Site-Directed Mutagenesis of a Potyvirus Coat Protein and its Assembly in Escherichia Coli

Jagadish, Mittur N.; Huang, Dexing; Ward, Colin W.

doi:10.1099/0022-1317-74-5-893

Cited by 62 publications

(24 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This is consistent with the behavior of various potyvirus core mutants that have been produced previously (10,35). As the core region is essential for virion formation (17), replication (10), and cellto-cell movement (9), we could assume that our core deletion mutant also disrupts viral encapsidation and causes dysfunctional viral movement.…”

Section: Discussionsupporting

confidence: 67%

A Nonviral Peptide Can Replace the Entire N Terminus of Zucchini Yellow Mosaic Potyvirus Coat Protein and Permits Viral Systemic Infection

Arazi¹,

Shiboleth²,

Gal‐On

2001

J Virol

View full text Add to dashboard Cite

Systematic deletion and peptide tagging of the amino-terminal domain (NT, ϳ43 amino acids) of an attenuated zucchini yellow mosaic potyvirus (ZYMV-AGII) coat protein (CP) were used to elucidate its role in viral systemic infection. Deletion mutants truncated by 8, 13, and 33 amino acid residues from the CP-NT 5 end were systemically infectious and produced symptoms similar to those of the AGII virus. Tagging these deletion mutants with either human c-Myc (Myc) or hexahistidine peptides maintained viral infectivity. Similarly, addition of these peptides to the intact AGII CP-NT did not affect viral life cycle. To determine which parts, if any, of the CP-NT are essential for viral systemic infection, a series of Myc-tagged mutants with 8 to 43 amino acids removed from the CP-NT were constructed. All Myc-tagged CP-NT deletion mutants, including those from which virtually all the viral CP-NT had been eliminated, were able to encapsidate and cause systemic infection. Furthermore, chimeric viruses with deletions of up to 33 amino acids from CP-NT produced symptoms indistinguishable from those caused by the parental AGII virus. In contrast to CP-NT Myc fusion, addition of the foot-and-mouth disease virus (FMDV) immunogenic epitope to AGII CP-NT did not permit systemic infection. However, fusion of the Myc peptide to the N terminus of the FMDV peptide restored the capability of the virus to spread systemically. We have demonstrated that all CP-NT fused peptides were exposed on the virion surface, masking natural CP immunogenic determinants. Our findings demonstrate that CP-NT is not essential for ZYMV spread and that it can be replaced by an appropriate foreign peptide while maintaining systemic infectivity.Zucchini yellow mosaic potyvirus (ZYMV) is a member of the Potyviridae family, the largest group of plant-infecting viruses (31). As in all potyviruses, the ZYMV genome consists of a single messenger-polarity RNA molecule of about 10 kb, encapsidated by ϳ2,000 subunits of coat protein (CP), forming a helical, flexuous filament particle about 750 nm long and 11 nm wide (8,19).Though there are no high-resolution X-ray diffraction data available on the structure of potyvirus CP, there is a considerable amount of information about its topology. Structure predictions, together with immunological studies (7, 29) of potyvirus CPs, have demonstrated structural features similar to those of the CP of tobacco mosaic virus (21) and potato virus X (26). Like those proteins, potyviral CP is a three-domain protein with variable N-and C-terminal regions exposed on the virion surface and a conserved core domain that probably interacts with viral RNA (1, 29). ZYMV CP (279 amino acids [aa]) is composed of a 214-aa core domain flanked by 43-to 45-and 20-aa N-and C-terminal domains, respectively, as predicted by Shukla et al. (30). The putative trypsin protease motif of potyvirus CP, representing the end of the surface-exposed N-terminal domain (NT), is presumed to be positioned between amino acids Lys and Asp, located in the KDKD motif ...

show abstract

Section: Discussionsupporting

confidence: 67%

A Nonviral Peptide Can Replace the Entire N Terminus of Zucchini Yellow Mosaic Potyvirus Coat Protein and Permits Viral Systemic Infection

Arazi¹,

Shiboleth²,

Gal‐On

2001

J Virol

View full text Add to dashboard Cite

show abstract

“…1). For ∆RQCP and ∆DCP nucleotides encoding the charged amino acids R##!, Q##" and D#'%, located in the core of the PPV CP and shown to be involved in VLP formation of another potyvirus CP (Jagadish et al, 1991(Jagadish et al, , 1993, were deleted.…”

Section: Effect Of Cp Structure On Formation Of Vlpsmentioning

confidence: 99%

Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants.

