The Open Virology Journal




(Discontinued)

ISSN: 1874-3579 ― Volume 15, 2021

The E89K Mutation in the Matrix Protein of the Measles Virus Affects In Vitro Cell Death and Virus Replication Efficiency in Human PBMC



Jianbao Dong1, 2, 3, Wei Zhu1, Akatsuki Saito1, Yoshitaka Goto1, Hiroyuki Iwata4, Takeshi Haga*, 1
1 Department of Veterinary Microbiology, University of Miyazaki, Miyazaki 889-2192, Japan
2 Research Fellow DC of the Japan Society for the Promotion of Science, Yamaguchi University, Yamaguchi 753-8511, Japan
3 The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi 753-8511, Japan
4 Department of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8511, Japan

Abstract

Matrix protein is known to have an important role in the process of virus assembly and virion release during measles virus replication. In the present in vitro study, a single mutation of E89K in the matrix protein was shown to affect cell death and virus replication efficiency in human PBMC. One strain with this mutation caused less cell death than the parental virus, and possessed high virus replication efficiency. Moreover, by Annexin V-FITC staining, polycaspase FLICA staining, and double labeling with poly-caspase FLICA and the Hoechst stain, the cell death seen was shown to be apoptosis.

Keywords: Measles virus, human PBMC, E89K mutation, cell death, replication efficiency..

Article Information


Identifiers and Pagination:

Year: 2012
Volume: 6
First Page: 68
Last Page: 72
Publisher Id: TOVJ-6-68
DOI: 10.2174/1874357901206010068

Article History:

Received Date: 27/1/2012
Revision Received Date: 14/3/2012
Acceptance Date: 29/3/2012
Electronic publication date: 8/6/2012
Collection year: 2012

© Dong et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.


* Address correspondence to this author at the Department of Veterinary Microbiology, University of Miyazaki, 1-1 Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan; Tel: +81-985-58-7575; Fax: +81-985-58-7575; E-mail: a0d518u@cc.miyazaki-u.ac.jp



INTRODUCTION

Measles, which is caused by the measles virus (MeV), remains a major contributor to childhood morbidity and mortality, particularly in developing countries with low vaccine coverage rates. MeV is an enveloped virus belonging to the genus Morbillivirus of the family Paramyxoviridae having a nonsegmental negative-strand RNA genome of 15,894 nucleotides in length. The MeV genome contains six sequential genes that code for structural proteins: nucleocapsid (N), phospho- (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) proteins (5’-N-P-M-F-H-L-3’). In addition, the MeV virion contains two nonstructural proteins, V and C. The V protein is generated from an edited mRNA that contains an extra G residue, while the C protein is alternatively translated from a downstream start signal [1 Knipe DM, Howley PM, Eds. Fields Virology Philadelphia: Lippincott Williams & Wilkins 2007.].

It is well known that the M protein of MeV and other members of the Paramyxoviridae family plays an important role in the process of virus assembly and virion release, which takes place at the plasma membrane [2Cathomen T, Naim HY, Cattaneo R. Measles viruses with altered envelope protein cytoplasmic tails gain cell fusion competence J Virol 1998; 72: 1224-34.-5 Takimoto T, Portner A. Molecular mechanism of paramyxovirus budding Virus Res 2004; 106: 133-45.]. In addition, many other functions have been discovered and confirmed. For example, M protein can inhibit polymerase activity and viral transcription during virus replication [6 Reuter T, Weissbrich B, Schneider-Schaulies S, et al. RNA interference with measles virus N P and L mRNAs efficiently prevents and with matrix protein mRNA enhances viral transcription J Virol 2006; 80: 5951-7., 7 Suryanarayana K, Baczko K, ter Meulen V, et al. Transcription inhibition and other properties of matrix proteins expressed by M genes cloned from measles viruses and diseased human brain tissue J Virol 1994; 68: 1532-43.]. It also can affect efficient growth and cell-cell fusion [8 Tahara M, Takeda M, Yanagi Y. Altered interaction of the matrix protein with the cytoplasmic tail of hemagglutinin modulates measles virus growth by affecting virus assembly and cell-cell fusion J Virol 2007; 81: 6827-36., 9 Tahara M, Takeda M, Yanagi Y. Contributions of matrix and large protein genes of the measles virus edmonston strain to growth in cultured cells as revealed by recombinant viruses J Virol 2005; 79: 15218-25.]. A previous study revealed that the E89K substitution mutation in M protein plays a key role in wild-type MeV adaptation in the cotton rat and cotton rat lung (CRL) cells [10 Dong JB, Saito A, Mine Y, et al. Adaptation of wild-type measles virus to cotton rat lung cells: E89K mutation in matrix protein contributes to its fitness Virus Genes 2009; 39: 330-4.].

