Replication cycle of chikungunya: a re-emerging arbovirus

Abstract

Arboviruses (or arthropod-borne viruses), represent a threat for the new century. The 2005-2006 year unprecedented epidemics of chikungunya virus (CHIKV) in the French Reunion Island in the Indian Ocean, followed by several outbreaks in other parts of the world such as India, have attracted the attention of clinicians, scientists, and state authorities about the risks linked to this re-emerging mosquito-borne virus. CHIKV, which belongs to the Alphaviruses genus, was not previously regarded as a highly pathogenic arbovirus. However, this opinion was challenged by the death of several CHIKV-infected persons in Reunion Island. The epidemic episode began in December 2005 and four months later the seroprevalence survey report indicated that 236,000 persons, more than 30% of Reunion Island population, had been infected with CHIKV, among which 0.4-0.5% of cases were fatal. Since the epidemic peak, the infection case number has continued to increase to almost 40% of the population, with a total of more than 250 fatalities. Although information available on CHIKV is growing quite rapidly, we are still far from understanding the strategies required for the ecologic success of this virus, virus replication, its interactions with its vertebrate hosts and arthropod vectors, and its genetic evolution. In this paper, we summarize the current knowledge of CHIKV genomic organization, cell tropism, and the virus replication cycle, and evaluate the possibility to predict its future evolution. Such understanding may be applied in order to anticipate future epidemics and reduce the incidence by development and application of, for example, vaccination and antiviral therapy.

Fig. 1

A simplified phylogenetic tree of Alphaviruses assuming their New World origin, adapted from Powers et al. (2001). This figure illustrates the evolution of Alphaviruses generated from partial E1 envelope glycoprotein sequence and highlights the evolution of CHIKV. Only 6 of the 7 antigenic complexes are shown because MIDV, which forms an antigenic complex independent from the other using several phylogenetic analyses, fall into the group of CHIKV and other virus from the SF group when phylogenetic tree is deducted from partial E1 nucleotide sequence. Readers are invited to refer to the excellent paper by Powers et al. (2001) for more details. It is worth noting that WEEV arose by recombination between EEEV and the Sindbis virus (Hahn et al., 1988; Weaver et al., 1997). The recombinant virus contains the glycoprotein of the SINV-like parent but the nucleocapsid protein of the EEEV parent.

Fig. 2

Organization of the CHIKV genome and gene products. The CHIKV genome resembles eukaryotic mRNAs in that it possesses 5′ cap structures and 3′ poly(A) tail. The 5′ and 3′ proximal sequences of CHIKV genome carry non-translatable regions (NTR). The junction region (J) is also non-coding. A subgenomic positive-strand mRNA referred to as 26S RNA, is transcribed from a negative-stranded-RNA intermediate and serves as the mRNA for the synthesis of the viral structural proteins. The different non-structural proteins (nsP1–nsP4) and structural proteins (C, Capsid; E1, E2, E3, envelope; 6K) are generated after proteolytic cleavage of polyprotein precursors.

Fig. 3

Schematic representation of repeated sequence elements (RSE) and lengths of 3′-NTRs of CHIKV and related viruses (adapted from Pfeffer et al. (1998)). The downward-pointing arrow indicates the stop codon of the structural E1 glycoprotein gene region. Both the overall lengths of the 3′-NTR and repeats have been drawn to scale. