Susceptibility to infections is largely determined by host genetics. A case in point is the susceptibility of certain mice strains to persistence of Theiler’s virus (TMEV). When inoculated intracranially with TMEV, the SJL/J strain develops a persistent infection in their central nervous system (CNS), while the B10.S strain clears TMEV

following a 2 week acute infection. This difference in susceptibility is partly attributed to a locus at chromosome 10 Tmevp3. It contains a novel gene named NeST, which codes for a 934-nt noncoding RNA, NeST, that appears to regulate expression of the adjacent interferon-gamma (IFN-γ) gene.    

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Theiler’s Murine Encephalomyelitis Virus (TMEV)  

NeST, a long non-coding RNA, and persistence of Theiler’s virus!Jan Clement A. Santiago†; Jose Antonio Gomez‡; Michel Brahic, MD, PhD‡; Karla Kirkegaard, PhD‡ !

Loyola Marymount University, Los Angeles CA 90045†; Dept. of Microbiology & Immunology, Stanford University, Stanford, CA 94305‡!Introduction!

Results!

Discussion!I.  Strains B10.S, B.cong and B10 S-R were intracranially inoculated

with 106 PFU of TMEV. II.  Brains and spinal cords were dissected 7 and 57 days post

infection, during acute and persistent phases respectively. III.  Test homogenized tissues with:

A.  Plague Assay – to determine viral load B.  qRT-PCR (quantitative Reverse Transcriptase–Polymerase Chain

Reaction) – to determine RNA levels of: a.  NeST b.  IFN-γ c.  TMEV viral replication

Fig. 2 Polymorphisms in Tmevp3 locus explain difference in their susceptibility; NeST is a candidate gene at this locus!

B10.S –virus cleared after 2 weeks!

SJL/J – virus persists for life!

Hypotheses!1.  NeST up-regulates IFN-γ expression!

2.  Increased NeST expression increases susceptibility to Theiler’s virus persistence (B10.S-R, transgenic for NeST, will be susceptible)!

Fig. 3 Genotypes of mice strains, with SJL/J (a) represented by a white bar, and B10.S (b) by black. B.cong (c) or B10.S congenic is a strain with Tmevp3 locus of SJL/J introgressed into a B10.S background. B10.S-R (d) is a transgenic B10.S strain for NeST. In making transgenic lines, genes inserted in the nuclei of egg cells of the background strain tend to integrate themselves multiple times on a specific spot of a chromosome. Thus B10.S-R is expected to over-express NeST.!

(a) SJL/J!

(b) B10.S!

!(c) B.cong!

(d) B10.S-R!NeST"NeST"NeST"

NeST"NeST"

NeST"

NeST"

Tmevp3 locus!

NeST"

Materials & Methods!

Fig. 1 TMEV

levels in mice at

persistent phase of infection!

Fig. 5 TMEV infectivity (PFU per brain or spinal cord) during persistence!

Fig. 6 TMEV viral RNA levels in CNS during persistence!

No difference in viral titers was observed during acute phase of infection (not shown). During persistent phase (Fig. 5), TMEV was below the limit of detection for brains of B10.S and B10.S-R, and in spinal cord of B10.S, suggesting clearance. TMEV persisted in the spinal cord of Bcong, as expected. Interestingly, TMEV persisted also in the spinal cords of the B10S-R. There is good correlation between viral titers and levels of viral RNA in this experiment (Fig. 6).

Fig. 7 Levels of NeST and IFN-γ  RNA  during acute phase !

NeST levels in B10.S and B10.S-R were below the level of detection during the acute phase (Fig. 7). They rose during the persistent phase (Fig. 8). No NeST was detected at 7 days, while IFN- γ was at the same level in both mice. There was no difference in NeST amounts between the mice 67 days post infection. Remarkably IFN-γ expression levels during persistence (Fig. 8 – Brain) was very significantly different (p=0.0003) between B10.S and B10.S-R

Fig. 4 Plague assay!

Ø TMEV persisted in the NeST transgenic B10.S-R strain. Strain B10.S cleared TMEV, even when inoculated with 100x the usual infection dose. Ø  NeST was expressed in both B10.S and B10.S-R strains, only late after inoculation, not during early infection.

Ø  There was no correlation between the level of NeST expression and TMEV persistence.

Ø  The level of IFN-γ RNA in CNS was higher for B10.S than for B10.S-R, a result consistent with the known role if IFN -γ in clearing the infection.

Ø  Though during in vitro activation of CD8 T-cells the expression of IFN -γ is associated with high NeST expression, there appears to be an inverse relationship in our in vivo experiments. Since we haven’t determined which cells express NeST in CNS during TMEV infection, it is not yet possible to give a comprehensive interpretation of these results.

Acknowledgments ! I’m truly grateful to my mentors in Kirkegaard Lab, Antonio

Gomez and Michel Brahic, for their untiring support and patience, and especially Karla Kirkegaard, principal investigator, for this opportunity to participate on the exciting research on NeST. I also want to thank the program assistant Irene Onyeneho and program director Melanie Bocanegra for facilitating this wonderful program. I want to acknowledge the following institutions for their funding and/or support:

Murine NeST has an equivalent in the human genome (Fig. 9), and appears to also be involved in IFN-γ regulation. It will be important to determine the role this long non-coding RNA play, as IFN-γ has crucial anti-viral and immunoregulatory functions, as well as being an inflammatory cytokine when unregulated. Elucidating the role of NeST in IFN-γ expression will contribute to the nascent of field noncoding RNAs and the study of autoimmune diseases.

Mouse Strain!

TMEV persistence!

NeST Expression!

SJL/J! +! high!

B10.S! O! low!

Bcong! +! high!

B10.S-R! ?! ?!Table 1 A summary of susceptibility to TMEV persistence and NeST expression of mice strains!

Ifng"Il22"

Nettoyage de Salmonella et Theiler's (NeST)!

murine locus"NeST!

human homologous region"IFNG"IL22" NEST!

Fig. 9 Human homology of NeST!

Literature !Fiette L, Aubert C, Muller U, Huang S, Aguet M, Brahic M, Bureau

J F. Theiler’s virus infection of 129Sv mice that lack the interferon α/β or interferon-γ receptors, Journal of Experimental Medicine 1995; 181: 2069-2076.

Holgado E M, Vela J M, Martin A A, Guaza C. LPS/IFN-y cytotoxicity in oligodendroglial cells: role of nitric oxide and protection by the anti-inflammatory cytokine IL-10. [abstract] European Journal of Neuroscience. 2001;13(3):493-502.

Vigneau S, Rohrlich P S, Brahic M, Bureau J F. Tmevpg1, a Candidate gene for the control of theiler’s virus persistence, could be implicated in the regulation of gamma interferon, Journal of Virology 2003; 77 (10): 5632-5638.

Fig. 8 Levels of NeST and IFN-γ  RNA  during persistent phase !

p: N.S.!p = 0.0003!