Human peripheral blood mononuclear cells (PBMC) are the main target cells of natural MeV infection. In order to determine how the E89K-mutated M protein affects the phenotype of PBMC and virus, cell death and virus replication efficiency were analyzed in this in vitro study. Results indicate that the mutant virus causes less cell death than the parental virus, and possesses high replication efficiency in human PBMC.

MATERIALS AND METHODS

Cell Culture and Virus Infection

Adapted virus MV99YC7, which adapted well to CRL cells after serial passages, possesses a mutation in the M gene causing E89K substitution and one silent mutation in the L gene, as compared to the parental virus MV99Y [10 Dong JB, Saito A, Mine Y, et al. Adaptation of wild-type measles virus to cotton rat lung cells: E89K mutation in matrix protein contributes to its fitness Virus Genes 2009; 39: 330-4.]. Human PBMC were isolated from healthy adults using Ficoll-Paque PREMIUM (GE Healthcare Bio-science, Piscataway, NJ). Purified PBMC were stimulated with 5 μg/ml concanavalin A (ConA) and cultured in 10% FCS-RPMI medium [RPMI 1640 with 2 mM L-glutamine and sodium bicarbonate (Sigma, St Louis, MO) containing 10% heat-inactivated fetal calf serum (FCS) (JRH Bioscience, Overland Park, Kansas)] for 24 hours at 37 °C in 5% CO2. Non-adherent cells were harvested, washed three times with PBS and then infected with MV99Y or MV99YC7 at an MOI of 1.0 by incubation at 37 °C in 5% CO2 for 1 hour. Mock-infected PBMC served as negative controls.

Cell Labeling, Staining and Analysis

After infection, cells were washed three times with 37 °C RPMI 1640 to remove unattached virus and then cultured in 10% FCS-RPMI medium containing 5 μg/ml ConA; 100 μg/ml Virus Replication Inhibiting Peptide (Z- D- Phe- Phe- Gly; Peptide Institute Inc. Osaka, Japan) [11 Richardson CD, Scheid A, Choppin PW. Specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the N-termini of the F1 or HA2 viral polypeptides Virology 1980; 105: 205-2.] was added to the medium to block a second round of infection by progeny viruses. Cells were harvested 24, 48, 72, and 96 hours post-infection (hpi), and then were stained with Annexin V-FITC (MBL, Nagoya, Japan) and poly-caspases FLICA (Fluorochrome Inhibitor of Caspases; Immunochemistry Technologies, Bloomington, MN), respectively. The fluorescence was measured using FACSCanto II (BD Biosciences, San Jose, CA) according to the manufacturer’s instruction. Double labeling with poly-caspases FLICA and Hoechst stain was performed for cells harvested at different time points and observed under fluorescence microscope.

Assessment of Comparable Virus Input

In parallel experiments, viruses were harvested and titrated with B95a cells, the Epstein–Barr virus-transformed cotton-top tamarin B cell line [12 Kobune F, Sakata H, Sugiura A. Marmoset lymphoblastoid cells as a sensitive host for isolation of measles virus J Virol 1990; 64: 700-5.]. To determine whether an equal amount of input virus (MV99Y and MV99YC7 strains) had infected the PBMC, quantitative RT-PCR assays were performed using one pair of specific primers, based on the nucleotide sequences of the M gene: RtmF (5’-TACAACAACACCCCACTAAC-3’) and RtmR (5’-AAACAACACGGAACCTCTGC-3’). After undergoing the infection procedure described above, 5 × 105 infected PBMC cells were harvested and RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Viral RNA was quantified in six repeats of real-time RT-PCR using One Step SYBR PrimeScript PLUS RT-PCR Kit (Takara, Shiga, Japan).

