Research:

 

Overview

 

Parvoviruses are the smallest DNA-containing viruses. The classification of the family Parvoviridae relies on morphology and functional characteristics. Parvoviruses are common animal and insect pathogens. Based on the ability to infect vertebrate or invertebrate cells the Parvoviridae are divided into Parvovirinae and Densovirinae, respectively (1; 2). Parvovirinae are subdivided into three genera according to their transcription maps, the nature of the terminal repeats, and the ability to efficiently replicate either autonomously (genus Parvovirus), with helper virus (genus Dependovirus), or preferentially in erythroid cells (genus Erythrovirus).

Minute virus of mice (MVM) is a non-pathogenic mouse virus and belongs to the Parvovirus genus and serves as a parvovirus model. There are two strains of MVM, the MVM prototype which efficiently infects mouse fibroblasts, and the immunosuppressive infecting T-lymphocytes.

Human parvovirus B19 (B19V) was discovered 1975 by Yvonne Cossart (3), and by the year 2000, it was the only accepted member of human Erythrovirus (2). The virus replicates exclusively in erythroid progenitor cells. Little is known about the biology of B19V since a suitable cell culture system is missing. B19V is a global and common infectious pathogen in humans. The prevalence of IgG antibodies directed against B19V ranges from 2 to 15% in younger children, 15 to 60% in adults, and more than 85% in the geriatric population (4-7). The virus is endemic, epidemics may occur during late winter and spring. The transmission of B19V occurs in most cases via aerosols. However, it may also occur via the application of plasma derived products.

 

The virions has a simple structure composed of only two proteins (VP1 and VP2) and a linear, single-stranded DNA molecule of about 5 and 5.6 kDa (1), respectively. The nonenveloped viral particles are 22 to 26 nm in diameter and show icosahedral symmetry. Additionally to the sequence present in all structural proteins, the minor capsid proteins have an N-terminal extension of about 143 to 227 amino acids, called N-VP1.

During life cycle, receptors mediate the first contact of the virus with the host cell; the attachment to the plasma membrane represents the beginning of the infection. The receptor for B19V for instance has been shown to be one of the blood group antigens, the glycolipid globoside (9), whereas MVM binds to sialic acid (10).

 

 

The interest in parvoviruses is based on several aspects

At least one of the parvovirus family members, B19V, is a widespread human pathogen. Although a B19V infection is in most cases without any considerable pathogenic effect, it may be the cause of anemia, arthropathy and foetal death. Since B19V can be transmitted through the administration of plasma derived products, its nature and especially its inactivation are of sizeable interest for manufacturers of blood and plasma derived clinical products.

Several parvoviruses are the cause of animal diseases with economical impact: Porcine parvovirus (PPV) is a common cause of stillbirths, aleutian mink disease virus (AMDV), goose parvovirus (GPV) and several shrimp viruses are responsible for occasional epidemics with high mortality rates in farms and finally, canine and feline parvovirus (CPV, FPV) are a threat to pets.

Parvoviruses serve as gene delivery vectors in gene therapy. The applications focus on two areas: the treatment of cancer and monogenic hereditary diseases.

Parvoviruses serve as model viruses. On one hand, some animal parvoviruses serve as models for B19V in validation studies. On the other hand, parvoviruses are a model for very resistant viruses that have not been discovered yet or may emerge in future.

 

 

Research topics

 

Although B19V is a significant human pathogen displaying many different manifestations, the B19V infection cycle remains largely unknown. The research of our group is focused on the early events of parvovirus infection. Aspects already under investigation are virus binding on the cellsurface and viral intracellular trafficking.

Structural rearrangements occur during the life cycle of all viruses and many viruses use the low pH environment of the endosomal network to induce necessary rearrangement steps for the infection. In the case of parvoviruses, capsid rearrangements were also observed in the endosomes, although it is not clear whether some of them are necessary or degradative. The capsid rearrangements observed in the cell can be mimicked in vitro, revealing differences between MVM and B19V.

Endosomal Processing of MVM

Early events of an MVM infection comprise binding and endocytosis, endosomal trafficking, endosomal escape and nuclear targeting. During the endosomal trafficking, the capsid undergoes structural rearrangements, which can be mimicked in vitro. Three rearrangements were observed: The cleavage of VP2 to VP3, the externalisation of N-VP1 and the externalisation of the viral DNA. To date, neither the triggers, nor the purpose of these incidents has been elucidated, except for the externalisation of N-VP1, which has been shown to be necessary for endosomal escape and nuclear targeting. Although the passage through the endosomal pathway is mandatory for the virus, it is also a dead end for most of the particles. Indeed, the endosomal escape is so inefficient that it could not be detected.

 

Mechanism Underlying B19V Inactivation

The inactivation of B19V is an important issue for the safety of plasma-derived products. Therefore the inactivation of B19V is well studied. It is achieved with heat, low or high pH, UVC and photochemical reactions (11, 12, 13, 14, and 15). However, although inactivation conditions of B19V were known, the mechanism on the molecular level remained unexplained. The first step of inactivation of B19V is the separation of the DNA from the virus capsid, before capsid disintegration occurred. Comparing the ease of inactivation among parvoviruses, B19V turned out to be the weakest of the so far tested viruses. This is due to a lower stability of the B19V DNA in its encapsidated form with respect to other parvoviruses. B19V has a much higher disposition to externalise its DNA upon heat than other parvoviruses. This feature might be characteristic for Erythroviruses, since all the other tested viruses were of the genus Parvovirus and it exemplified that animal Parvoviruses are not the ideal model viruses for the inactivation of the Erythrovirus B19V (16). The dissociation upon heat of the viral genome from its capsid has been reported for B19V, MVM and other parvoviruses. In vitro, it is the cause for inactivation. However, the same externalisation of the viral DNA has been reported to happen during the endosomal trafficking of MVM. Although it is not known whether the externalised DNA is dissociated from the capsid or not, or whether this uncoating is degradative or necessary, it occurs without capsid disintegration as well. Besides, during the synthesis of parvoviruses, already assembled capsids are filled with DNA, illustrating as well that the DNA entry/exit is also possible in vivo with an intact capsid. Therefore, it seems probable that the uncoating of parvoviruses during the infection occurs without capsid disintegration by the release of the genome though the five fold axis, be it in the endosomes, at the nuclear pore or in the nucleus.

 

Conformational change of the N-VP1 and binding to receptor

B19V life cycle begins with the binding to a host cell receptor and internalization. The cellular receptor for B19V has been identified as globoside or erythrocyte P antigen (9). The virus infects and propagates mainly in erythroid progenitor cells leading to high viremia of up to 1012-1014 particles per ml. Some other cell types that are positive for globoside may also be infected but the virus replication is often aborted. It has been shown that globoside is necessary for Parvovirus B19 binding but not sufficient for virus entry into cells (18). Therefore, the presence of a coreceptor is needed. The role of α5β1-integrin as the cellular coreceptor for B19 infection was described (17). In addition, Ku80 has also been identified as a coreceptor for B19V (19).

The N-VP1 of B19V elicits a dominant immune response to humans. Additionally a phospholipase A2 (PLA2) motif has been identified in the N-VP1 that is required for infection. It has been shown that Parvoviruses without a PLA2 motif are not infective (20). In a recent publication (21) it was shown that the N-VP1 PLA2 motif is not accessible or has an inactive conformation in the native capsids, which can change into active conformation upon heating, low pH treatments or unknown cell factors. These results would indicate that N-VP1is a dynamic protein adopting different conformations in the native B19 capsid. We want to identify the cellular factor that triggers the exposure of N-VP1 and the role of N-VP1during the entry. We elucidate if the PLA2 motif is exposed at the cell surface or later in the endosomal compartment where it might function to allow the endosomal escape of the virus.

 

Cytoplasmatic trafficking and nuclear targeting

Studies about intracellular trafficking of Parvoviruses are so far only available for CPV, AAV and MVM. It has been shown that Parvoviruses follow the endocytic route via early and late endosomes and finally accumulate in the lysosomes (22, 23). The low endosomal pH and an intact endocytic network are necessary for infection. Thus, we study the infectious intracellular pathway of B19V.

