Susceptibility of human primary neuronal cells to Xenotropic Murine Leukemia Virus-related (XMRV) virus infection
© Ravichandran et al; licensee BioMed Central Ltd. 2011
Received: 1 August 2011
Accepted: 20 September 2011
Published: 20 September 2011
Xenotropic Murine Leukemia Virus-related (XMRV) virus is a recently identified mouse gammaretrovirus that has the ability to infect certain human cells. In this study, we investigated the susceptibility of primary neuronal cell types to infection with XMRV.
We observed that the human primary progenitors, progenitor-derived neurons, and progenitor-derived astrocytes supported XMRV multiplication. Interestingly, both progenitors and progenitor-derived neurons were more susceptible compared with progenitor-derived astrocytes. In addition, XMRV-infected Jurkat cells were able to transmit infection to neuronal cells.
These data suggest that neuronal cells are susceptible for XMRV infection.
KeywordsXMRV infection Primary neuronal cells Non-neuronal cells Nuclear factors
XMRV (108 copies/mL) was used to infect progenitors, PDN, and PDA for two hours, and then washed, replenished with appropriate fresh neuronal media. Four days after the infection, cells were washed four times, and harvested for total RNA isolation. Real-time quantitative PCR was performed with Master Mix (Quantitect Probe RT-PCR, Qiagen) using forward and reverse primers encoding gag region of XMRV, probe, RT-mix, and 1 ug of RNA in a total volume of 25 uL. The mixture was incubated at 50°C for 20 min, at 95°C for 10 min, and then cycled at 95°C for 15 sec and 60°C for 60 sec 40 times, using the Applied Biosystems 7500 sequence detection system. XMRV levels were quantitated using the XMRV clone VP62-pcDNA3.1 (GenBank accession no. EF185282; obtained through NIH AIDS Research and Reference Reagent Program) as a standard. Expression levels of XMRV were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA expression.
Comparison of XMRV infection
XMRV level (pg/ug total RNA)
Human prostate carcinoma
Human prostate adenocarcinoma
Human embryonic kidney
T-cell traffic into the central nervous system is thought to occur when activated T-cells cross the blood-brain barrier . The T-cells presumably act as a "Trojan horse" to store and transport infectious materials across the blood-brain barrier. This "Trojan horse" hypothesis has been well established in the pathogenesis of many viruses that infect the central nervous system [5–7]. Based on the assumption that XMRV infected lymphocytes could infiltrate and infect the neuronal cells, we conducted an in-vitro co-infection/co-culture study using XMRV-infected Jurkat cells with neuronal cells.
To produce XMRV-infected Jurkat cells, Jurkat cells were exposed with XMRV (1.0 × 108 copies of XMRV/mL) for two hours, washed and replenished with fresh media. Four weeks after the infection, cells were washed four times, and harvested for total RNA isolation. The XMRV levels were measured using Real-time PCR method as described above. The XMRV-infected Jurkat cells had levels around 500 pg of XMRV per ug of total RNA. For the co-infection/co-culture experiment, 1.0 × 106 XMRV-infected Jurkat cells (XMRV equivalent of 500 pg) were pelleted, treated with trypsin for 3 min at room temperature, washed and suspended in progenitor/PDN/PDA media; and added on to the appropriate adherent neuronal cell types (progenitors, PDN, or PDA) grown in 24 well plate (2.5 × 105 cells/well). Four hours after co-infection/co-culture, XMRV-infected Jurkat cells were removed, and the adherent neuronal cells were washed with appropriate neuronal media. To remove any adhered Jurkat cells from neuronal cells, each culture well was treated with trypsin, and just before the cells were ready to detach (after about 3 min), fresh neuronal media were added and washed three times using neuronal media. The resulting adherent neuronal cells were free from Jurkat cells, and continued to grow with appropriate neuronal media. After four days, the neuronal cells were harvested for total RNA isolation, and XMRV levels were quantified.
As in the neuronal infection study (Figure 2A), the co-culture/co-infection of XMRV-infected Jurkat (XMRV equivalent of 500 pg) with neuronal cells resulted in proportionate XMRV infection in neuronal cells (Figure 2B). In a separate co-culture/co-infection experiment, the XMRV-infected Jurkats (XMRV equivalent of 4.8 pg) were added with individual neuronal cells (progenitor, PDN, or PDA) as described above, continued for four days. We observed that even the small amount of XMRV in the XMRV-infected Jurkats could release virus and infect the neuronal cells (results not shown). Interestingly, in the co-cultures/co-infection study, we co-culture the XMRV-infected Jurkats with neuronal cells only for four hours. The released virus during this time was able to infect the bystander neuronal cells. These observations support the possibility that the infiltrated XMRV-infected T-cells could infect neuronal cells.