Apoptosis Assessment

In order to determine whether viral replication contributes to apoptosis induction, UV-inactivated MV99Y and MV99YC7 were generated by ultraviolet (UV) irradiation at 2 J/cm2 as described in a previous report [13 Haga T, Murayama N, Shimizu Y, et al. Analysis of antibody response by temperature-sensitive measles vaccine strain in the cotton rat model Comp Immunol Microbiol Infect Dis 2009; 32: 395-406.]. PBMC were infected, as described above, with these at an MOI of 1.0, harvested at 24, 48, 72, and 96 hpi, stained with Annexin V-FITC and poly-caspase FLICA, and then analyzed by flow cytometry.

Statistical Analysis

Data were analyzed using Excel software (Microsoft). Variance analysis was performed with single factor (One Way) ANOVA tests.

RESULTS

MeV-infected PBMC cell death was detected with Annexin V staining and flow cytometric analysis. The mutant MV99YC7 strain caused significantly (P<0.01) less cell death than the parental strain MV99Y at all time points studied (Fig. 1). Compared to uninfected control cells, MV99Y-infected cells were not different at 24 hpi, but a highly significant difference (P<0.01) emerged between 48 and 96 hpi.

To determine whether cell death was caused by viral replication, UV-inactivated MeV infection was carried out; no significant differences were found among UV-inactivated MV99Y-infected, UV-inactivated MV99YC7-infected cells and uninfected PBMC (Fig. 1), suggesting that viral replication contributes to the cell death.

Virus replication kinetics are shown in Fig. (2); the MV99YC7 strain possessed higher replication efficiency than MV99Y at 48, 72 and 96 hpi (P<0.01). Quantitative real-time RT-PCR assays were performed out to assess the virus particle input, including receptor-bound virus. No significant differences were found between MV99Y- and MV99YC7-infected cells. Considered together, these results indicated that the mutant possessed higher growth efficiency than the parental strain in this study.

Cell death has been divided into two main types: apoptosis and necrosis. In apoptosis, caspases are well known to participate in a series of reactions triggered in response to proapoptotic signals, resulting in cleavage of protein substrates, causing cell disassembly [14 Slee EA, Adrain C, Martin SJ. Serial killers: ordering caspase activation events in apoptosis Cell Death Differ 1999; 6: 1067-74.]. In order to identify the type of cell death caused by MeV in this study, double labeling with poly-caspase FLICA and the Hoechst stain (which reacts with the condensed nuclei of dying cells) were carried out. Only a few positive cells were seen in the mock-infected PBMC. In contrast, PBMC infected with MV99Y and MV99YC7 were definitely positive for both labels (Fig. 3), indicating that the cell death seen in the present study was due to apoptosis.

Single staining with poly-caspase FLICA was also performed. Flow cytometric analysis revealed a low percentage (3.9 to 6.9 %) of poly-caspase positive mock-infected cells at all time points. At 24 hpi, a low percentage of MV99Y- and MV99YC7-infected cells was detected (7.85% and 6.8%, respectively; P>0.05); both were significantly different (P<0.01) than mock-infected cells (3.9%). Between 48 and 96 hpi, significant differences within the MV99Y- and MV99YC7-infected cultures emerged; infected cell numbers increased from 36.4% to 56.7 % and from 16.3% to 37.8 %, respectively. In addition, during that time period differences between parental and mutant infections were significant (P<0.01) (Fig. 4).

UV-inactivated MV99Y and MV99YC7 were utilized to determine whether viral replication contributes to apoptosis induction. A low percentage of caspase-positive cells was detected in UV-inactivated parental- and mutant-infected cells at all time points (Fig. 4), and there was no significant difference among them and mock-infected cells. This indicates indirectly that viral replication contributed to apoptosis in the present study.

