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Expression of Chikungunya virus-like particles
R. A. Velez*, A. P. Alves de Matos**, R. Parreira*, J. Piedade, B. Matos***, C. Correia*** and A.
Esteves*
* Grupo de Virologia, Unidade de Microbiologia Médica, UPMM, Instituto de Higiene e Medicina
Tropical, UNA, 1349-008 Lisboa, Portugal
** Centro Hospitalar de Lisboa Central – HCC, Serviço de Anatomia Patológica, R. da Beneficência
8, 1069-166 Lisboa and Centro de Estudos do Ambiente e do Mar (CESAM) – Faculdade de Ciências
da Universidade de Lisboa, Campo Grande, 1149-016, Lisboa, Portugal
*** Centro Hospitalar de Lisboa Central – HCC, Serviço de Anatomia Patológica, R. da Beneficência
8, 1069-166 Lisboa, Portugal
Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus responsible for painful disabling
polyarthritis, undergoing re-emergence in the Indian Ocean and the Asian continent[1]. Travelling and
changing patterns of vector distribution, and abundance due to climate changes, make CHIKV a global
threat without effective control strategies. One approach to reduce the CHIKV burden is the
development of a vaccine. Virus-like particles (VLPs) are a safe and highly effective class of subunit
vaccines that mimic the overall structure of virus particles. Accordingly, we aim to produce CHIKV
VLPs in a baculovirus-insect cell system, well known for allowing high yields of recombinant protein
expression, to be used as a vaccine preparation.
The ORF coding the CHIKV structural proteins is 3.8 kb long, and therefore there is a chance for
generating recombinant baculoviruses with spurious stop codons leading to truncated polyproteins. To
minimize this possibility, the CHIKV structural sequence was amplified by RT-PCR as a SacI-NotI
fragment, and cloned into the mammalian expression vector pLEXm (a gift from R. Aricescu, Oxford,
UK) downstream the strong chick beta actin promoter. Several clones with the correct insert size were
obtained and transfected into HEK293T cells using polyethylenimine as the transfection reagent[2].
Five recombinant vectors were shown to express CHIKV proteins by immunofluorescence (IFA) and
Western blotting (WB) analyses using a polyclonal serum against CHIKV (Figure 1A). One clone,
with high levels of IFA labelling, coded for a truncated viral polyprotein (WB data), while three
others, with a lower IFA signal, originated low levels of viral envelope glycoproteins correctly
processed. Cells transfected with clone p67 showed IFA staining and viral glycoprotein pattern on WB
similar to cells infected with CHIKV (Figure 1A compare lanes I and p67). Precipitation of clarified
cell culture medium from p67 transfected cells, and from CHIKV infected cells, with PEG 8000
generated pellets with an identical content of viral glycoproteins on WB (Figure 1B). These results
strongly indicate that p67 transfected cells express CHIKV structural proteins that are assembled into
VLPs. Transmission electron microscopy analysis of p67 transfected cells (Figure 3) confirmed the
assembly of VLPs with a morphology similar to the virus particles produced in CHIKV infected cells
(Figure 2). Quite unexpectedly, transfected cells expressing only the CHIKV structural proteins
(Figure 3) exhibit cytoplasmic membrane rearrangements similar to the vesicle arrays observed in
CHIKV infected cells (Figure 2) and reported as induced by non-structural viral proteins. In
conclusion, p67 will be used as a source of CHIKV structural genes for construction of recombinant
baculoviruses.
References
1. Townson H. and Nathan M.B., Trans R Soc Trp Med Hyg 102: 308-309, 2008
2. Aricescu et al., Acta Cryst. D62: 1243-1250, 2006
59
doi:10.1017/S1431927612012950
Microsc. Microanal. 18 (Suppl 5), 2012
© Microscopy Society of America 2012
Figure 1. Western blot analysis of CHIKV proteins. Cells were infected with CHIKV (I) or transfected
with the indicated recombinant vectors. Cell lysates (A) and PEG-pelleted supernatants (B) were
probed with mouse anti-CHIKV polyclonal sera. The predicted viral proteins (E1, E2 and p62) are
indicated. NI, non-infected cells. V, cells transfected with the pLEXm vector alone.
Figure 2. HEK 293T cells infected with CHIKV
for 24h. a) Array of vesicles in the cytoplasm of
the infected cells. b) Extracellular CHIKV
particles (arrows)
Figure 3. HEK 293T cells transfected with
recombinant plasmid clone p67 for 48h. a)
Array of vesicles in the cytoplasm of the
transfected cells. b) Extracellular CHIKV VLPs
(arrows).
A
B
I V
p
28
p
45
p
67
p
76 I NI V
p
28
p
45
p
62
p
67
p
69
p
76
E1/E2
E1/E2
p62
Microsc. Microanal. 18 (Suppl 5), 201260