 

Virus cell interaction RBC

 

 

Future studies

inactivation studies B19V, MVM

References

 

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3.)    Cossart, Y. E., A. M. Field, B. Cant, and D. Widdows. 1975. Parvovirus-like particles in human sera. Lancet i:7273.

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9.)    K. E. Brown, S. M. Anderson, and N. S. Young. Erythrocyte P antigen: cellular receptor for B19 parvovirus. Science, 262(5130):114117, Oct 1993.

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11.)                       N. Boschetti, K. Wyss, A. Mischler, T. Hostettler, and C. Kempf. Stability of minute virus of mice against temperature and sodium hydroxide. Biologicals, 31(3):181185, 2003.

12.)                       N. Boschetti, I. Niederhauser, C. Kempf, A. Stuhler, J. Lower, and J. Blumel. Different susceptibility of B19 virus and mice minute virus to low pH treatment. Transfusion, 44(7):10791086, 2004.

13.)                       P. Caillet-Fauquet, M. Di Giambattista, M. L. Draps, F. Sandras, T. Branckaert, Y. de Launoit, and R. Laub. Continuous-flow UVC irradiation: a new, effective, protein activity-preserving system for inactivating bacteria and viruses, including erythrovirus B19. J. Virol. Methods, 118(2):131139, 2004.

14.)                       L. Lin, C. V. Hanson, H. J. Alter, V. Jauvin, K. A. Bernard, K. K. Murthy, P. Metzel, and L. Corash. Inactivation of viruses in platelet concentrates by photochemical treatment with amotosalen and long-wavelength ultraviolet light. Transfusion, 45(4):580590, 2005.

15.)                       M. Yunoki, M. Tsujikawa, T. Urayama, Y. Sasaki, M. Morita, H. Tanaka, S. Hattori, K. Takechi, and K. Ikuta. Heat sensitivity of human parvovirus B19. Vox Sang., 84(3):164169, 2003.

16.)                       F. Kasermann, C. Kempf, and N. Boschetti. Strengths and limitations of the model virus concept. PDA. J. Pharm. Sci. Technol., 58(5):244249, 2004.

17.)                       KA Weigel-Kelley, MC Yoder, A Srivastava. α5β1-integrin as a cellular co-receptor for human parvovirus B19: requirement of functional activation of {beta} 1 integrin for viral entry. Blood. 2003; 102:3927-3933.

18.)                       K.A. Weigel-Kelley, M.C Yoder., A Srivastava. 2001. Recombinant Human Parvovirus B19 Vectors: Erythrocyte P Antigen Is Necessary but not Sufficient for Successful Transduction of Human Hematopoietic Cells. J. Virol. 75 (9):4110-4116

19.)                       Y Munakata, I Kato T, Saito, T Kodera, KK Ishi, T Sasaki: Human parvovirus B19 infection of monocytic cell line U937 and antibody-dependent enhancement. Virology 2005.

20.)                       Zadori Z., J. Szelei, M. C. Lacoste, Y. Li, S. Gariepy, P. Raymond, M. Allaire, I. R. Nabi, and P. Tijssen. A viral phospholipase a2 is required for parvovirus infectivity. Dev. Cell, 1(2):291302, 2001.

21.)                       Ros C, Baltzer C, Mani B, Kempf C: Parvovirus uncoating in vitro reveals a mechanism of DNA release without capsid disassembly and striking differences in encapsidated DNA stability. Virol 2006; 345:137-147.

22.)                       Suikkanen S., M. Antila, A. Jaatinen, M. Vihinen-Ranta, and M. Vuento. Release of canine parvovirus from endocytic vesicles. Virology, 316(2):267280, 2003.

23.)                       Mani B., C. Baltzer, N. Valle, J.M. Almendral, Ch. Kempf, and C. Ros. Low pH dependent endosomal processing of the incoming parvovirus minute virus of mice virion leads to externalization of the VP1 N-terminal sequence (N-VP1), N-VP2 cleavage, and uncoating of the full-length genome. J Virol, 80(2):10151024, Jan 2006.