JVI Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gasparovic, M. L.
Right arrow Articles by Atwood, W. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gasparovic, M. L.
Right arrow Articles by Atwood, W. J.

 Previous Article  |  Next Article 

Journal of Virology, November 2006, p. 10858-10861, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.01298-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

JC Virus Minor Capsid Proteins Vp2 and Vp3 Are Essential for Virus Propagation

M. L. Gasparovic,2 G. V. Gee,3 and W. J. Atwood1,2*

Department of Molecular Biology, Cell Biology and Biochemistry,1 Graduate Program in Molecular Biology, Cell Biology and Biochemistry,2 Department of Ecology and Evolutionary Biology, Brown University, Providence, Rhode Island 029123

Received 20 June 2006/ Accepted 7 August 2006


   ABSTRACT
Top
 Abstract
Text
 References
 
Virus-encoded capsid proteins play a major role in the life cycles of all viruses. The JC virus capsid is composed of 72 pentamers of the major capsid protein Vp1, with one of the minor coat proteins Vp2 or Vp3 in the center of each pentamer. Vp3 is identical to two-thirds of Vp2, and these proteins share a DNA binding domain, a nuclear localization signal, and a Vp1-interacting domain. We demonstrate here that both the minor proteins and the myristylation site on Vp2 are essential for the viral life cycle, including the proper packaging of its genome.


   TEXT
Top
 Abstract
 Text
References
 
JC virus (JCV) is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) (22). PML develops from a lytic infection of the myelin-producing oligodendrocytes in the central nervous system. JCV is widespread in the human population, where it is estimated that 70% of people are seropositive for the virus (21). JCV is generally latent but can traffic to the central nervous system upon immunosupression, lytically infecting oligodendrocytes and causing PML (1, 15, 24).

JCV contains a small, nonenveloped, double-stranded DNA genome that is organized into early and late coding regions. These regions are divided by the regulatory region containing a bidirectional promoter and the viral origin of DNA replication. The late region contains agno, Vp1, Vp2, and Vp3. The V antigens make up the virus capsid, consisting of 360 molecules of the major coat protein Vp1. These are arranged in 72 pentamers, creating the icosahedral shape. One of the minor coat proteins, Vp2 or Vp3, lies in the center of each pentamer. Vp3 is identical to the C'-terminal two-thirds of Vp2; this shared domain is comprised of the nuclear localization signal (NLS), the DNA binding domain, and the Vp1-interacting domain (2, 4, 5, 8) (Fig. 1B). Vp2 is also modified by a myristylation moiety on its N' terminus. Myristylation is the cotranslational addition of a fatty acid. Myristyl proteins may be either cytoplasmic or membrane associated. The myristyl group is transferred by the enzyme N-myristyl transferase after the methionine is removed and an N'-terminal glycine residue is recognized (23).


Figure 1
View larger version (20K):
[in this window]
[in a new window]
  		FIG. 1. (A) Diagram of capsid proteins that will remain after site-directed mutagenesis. (B) Diagram of Vp2 and Vp3 and their functional domains.

 
The viral DNA is packaged with histones H2A, H2B, H3, and H4 and creates a minichromosome structure that is almost indistinguishable from the host's chromatin (20). The host cell type specificity can be controlled through transcriptional blocks to infection as well as virus-receptor interactions (3, 7, 19).

Previous work evaluating the role of minor coat proteins in the related polyomavirus simian virus 40 (SV40) found that Vp2 was dispensable but that Vp3 was necessary for infection. It also suggested that the importance of Vp3 could lie in its activation of poly(ADP-ribose) polymerase (9). This overactivation of poly(ADP-ribose) polymerase is thought to deplete intracellular ATP and cause necrosis, thereby releasing the virus (9, 10). The SV40 minor proteins have also been shown to contain lytic properties in bacteria that could aid in virus release from the cell (6).