DISCUSSION

It is known that MeV infection can cause lymphopenia and degradation of cell-mediated immunity, and that virulent strains usually cause more severe immunosuppression than strains used in vaccines. However, the precise mechanisms involved remain unclear, although motifs of MeV proteins might be pertinent. Compared with the wild-type Edmoston strain, all vaccine strains (AIK-C, Moraten, Schwarz, Rubeovax, Zagreb, CAM-70, Changchun-47, Leningrad-4 and Shanghai-191) possess the E89K mutant M protein [15 Bankamp B, Takeda M, Zhang Y, et al. Genetic characterization of measles vaccine strains J Infect Dis 2011; 204(Suppl 1 ): S533-48., 16 Parks CL, Lerch RA, Walpita P, et al. Comparison of predicted amino acid sequences of measles virus strains in the Edmonston vaccine lineage J Virol 2001; 75: 910-20.]. It has remained unclear whether this mutation is associated with MeV virulence. Results of the present study indicate that the mutant virus caused less cell death than the parent. Since only the infection on monkey model can show attenuation of MeV strain. Therefore, further investigation is necessary to clarify the relationship between virulence and the E89K mutation.

P64S and E89K M protein substitutions were reported to be responsible for efficient virus growth in Vero cells [9 Tahara M, Takeda M, Yanagi Y. Contributions of matrix and large protein genes of the measles virus edmonston strain to growth in cultured cells as revealed by recombinant viruses J Virol 2005; 79: 15218-25.]. However, recombinant viruses with these mutations had a growth disadvantage in CD150-positive B95a cells [9 Tahara M, Takeda M, Yanagi Y. Contributions of matrix and large protein genes of the measles virus edmonston strain to growth in cultured cells as revealed by recombinant viruses J Virol 2005; 79: 15218-25.]. Moreover, interactions of the M protein with the cytoplasmic tails of the H protein modulated MeV growth in different cell types [8 Tahara M, Takeda M, Yanagi Y. Altered interaction of the matrix protein with the cytoplasmic tail of hemagglutinin modulates measles virus growth by affecting virus assembly and cell-cell fusion J Virol 2007; 81: 6827-36.]. In this study, we presented E89K substitution in the M protein contributed to virus replication efficiency in human PMBC.

The E89K mutation in the M protein has been shown to play a key role in wild-type MeV adaptation in cotton rat and CRL cells [10 Dong JB, Saito A, Mine Y, et al. Adaptation of wild-type measles virus to cotton rat lung cells: E89K mutation in matrix protein contributes to its fitness Virus Genes 2009; 39: 330-4.]. This may be explained by the possibility that the mutation facilitates a strong interaction between the M protein and the cytoplasmic tail of the H protein, thus enhancing assembly of virus particles [8 Tahara M, Takeda M, Yanagi Y. Altered interaction of the matrix protein with the cytoplasmic tail of hemagglutinin modulates measles virus growth by affecting virus assembly and cell-cell fusion J Virol 2007; 81: 6827-36., 9 Tahara M, Takeda M, Yanagi Y. Contributions of matrix and large protein genes of the measles virus edmonston strain to growth in cultured cells as revealed by recombinant viruses J Virol 2005; 79: 15218-25.]. Interestingly, the result of the present study provide another presumed mechanism that the E89K mutation inhibited the cell apoptosis to aid virus adaption. Further studies are required to elucidate the precise mechanisms involved.

In conclusion, compared with the parental strain MV99Y, the MV99YC7 mutant strain caused less cell death and possessed higher replication efficiency in human PBMC in vitro. Furthermore, the cell death seen was shown to be due to apoptosis.

ACKNOWLEDGEMENTS

This work was partly supported by the Project for Zoonoses Education and Research, University of Miyazaki, and by a grant from the Japan Society for the Promotion of Science (No. KAKENHI22780272).

CONFLICT OF INTEREST

Declared none.

