The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1

doi:10.1016/0042-6822(92)90615-V

Virology

Volume 189, Issue 2, August 1992, Pages 821-827

https://doi.org/10.1016/0042-6822(92)90615-V Get rights and content

Abstract

A nuclear localization signal (NLS) has been identified in the N-terminal (Ala¹-Pro-Lys-Arg-Lys-Ser-Gly-Val-SerLys-Cys¹¹) amino acid sequence of the polyomavirus major capsid protein VP1. The importance of this amino acid sequence for nuclear transport of VP1 protein was demonstrated by a genetic “subtractive” study using the constructs pSG5VP1 (full-length VP1) and pSG5Δ5′VP1 (truncated VP1, lacking amino acids Ala¹-Cys¹¹). These constructs were used to transfect COS-7 cells, and expression and intracellular localization of the VP1 protein was visualized by indirect immunofluorescence. These studies revealed that the full-length VP1 was expressed and localized in the nucleus, while the truncated VP1 protein was localized in the cytoplasm and not transported to the nucleus. These findings were substantiated by an “additive” approach using FITC-labeled conjugates of synthetic peptides homologous to the NLS of VP1 cross-linked to bovine serum albumin or immunoglobulin G. Both conjugates localized in the nucleus after microinjection into the cytoplasm of 3T6 cells. The importance of individual amino acids found in the basic sequence (Lys³-Arg-Lys⁵) of the NLS was also investigated. This was accomplished by synthesizing three additional peptides in which lysine-3 was substituted with threonine, arginine-4 was substituted with threonine, or lysine-5 was substituted with threonine. It was found that lysine-3 was crucial for nuclear transport, since substitution of this amino acid with threonine prevented nuclear localization of the microinjected, FITC-labeled conjugate.

References (52)

J. Clever et al.
Virology
(1991)
P. Deininger et al.
Cell
(1979)
C. Dingwall et al.
Trends Biochem. Sci.
(1991)
R.L. Garcea et al.
Virology
(1983)
W. Gibson
Virology
(1974)
Y. Gluzman
Cell
(1981)
F.L. Graham et al.
Virology
(1973)
D. Kalderon et al.
Cell
(1984)
R.E. Lanford et al.
Cell
(1984)
R.E. Lanford et al.
Cell
(1986)

A.D. Leavitt et al.

J. Biol. Chem.

(1985)

R.B. Moreland et al.

Virology

(1991)

E.A. Nigg et al.

Cell

(1991)

R. Peters

Biochim. Biophys. Acta.

(1986)

W.D. Richardson et al.

Cell

(1986)

H.-P. Rihs et al.

FEBS Lett.

(1991)

B. Roberts

Biochim. Biophys. Acta.

(1989)

P.A. Silver

Cell

(1991)

L.K.C. Yuen et al.

Virology

(1985)

S.A. Adam et al.

Nature (London)

(1989)

S.A. Adam et al.

J. Cell Biol.

(1990)

J.N. Brady et al.

J. Virol.

(1978)

T.R. Burglin et al.

EMBO J.

(1987)

D. Chelsky et al.

Mol. Cell. Biol.

(1989)

W.H. Colledge et al.

Mol. Cell. Biol.

(1986)

S.I. Dworetzky et al.

J. Cell Biol.

(1988)

Cited by (46)