Similar work has also been performed for mouse polyomavirus where Vp2, Vp3, and Vp2 myristylation mutants have been produced by two different labs. Both labs determined that Vp2 and Vp3 are essential for virus production and infection. The myristylation site is also necessary and has been postulated to be important for both exit from the cell and reinfection (16, 25).

To determine the role of the minor proteins in the human polyomavirus JCV, we used site-directed mutagenesis to eliminate Vp2 and Vp3 and the myristylation site on Vp2. The Mad1-SVE{Delta} strain of JCV was linearized at the BamHI sites and subcloned into pUC19 for viral DNA propagation in bacteria. The start sites of Vp2 and Vp3 were replaced by alanine residues by site-directed mutagenesis using GeneEditor (Promega). The results of the mutagenesis were confirmed by sequencing (the primers used for mutagenesis were 5'-gtgttttcaggttcGCgggtgccgcacttg-3' [Vp2] and 5'-cagcagccagctGCggctttacaatta-3' [Vp3], where mismatched nucleotides are shown in uppercase). The double mutant was also created, using both primers to eliminate both Vp2 and Vp3. Additional mutants were created by the removal of the myristylation site at position 2 of Vp2 by changing the glycine to either an alanine, glutamate, glutamine, or histidine (the primers used for mutagenesis were 5'-ggttcatcgCtgccgcac-3' [G2A], 5'-gttttcaggttcatgGAAgccgcacttgcac-3' [G2E], 5'-gttttcaggttcatgCATgccgcacttgcac-3' [G2H], and 5'-gttttcaggttcatgCAAgccgcacttgcac-3' [G2Q], where mismatched nucleotides are shown in uppercase) (Fig. 1A).

Mutant and wild-type genomes were linearized, and equal amounts of viral DNA were transfected into permissive SVG-A cells by using Lipofectamine (Invitrogen) and Plus (Invitrogen) reagents. They were scored at 84 h posttransfection for viral expression by indirect immunofluorescence assay of Vp1. At 84 h posttransfection, Vp1 was expressed in approximately 5% of the cells, which is consistent with the transfection efficiency of these cells. Vp1 has a weak monopartite NLS, in contrast to the strong bipartite NLSs of its family members SV40 and BK virus (11, 26). JCV therefore relies on the presence of the minor proteins for efficient nuclear localization. All samples containing one minor protein correctly localized Vp1 to the nucleus, indicating the mutant DNA is able to be expressed and that only one minor protein is needed for Vp1 import (Fig. 2A). The double mutant (Vp2–/Vp3–) did not localize Vp1 to the nucleus as efficiently as either of the single mutants, which is consistent with the weak NLS in JCV Vp1 (Fig. 2A). The infection was followed for 19 days, and the mutants were unable to efficiently propagate the infection. In contrast, wild-type virus spread to 85% of the cells (Fig. 2B and C). This suggests that JCV requires Vp2, the myristylation site on Vp2, and Vp3 for completion of its life cycle. This also shows that Vp2 and Vp3 have distinct functions in the viral life cycle, as neither can be complemented by the other protein.


Figure 2
View larger version (46K):
[in this window]
[in a new window]
  		FIG. 2. Viral propagation requires the minor proteins. (A) Nuclear localization was monitored by indirect immunofluorescence of Vp1. All mutants except the double mutant properly localize Vp1. (B) Viral growth was monitored by indirect immunofluorescence of Vp1. All mutants are expressed at 4 days posttransfection but fail to grow over time. (C) Growth curves were created for wild-type (WT) and mutant viruses by determining the average number of Vp1-positive cells/250 cells counted.

