:10.1016/j.isci.2018.02.005

Cited by 29 publications

(31 citation statements)

References 52 publications

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“…Similar results have been also found in both WNV and tick-borne encephalitis virus (TBEV) (59,66). Moreover, this glycosylation is critical for ZIKV infection of mammalian and mosquito hosts (71,72) and antagonizing to the vector immune defense (73). In the present study, we demonstrate that nonglycosylation on the E protein of TMUVs results in limited virus replication (not systemic infection in ducks) and abrogation of virus transmission.…”

Section: Figsupporting

confidence: 87%

A Single Mutation at Position 156 in the Envelope Protein of Tembusu Virus Is Responsible for Virus Tissue Tropism and Transmissibility in Ducks

Yan

Shi

Wang

et al. 2018

J Virol

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Duck Tembusu virus (TMUV), like other mosquito-borne flaviviruses, such as Japanese encephalitis virus, West Nile virus, and Bagaza virus, is able to transmit vector-independently. To date, why these flaviviruses can be transmitted without mosquito vectors remains poorly understood. To explore the key molecular basis of flavivirus transmissibility, we compared virus replication and transmissibility of an early and a recent TMUV in ducks. The recent TMUV strain FX2010 replicated systemically and transmitted efficiently in ducks, while the replication of early strain MM1775 was limited and did not transmit among ducks. The TMUV envelope protein and its domain I were responsible for tissue tropism and transmissibility. The mutation S156P in the domain I resulted in disruption of N-linked glycosylation at amino acid 154 of the E protein and changed the conformation of "150 loop" of the E protein, which reduced virus replication in lungs and abrogated transmission in ducks. These data indicate that the 156S in the envelope protein is critical for TMUV tissue tropism and transmissibility in ducks in the absence of mosquitos. Our findings provide novel insights on understanding TMUV transmission among ducks. Tembusu virus, similar to other mosquito-borne flaviviruses such as WNV, JEV, and BAGV, can be transmitted without the presence of mosquito vectors. We demonstrate that the envelope protein of TMUV and its amino acid (S) at position 156 is responsible for tissue tropism and transmission in ducks. The mutation S156P results in disruption of N-linked glycosylation at amino acid 154 of the E protein and changes the conformation of "150 loop" of the E protein, which induces limited virus replication in lungs and abrogates transmission between ducks. Our findings provide new knowledge about TMUV transmission among ducks.

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Section: Figsupporting

confidence: 87%

A Single Mutation at Position 156 in the Envelope Protein of Tembusu Virus Is Responsible for Virus Tissue Tropism and Transmissibility in Ducks

Yan

Shi

Wang

et al. 2018

J Virol

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“…By using this plasmid and also choosing a viral strain (MR766) that grows to high titer in cell culture, we were able to generate viral libraries that effectively sampled all amino-acid mutations to E protein. This library diversity contrasts to two other recent ZIKV deep mutational scanning studies 21,22 : both of these studies revealed important biological insights about host adaptation, but neither produced a complete map of amino-acid level selection due to an inability to generate and maintain the viral library diversity to sample all amino acids. Of course, our experiments were still subject to noise, probably because even our high-efficiency reversegenetics had some bottlenecking of viral diversity.…”

Section: Discussioncontrasting

confidence: 76%

“…To validate the antibody-escape mutants, we chose two mutations expected to strongly affect sensitivity to each anti-ZIKV antibody (A333T and T335E for ZKA64, and D67A and K118R for ZKA185). We performed neutralization assays against viruses engineered to contain these mutations, using a previously described approach 14,22,32 . As expected from the mutational antigenic profiling, each mutation greatly reduced neutralization sensitivity to the antibody that selected it, but not to the other antibody (Fig 5E,F,G).…”

