1. 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 AA, 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!