REFERENCES
[1] Knipe DM, Howley PM, Eds. Fields Virology Philadelphia: Lippincott Williams & Wilkins 2007.
[2] Cathomen T, Naim HY, Cattaneo R. Measles viruses with altered envelope protein cytoplasmic tails gain cell fusion competence J Virol 1998; 72: 1224-34.
[3] Garoff H, Hewson R, Opstelten DJ. Virus maturation by budding Microbiol Mol Biol Rev 1998; 62: 1171-90.
[4] Schmitt AP, He B, Lamb RA. Involvement of the cytoplasmic domain of the hemagglutinin-neuraminidase protein in assembly of the paramyxovirus simian virus 5 J Virol 1999; 73: 8703-12.
[5] Takimoto T, Portner A. Molecular mechanism of paramyxovirus budding Virus Res 2004; 106: 133-45.
[6] Reuter T, Weissbrich B, Schneider-Schaulies S, et al. RNA interference with measles virus N P and L mRNAs efficiently prevents and with matrix protein mRNA enhances viral transcription J Virol 2006; 80: 5951-7.
[7] Suryanarayana K, Baczko K, ter Meulen V, et al. Transcription inhibition and other properties of matrix proteins expressed by M genes cloned from measles viruses and diseased human brain tissue J Virol 1994; 68: 1532-43.
[8] Tahara M, Takeda M, Yanagi Y. Altered interaction of the matrix protein with the cytoplasmic tail of hemagglutinin modulates measles virus growth by affecting virus assembly and cell-cell fusion J Virol 2007; 81: 6827-36.
[9] Tahara M, Takeda M, Yanagi Y. Contributions of matrix and large protein genes of the measles virus edmonston strain to growth in cultured cells as revealed by recombinant viruses J Virol 2005; 79: 15218-25.
[10] Dong JB, Saito A, Mine Y, et al. Adaptation of wild-type measles virus to cotton rat lung cells: E89K mutation in matrix protein contributes to its fitness Virus Genes 2009; 39: 330-4.
[11] Richardson CD, Scheid A, Choppin PW. Specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the N-termini of the F1 or HA2 viral polypeptides Virology 1980; 105: 205-2.
[12] Kobune F, Sakata H, Sugiura A. Marmoset lymphoblastoid cells as a sensitive host for isolation of measles virus J Virol 1990; 64: 700-5.
[13] Haga T, Murayama N, Shimizu Y, et al. Analysis of antibody response by temperature-sensitive measles vaccine strain in the cotton rat model Comp Immunol Microbiol Infect Dis 2009; 32: 395-406.
[14] Slee EA, Adrain C, Martin SJ. Serial killers: ordering caspase activation events in apoptosis Cell Death Differ 1999; 6: 1067-74.
[15] Bankamp B, Takeda M, Zhang Y, et al. Genetic characterization of measles vaccine strains J Infect Dis 2011; 204(Suppl 1 ): S533-48.
[16] Parks CL, Lerch RA, Walpita P, et al. Comparison of predicted amino acid sequences of measles virus strains in the Edmonston vaccine lineage J Virol 2001; 75: 910-20.
Track Your Manuscript:


Endorsements



"Open access will revolutionize 21st century knowledge work and accelerate the diffusion of ideas and evidence that support just in time learning and the evolution of thinking in a number of disciplines."


Daniel Pesut
(Indiana University School of Nursing, USA)

"It is important that students and researchers from all over the world can have easy access to relevant, high-standard and timely scientific information. This is exactly what Open Access Journals provide and this is the reason why I support this endeavor."


Jacques Descotes
(Centre Antipoison-Centre de Pharmacovigilance, France)

"Publishing research articles is the key for future scientific progress. Open Access publishing is therefore of utmost importance for wider dissemination of information, and will help serving the best interest of the scientific community."


Patrice Talaga
(UCB S.A., Belgium)

"Open access journals are a novel concept in the medical literature. They offer accessible information to a wide variety of individuals, including physicians, medical students, clinical investigators, and the general public. They are an outstanding source of medical and scientific information."


Jeffrey M. Weinberg
(St. Luke's-Roosevelt Hospital Center, USA)

"Open access journals are extremely useful for graduate students, investigators and all other interested persons to read important scientific articles and subscribe scientific journals. Indeed, the research articles span a wide range of area and of high quality. This is specially a must for researchers belonging to institutions with limited library facility and funding to subscribe scientific journals."


Debomoy K. Lahiri
(Indiana University School of Medicine, USA)

"Open access journals represent a major break-through in publishing. They provide easy access to the latest research on a wide variety of issues. Relevant and timely articles are made available in a fraction of the time taken by more conventional publishers. Articles are of uniformly high quality and written by the world's leading authorities."


Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)

Browse Contents




Webmaster Contact: info@benthamopen.net
Copyright © 2023 Bentham Open