Section: Resultsmentioning

confidence: 99%

“…Second, Vero cells are a relatively permissive monkey-derived cell line, whereas actual ZIKV evolves under selection to efficiently replicate in actual humans and mosquitoes. Indeed, prior deep mutational scanning studies of ZIKV 21,22 and other viruses 37,38 have identified numerous mutations with effects that depend on the host species and level of innate-immune function in the cells used to grow the virus. Therefore, our map should be interpreted as providing a baseline estimate of the functional effects of mutations to the E protein in a relatively permissive context.…”

Section: Discussionmentioning

confidence: 99%

“…This approach has been used to assess the effects of all viral mutations on the function and antigenicity of key proteins from influenza virus 16–18 and HIV 19,20 . Two studies have applied deep mutational scanning to ZIKV 21,22 , but these studies did not systematically map the mutational tolerance or antigenicity of all amino-acid mutations. Instead, they used libraries comprised of a subset of E protein variants to identify mutations that differentially affect viral growth in mammalian and insect cells.…”

Section: Introductionmentioning

confidence: 99%

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Deep mutational scanning comprehensively maps how Zika envelope protein mutations affect viral growth and antibody escape

Sourisseau

Lawrence

Schwarz

et al. 2019

Preprint

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Functional constraints on viral proteins are often assessed by examining sequence conservation among natural strains, but this approach is relatively ineffective for Zika virus because all known sequences are highly similar. Here we take an alternative approach to map functional constraints on Zika virus’s envelope (E) protein by using deep mutational scanning to measure how all amino-acid mutations to the protein affect viral growth in cell culture. The resulting sequence-function map is consistent with existing knowledge about E protein structure and function, but also provides insight into mutation-level constraints in many regions of the protein that have not been well characterized in prior functional work. In addition, we extend our approach to completely map how mutations affect viral neutralization by two monoclonal antibodies, thereby precisely defining their functional epitopes. Overall, our study provides a valuable resource for understanding the effects of mutations to this important viral protein, and also offers a roadmap for future work to map functional and antigenic selection to Zika virus at high resolution. Importance Zika virus has recently been shown to be associated with severe birth defects. The virus’s E protein mediates its ability to infect cells, and is also the primary target of the antibodies that are elicited by natural infection and vaccines that are being developed against the virus. Therefore, determining the effects of mutations to this protein is important for understanding its function, its susceptibility to vaccine-mediated immunity, and its potential for future evolution. We completely mapped how amino-acid mutations to E protein affected the virus’s ability to grow in cells in the lab and escape from several antibodies. The resulting maps relate changes in the E protein’s sequence to changes in viral function, and therefore provide a valuable complement to existing maps of the physical structure of the protein.

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Zika virus: Molecular responses and tissue tropism in the mammalian host

Shaily

Upadhya

2019

Reviews in Medical Virology

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Zika virus (ZIKV) outbreaks have raised alarm because of reports of congenital Zika virus syndrome in infants. The virus is also known to cause the debilitating Guillain-Barré syndrome in adults. As a result, extensive research has been carried out on the virus over the past few years. To study the molecular responses of viral infectivity in mammals, in vitro two-dimensional and three-dimensional cellular models have been employed. The in vivo models of mouse, pig, chicken, and nonhuman primates are primarily used to investigate the teratogenicity of the virus, to study effects of the virus on specific tissues, and to study the systemic effects of a proposed antiviral agent. The virus exhibits wide tissue tropism in the mammalian host.

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High-Throughput Fitness Profiling of Zika Virus E Protein Reveals Different Roles for Glycosylation during Infection of Mammalian and Mosquito Cells

Cited by 29 publications

References 52 publications

A Single Mutation at Position 156 in the Envelope Protein of Tembusu Virus Is Responsible for Virus Tissue Tropism and Transmissibility in Ducks

A Single Mutation at Position 156 in the Envelope Protein of Tembusu Virus Is Responsible for Virus Tissue Tropism and Transmissibility in Ducks

Deep mutational scanning comprehensively maps how Zika envelope protein mutations affect viral growth and antibody escape

Zika virus: Molecular responses and tissue tropism in the mammalian host

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