 
The lack of the minor proteins could affect many aspects of the viral life cycle. These proteins have been shown to localize to ND10 domains in the nucleus, so their absence could cause mislocalization and therefore assembly defects (27). To address an assembly defect in the life cycle, a DNase protection assay was performed. Virus was harvested from transfected cells at 4, 13, and 22 days posttransfection, and genome protection by the capsid was tested with DNase treatment. The virus was harvested by freeze-thawing, sonication, membrane lysis with 2.5% deoxycholate, and centrifugation to remove cell debris. Equal amounts of virus harvested from the cells were treated with a 20-fold excess of DNase I or were mock treated with water. The capsids were removed using proteinase K, and the genomes were purified and amplified by PCR (primers JCVfor401 [5'-GTG AAG ACA GTG TAG ACG G-3'] and JCVrev1070 [5'-GAA TTT CCT GAG AGG TTA AGC-3']). Cells that received no viral DNA were used as a control to show that cellular DNA was not being amplified. At all of the time points, wild-type virus showed protection of its DNA, whereas the mutants were unable to protect their DNA (Fig. 3, compare lanes 3, 5, 8, and 10). This lack of amplification in the mutant samples is not due to a lack of viral DNA as shown by the mock-treated lanes. (Fig. 3, compare lanes 4, 6, 9, and 11). At the 4-day time point the wild type and mutants were expressed equally, suggesting that the result is not due to excess wild-type virus.


Figure 3
View larger version (76K):
[in this window]
[in a new window]
  		FIG. 3. DNase sensitivity assay. Equal amounts of virus was obtained for each time point. The virus samples were subjected to treatment with a 20-fold excess of DNase I or were mock treated with water. The capsids were then removed by proteinase K treatment and the genomes amplified by PCR. Control, uninfected cells; N, PCR control that received no input DNA.

 
From these results we concluded that both of the minor proteins and the myristylation of JCV Vp2 are necessary for efficient propagation of the virus. Neither minor protein is able to functionally substitute for the other. Both minor proteins and the myristylation site are needed for the correct packaging of the virus. Studies to further understand the assembly defects are under way in our lab. These studies will continue to evaluate whether there is a difference in the localization of the capsid proteins or the genome in the mutant viruses.

Our results are also consistent with recent findings using mouse polyomavirus. When the myristylation moiety of polyomavirus is mutated to histidine, glutamine, and glutamate, the viruses have a lower viral burst and therefore have fewer infected cells over time (16). Also, when the myristylation site is mutated to a glutamate, electron microscopy studies show an altered morphology. These mutants were able to create capsid-like structures but were less regular and less compact (14).

Myristylated proteins have been known to play a key role in assembly for other nonenveloped viruses, such as poliovirus (17, 18). It is possible that the myristyl moiety makes key contacts within the virion that are necessary for the structural integrity of the virus. Additionally, the assembly defects could be because of unique protein interactions between the minor proteins and cellular chaperones.

The inability of the virus to protect DNA when it is lacking the minor proteins is also consistent with studies using virus-like particles (VLPs). One study clearly showed that JCV VLPs, consisting of Vp1 alone, were unable to protect genome-size DNA from DNase degradation (28). It has also been shown that under physiological conditions, Vp2 and VP3 are both required for SV40 Vp1 to form VLPs (12, 13).


   ACKNOWLEDGMENTS
 
We thank all the members of the Atwood laboratory for critical discussion during the course of this work.

Work in our laboratory was supported by a grant from the National Cancer Institute (R01 CA71878) and by a grant from the National Institute of Neurological Disorders and Stroke (R01 NS43097) to W.J.A. M.L.G. is supported by a Ruth L. Kirschstein predoctoral fellowship (no. 1F31NS053340-01).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, 70 Ship Street, Box G-E434, Providence, RI 02903. Phone: (401) 863-3116. Fax: (401) 863-9653. E-mail: Walter_Atwood{at}Brown.edu. Back