Jacquet¹,

Delécolle²,

Raccah³

et al. 1998

Journal of General Virology

View full text Add to dashboard Cite

Aphid transmission of a non-aphid-transmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) occurs in transgenic plants expressing the plum pox potyvirus (PPV) coat protein (CP) gene. Heteroencapsidation has been shown to be responsible for this modification in the epidemiological characteristics of the infecting virus. In order to prevent this biological risk, several modified PPV CP constructs were produced that were designed to interfere with heteroencapsidation itself or to block aphid transmission of heteroencapsidated virions. These constructs were first expressed in Escherichia coli in order to check for the accumulation of

show abstract

“…Johnson grass mosaic virus (JGMV) coat protein (CP) has been shown to self-assemble to form rod-shaped virus-like particles (VLPs) even in the absence of the viral RNA (7). In the present study, peptide sequences chosen from JEV E protein, with a potential to induce anti-JEV neutralizing antibodies, were fused to the C terminus of JGMV CP by using recombinant DNA methods.…”

mentioning

confidence: 99%

A Japanese Encephalitis Virus Peptide Present on Johnson Grass Mosaic Virus-Like Particles Induces Virus-Neutralizing Antibodies and Protects Mice against Lethal Challenge

Saini

Vrati

2003

J Virol

View full text Add to dashboard Cite

Protection against Japanese encephalitis virus (JEV) is antibody dependent, and neutralizing antibodies alone are sufficient to impart protection. Thus, we are aiming to develop a peptide-based vaccine against JEV by identifying JEV peptide sequences that could induce virus-neutralizing antibodies. Previously, we have synthesized large amounts of Johnson grass mosaic virus (JGMV) coat protein (CP) in Escherichia coli and have shown that it autoassembled to form virus-like particles (VLPs). The envelope (E) protein of JEV contains the virus-neutralization epitopes. Four peptides from different locations within JEV E protein were chosen, and these were fused to JGMV CP by recombinant DNA methods. The fusion protein autoassembled to form VLPs that could be purified by sucrose gradient centrifugation. Immunization of mice with the recombinant VLPs containing JEV peptide sequences induced anti-peptide and anti-JEV antibodies. A 27-amino-acid peptide containing amino acids 373 to 399 from JEV E protein, present on JGMV VLPs, induced virus-neutralizing antibodies. Importantly, these antibodies were obtained without the use of an adjuvant. The immunized mice showed significant protection against a lethal JEV challenge.Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus responsible for acute encephalitis in humans, with fatality rates ranging from 20% to as high as 50%. The virus is active in a vast geographic area that includes India, China, Japan, and almost all of Southeast Asia. As many as 50,000 cases of JEV infection are reported from these areas every year, of which about 10,000 result in fatality. A high proportion of survivors have serious neurologic and psychiatric sequelae (34). A mouse brain-grown, formalin-inactivated vaccine is available internationally that has many limitations; it is expensive to produce, does not provide long-term immunity, and may cause allergic reactions because of the inclusion of murine encephalogenic basic protein or gelatin stabilizer (1,20,25,27). Thus, there is a need to produce an alternate vaccine that may be safer and cheaper.JEV infection of host cells produces three structural and seven nonstructural proteins. One of these, the E protein, is the major envelope protein of the virion. This protein is believed to play an important role in a number of processes, including viral attachment, membrane fusion, and entry into the host cell. In response to JEV infection, the host produces virus neutralizing antibodies and cytotoxic T cells (CTLs). The principal target for the neutralizing antibodies is the E protein.It has been shown that protection against JEV infection is mainly antibody dependent, and virus-neutralizing antibodies alone are sufficient to impart protection (10, 18). Peptide(s) from JEV E protein that forms the virus-neutralizing epitope(s) could, therefore, be used for inducing JEV-neutralizing antibodies.Recent findings suggest that presentation of peptides in a highly ordered aggregate form can result in enhanced immune responses (12). Johnson grass m...

show abstract

Site-Directed Mutagenesis of a Potyvirus Coat Protein and its Assembly in Escherichia Coli

Cited by 62 publications

References 9 publications

A Nonviral Peptide Can Replace the Entire N Terminus of Zucchini Yellow Mosaic Potyvirus Coat Protein and Permits Viral Systemic Infection

A Nonviral Peptide Can Replace the Entire N Terminus of Zucchini Yellow Mosaic Potyvirus Coat Protein and Permits Viral Systemic Infection

Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants.

A Japanese Encephalitis Virus Peptide Present on Johnson Grass Mosaic Virus-Like Particles Induces Virus-Neutralizing Antibodies and Protects Mice against Lethal Challenge

Contact Info

Product

Resources

About