Production and biomedical applications of virus-like particles derived from polyomaviruses
2013, Journal of Controlled Release
Citation Excerpt :
The positively charged residues among the first 12 amino acids (aas) are essential for this interaction [25]. Overlapping the VP1 DNA binding domain lies a nuclear localization signal (NLS) [26–28]. The hamster polyomavirus (HaPyV) has an additional N-terminally extended form.
Virus-like particles (VLPs), aggregates of capsid proteins devoid of viral genetic material, show great promise in the fields of vaccine development and gene therapy. These particles spontaneously self-assemble after heterologous expression of viral structural proteins. This review will focus on the use of virus-like particles derived from polyomavirus capsid proteins. Since their first recombinant production 27 years ago these particles have been investigated for a myriad of biomedical applications. These virus-like particles are safe, easy to produce, can be loaded with a broad range of diverse cargos and can be tailored for specific delivery or epitope presentation. We will highlight the structural characteristics of polyomavirus-derived VLPs and give an overview of their applications in diagnostics, vaccine development and gene delivery.
Nuclear entry mechanism of the human polyomavirus JC virus-like particle: Role of importins and the nuclear pore complex
2004, Journal of Biological Chemistry
JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. Although transport of virions to the nucleus is an important step in JCV infection, the mechanism of this process has remained unclear. The outer shell of the JCV virion comprises the major capsid protein VP1, which possesses a putative nuclear localization signal (NLS), and virus-like particles (VLPs) consisting of recombinant VP1 exhibit a virion-like structure and physiological functions (cellular attachment and intracytoplasmic trafficking) similar to those of JCV virions. We have now investigated the mechanism of nuclear transport of JCV with the use of VLPs. Wild-type VLPs (wtVLPs) entered the nucleus of most HeLa or SVG cells. The virion structure of VLPs was preserved during transport to the nucleus as revealed by confocal microscopy of cells inoculated with fluorescein isothiocyanate-labeled wtVLPs containing packaged Cy3. The nuclear transport of wtVLPs in digitonin-permeabilized cells was dependent on the addition of importins α and β and was prevented by wheat germ agglutinin or by antibodies to the nuclear pore complex. The nuclear entry of VLPs composed of VP1 with a mutated NLS was greatly inhibited, compared with that of wtVLPs, in both intact and permeabilized cells. Unlike wtVLPs, the mutant VLPs did not bind to importins α or β. Limited proteolysis analysis revealed that the NLS of VP1 was exposed on the surface of wtVLPs. These results suggest that JCV VLPs bind to cellular importins via the NLS of VP1 and are transported into the nucleus through the nuclear pore complex.
Characterization of self-assembled virus-like particles of human polyomavirus BK generated by recombinant baculoviruses
2003, Virology
The major structural protein of the human polyomavirus BK (BKV), VP1, was expressed by using recombinant baculoviruses. A large amount of protein with a molecular mass of about 42 kDa was synthesized and identified by Western blotting. The protein was detected exclusively in the nuclei by immunofluorescent analysis and it was released into culture medium. The expressed BKV VP1 protein was self-assembled into virus-like particles (BK-VLPs) with two different sizes (50 and 26 nm in diameter), which migrated into four different bands in CsCl gradient with buoyant densities of 1.29, 1.30, 1.33, and 1.35 g/cm³. The immunological studies on the BK-VLPs suggested that they have similar antigenicity with those of authentic BKV particles. Cryoelectron microscopy and 3D image analysis further revealed that the larger BK-VLPs were composed of 72 capsomers which all were pentamers arranged in a T = 7 surface lattice. This system provides useful information for detailed studies of viral morphogenesis and the structural basis for the antigenicity of BKV.
Involvement of minor structural proteins in recombination of polyoma virus DNA
2000, Virology
We have previously observed that a polyoma-mouse chimeric DNA molecule (RmI) in which the murine DNA insert is flanked by directly repeated viral sequences is effectively converted into unit-length polyoma DNA upon transfection of permissive mouse cells. This intramolecular recombination event appears to be dependent on VmP1, a protein encoded by RmI which includes the 328 N-terminal amino acids of polyoma VP1, and nine amino acids of murine origin carrying the C-terminus of the protein. We report here that introducing mutations into the VP2/VP3 coding sequence reduces the ability of RmI to generate polyoma DNA, even though the same mutations seem to exert little or no effect on the ability of polyoma DNA to either replicate or accumulate inside transfected cells. A mutation affecting VP2 alone being as effective as one that affects both VP2 and VP3, VP2 appears to be playing a critical role in recombination.
Use of electron microscopic and immunogold labeling techniques to determine polyomavirus recombinant VP1 capsid-like particles entry into mouse 3T6 cell nucleus
2000, Journal of Virological Methods
Murine polyomavirus major structural protein VP1 could assemble into capsid-like particles when expressed in the baculovirus system. The recombinant capsid-like particles that were purified by CsCl density gradient centrifugation were capable of packaging host DNA. Electron microscopic and immunogold labeling techniques were used to study the entry of these VP1 recombinant capsid-like particles into mouse 3T6 cells. It was found that these VP1 recombinant capsid-like particles, which lack polyomavirus minor structural proteins (VP2 and VP3), use the same mechanism to enter mouse 3T6 cell cytoplasm and nucleus as that used by native polyomavirus virions.
Murine polyomavirus infection of 3T6 mouse cells shows evidence of predominant necrosis as well as limited apoptosis
2000, Virus Research
The current study was developed to determine if polyomavirus infected 3T6 mouse cells evoked an apoptotic or a necrotic mechanism during infection. Infected cells were analyzed by flow cytometry, transmission electron microscopy (TEM), DNA electrophoresis and by measuring caspase-3 enzymatic activity. Infected cells that were analyzed at 72 h post-infection showed the following: flow cytometry analysis revealed a 5% increase in apoptotic cells and a 46% increase in necrotic cells when compared to uninfected cells; electron microscopy showed 10% cells with characteristic apoptotic morphology and 40% with necrotic appearance; caspase-3 activity was found to increase two fold when compared to uninfected cells and DNA fragmentation (laddering) was clearly evident late in infection. It was concluded that infected cells predominantly showed necrosis, although some cells showed apoptosis in late infection. Recombinant capsid-like particles composed of the polyomavirus structural proteins were not able to induce cell death.

View all citing articles on Scopus

^☆: Contribution 92-47-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kansas 66506.

View full text

Short communicationThe use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1☆

Abstract

Virology

Cell

Trends Biochem. Sci.

Virology

Virology

Cell

Virology

Cell

Cell

Cell

J. Biol. Chem.

Virology

Cell

Biochim. Biophys. Acta.

Cell

FEBS Lett.

Biochim. Biophys. Acta.

Cell

Virology

Nature (London)

J. Cell Biol.

J. Virol.

EMBO J.

Mol. Cell. Biol.

Mol. Cell. Biol.

J. Cell Biol.

Short communication
The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1☆