   REFERENCES
Top
 Abstract
 Text
 References
 

  1. Astrom, K. E., E. L. Mancall, and E. P. Richardson, Jr. 1958. Progressive multifocal leuko-encephalopathy; a hitherto unrecognized complication of chronic lymphatic leukaemia and Hodgkin's disease. Brain 81:93-111.[Free Full Text]
  2. Barouch, D. H., and S. C. Harrison. 1994. Interactions among the major and minor coat proteins of polyomavirus. J. Virol. 68:3982-3989.[Abstract/Free Full Text]
  3. Chen, B. J., and W. J. Atwood. 2002. Construction of a novel JCV/SV40 hybrid virus (JCSV) reveals a role for the JCV capsid in viral tropism. Virology 300:282-290.[CrossRef][Medline]
  4. Clever, J., D. A. Dean, and H. Kasamatsu. 1993. Identification of a DNA binding domain in simian virus 40 capsid proteins Vp2 and Vp3. J. Biol. Chem. 268:20877-20883.[Abstract/Free Full Text]
  5. Clever, J., and H. Kasamatsu. 1991. Simian virus 40 Vp2/3 small structural proteins harbor their own nuclear transport signal. Virology 181:78-90.[CrossRef][Medline]
  6. Daniels, R., N. M. Rusan, A. K. Wilbuer, L. C. Norkin, P. Wadsworth, and D. N. Hebert. 2006. Simian virus 40 late proteins possess lytic properties that render them capable of permeabilizing cellular membranes. J. Virol. 80:6575-6587.[Abstract/Free Full Text]
  7. Gee, G. V., K. Manley, and W. J. Atwood. 2003. Derivation of a JC virus-resistant human glial cell line: implications for the identification of host cell factors that determine viral tropism. Virology 314:101-109.[CrossRef][Medline]
  8. Gharakhanian, E., and H. Kasamatsu. 1990. Two independent signals, a nuclear localization signal and a Vp1-interactive signal, reside within the carboxy-35 amino acids of SV40 Vp3. Virology 178:62-71.[CrossRef][Medline]
  9. Gharakhanian, E., L. Munoz, and L. Mayorca. 2003. The simian virus 40 minor structural protein Vp3, but not Vp2, is essential for infectious virion formation. J. Gen. Virol. 84:2111-2116.[Abstract/Free Full Text]
  10. Gordon-Shaag, A., Y. Yosef, M. Abd El-Latif, and A. Oppenheim. 2003. The abundant nuclear enzyme PARP participates in the life cycle of simian virus 40 and is stimulated by minor capsid protein VP3. J. Virol. 77:4273-4282.[Abstract/Free Full Text]
  11. Ishii, N., N. Minami, E. Y. Chen, A. L. Medina, M. M. Chico, and H. Kasamatsu. 1996. Analysis of a nuclear localization signal of simian virus 40 major capsid protein Vp1. J. Virol. 70:1317-1322.[Abstract]
  12. Kanesashi, S. N., K. Ishizu, M. A. Kawano, S. I. Han, S. Tomita, H. Watanabe, K. Kataoka, and H. Handa. 2003. Simian virus 40 VP1 capsid protein forms polymorphic assemblies in vitro. J. Gen. Virol. 84:1899-1905.[Abstract/Free Full Text]
  13. Kawano, M. A., T. Inoue, H. Tsukamoto, T. Takaya, T. Enomoto, R. U. Takahashi, N. Yokoyama, N. Yamamoto, A. Nakanishi, T. Imai, T. Wada, K. Kataoka, and H. Handa. 2006. The VP2/VP3 minor capsid protein of simian virus 40 promotes the in vitro assembly of the major capsid protein VP1 into particles. J. Biol. Chem. 281:10164-10173.[Abstract/Free Full Text]
  14. Krauzewicz, N., C. H. Streuli, N. Stuart-Smith, M. D. Jones, S. Wallace, and B. E. Griffin. 1990. Myristylated polyomavirus VP2: role in the life cycle of the virus. J. Virol. 64:4414-4420.[Abstract/Free Full Text]
  15. Major, E. O., K. Amemiya, C. S. Tornatore, S. A. Houff, and J. R. Berger. 1992. Pathogenesis and molecular biology of progressive multifocal leukoencephalopathy, the JC virus-induced demyelinating disease of the human brain. Clin. Microbiol. Rev. 5:49-73.[Abstract/Free Full Text]