Brain cells are protected from pathogens by the blood-brain barrier. Even though neuronal cells are susceptible for XMRV infection, this virus should first cross the blood-brain barrier to inflict any damage to the brain cells in vivo. After XMRV is transported to neuronal cells, the virus needs favorable multiplication environment for its multiplication. For example, we have observed the requirement of nuclear factor I-A (NF-IA) for the XMRV multiplication in Jurkat cells (unpublished observation). Interestingly, both progenitors and neurons express higher levels of NF-IA , and this could account for the optimal XMRV multiplication in these cells. In addition, PDA, which is shown to express low NF-IA level compared to progenitor and PDN , showed lower susceptibility to XMRV.
In a recent animal study, XMRV was administered to rhesus macaques through intravenous inoculation . Interestingly, 291 days post-infection, infected cells were detectable by fluorescence in situ hybridization (FISH) in brain, showing the possibility of XMRV infection in the brain. This study further substantiates our speculation that XMRV could infect brain cells. Further in-vitro and in-vivo studies are warranted to identify and characterize the XMRV, and its role if any, in the brain cells.
We thank Dr. Xue Wang, Dr. Mingjie Zhang, Dr. Robin Biswas, and Dr. Chintamani D. Atreya for critical review of the article. The findings and conclusions in this paper have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.
- Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, Mikovits JA: Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science 2009, 326: 585-9. 10.1126/science.1179052View ArticlePubMedGoogle Scholar
- Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, Klein EA, Malathi K, Magi-Galluzzi C, Tubbs RR, Ganem D, Silverman RH, DeRisi JL: Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. Plos Pathogens 2006, 2: e25. 10.1371/journal.ppat.0020025PubMed CentralView ArticlePubMedGoogle Scholar
- Messam CA, Hou J, Gronostajski RM, Major EO: Lineage pathway of human brain progenitor cells identified by JC virus susceptability. Annals of Neurology 2003, 53: 636-646. 10.1002/ana.10523View ArticlePubMedGoogle Scholar
- Galea I, Bernardes-Silva M, Forse PA, van Rooijen N, Liblau RS, Perry VH: An antigen-specific pathway for CD8 T cells across the blood-brain barrier. J Exp Med 2007, 204: 2023-30. 10.1084/jem.20070064PubMed CentralView ArticlePubMedGoogle Scholar
- Houff SA, Berger JR: The bone marrow, B cells, and JC virus. Journal of Neurovirology 2008, 14: 341-3. 10.1080/13550280802348222View ArticlePubMedGoogle Scholar
- Houff SA, Major EO, Katz DA, Kufta CV, Sever JL, Pittaluga S, Roberts JR, Gitt J, Saini N, Lux W: Involvement of JC virus-infected mononuclear cells from the bone marrow and spleen in the pathogenesis of progressive multifocal leukoencephalopathy. N Engl J Med 1988, 318: 301-5. 10.1056/NEJM198802043180507View ArticlePubMedGoogle Scholar
- Monaco MC, Atwood GWJ, Gravell M, Tornatore CS, Major EO: JC virus infection of hematopoietic progenitor cells, primary B lymphocytes, and tonsillar stromal cells: Implications for viral latency. Journal of Virology 1996, 70: 7004-7012.PubMed CentralPubMedGoogle Scholar
- Ravichandran V, Major EO: DNA-binding transcription factor NF-1A negatively regulates JC virus multiplication. Journal of General Virology 2008, 89: 1396-401. 10.1099/vir.0.2008/000059-0PubMed CentralView ArticlePubMedGoogle Scholar
- Onlamoon N, Das Gupta J, Sharma P, Rogers K, Suppiah S, Rhea J, Molinaro RJ, Gaughan C, Dong B, Klein EA, Qiu X, Devare S, Schochetman G, Hackett J Jr, Silverman RH, Villinger F: Infection, Viral Dissemination, and Antibody Responses of Rhesus Macaques Exposed to the Human Gammaretrovirus XMRV. J Virol 2011, 85: 4547-57. 10.1128/JVI.02411-10PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.