  16. Mannova, P., D. Liebl, N. Krauzewicz, A. Fejtova, J. Stokrova, Z. Palkova, B. E. Griffin, and J. Forstova. 2002. Analysis of mouse polyomavirus mutants with lesions in the minor capsid proteins. J. Gen. Virol. 83:2309-2319.[Abstract/Free Full Text]
  17. Marc, D., M. Girard, and S. van der Werf. 1991. A Gly1 to Ala substitution in poliovirus capsid protein VP0 blocks its myristoylation and prevents viral assembly. J. Gen. Virol. 72:1151-1157.[Abstract/Free Full Text]
  18. Marc, D., G. Masson, M. Girard, and S. van der Werf. 1990. Lack of myristoylation of poliovirus capsid polypeptide VP0 prevents the formation of virions or results in the assembly of noninfectious virus particles. J. Virol. 64:4099-4107.[Abstract/Free Full Text]
  19. Monaco, M. C., B. F. Sabath, L. C. Durham, and E. O. Major. 2001. JC virus multiplication in human hematopoietic progenitor cells requires the NF-1 class D transcription factor. J. Virol. 75:9687-9695.[Abstract/Free Full Text]
  20. Muller, U., H. Zentgraf, I. Eicken, and W. Keller. 1978. Higher order structure of simian virus 40 chromatin. Science 201:406-415.[Abstract/Free Full Text]
  21. Padgett, B. L., and D. L. Walker. 1973. Prevalence of antibodies in human sera against JC virus, an isolate from a case of progressive multifocal leukoencephalopathy. J. Infect. Dis. 127:467-470.[Medline]
  22. Padgett, B. L., D. L. Walker, G. M. ZuRhein, R. J. Eckroade, and B. H. Dessel. 1971. Cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy. Lancet i:1257-1260.
  23. Resh, M. D. 1999. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim. Biophys. Acta 1451:1-16.[Medline]
  24. Richardson, E. P., Jr. 1961. Progressive multifocal leukoencephalopathy. N. Engl. J. Med. 265:815-823.[Medline]
  25. Sahli, R., R. Freund, T. Dubensky, R. Garcea, R. Bronson, and T. Benjamin. 1993. Defect in entry and altered pathogenicity of a polyoma virus mutant blocked in VP2 myristylation. Virology 192:142-153.[CrossRef][Medline]
  26. Shishido-Hara, Y., Y. Hara, T. Larson, K. Yasui, K. Nagashima, and G. L. Stoner. 2000. Analysis of capsid formation of human polyomavirus JC (Tokyo-1 strain) by a eukaryotic expression system: splicing of late RNAs, translation and nuclear transport of major capsid protein VP1, and capsid assembly. J. Virol. 74:1840-1853.[Abstract/Free Full Text]
  27. Shishido-Hara, Y., S. Ichinose, K. Higuchi, Y. Hara, and K. Yasui. 2004. Major and minor capsid proteins of human polyomavirus JC cooperatively accumulate to nuclear domain 10 for assembly into virions. J. Virol. 78:9890-9903.[Abstract/Free Full Text]
  28. Wang, M., T. H. Tsou, L. S. Chen, W. C. Ou, P. L. Chen, C. F. Chang, C. Y. Fung, and D. Chang. 2004. Inhibition of simian virus 40 large tumor antigen expression in human fetal glial cells by an antisense oligodeoxynucleotide delivered by the JC virus-like particle. Hum. Gene Ther. 15:1077-1090.[CrossRef][Medline]

Journal of Virology, November 2006, p. 10858-10861, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.01298-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gasparovic, M. L.
Right arrow Articles by Atwood, W. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gasparovic, M. L.
Right arrow Articles by Atwood, W. J.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Mol. Cell. Biol. Microbiol. Mol. Biol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS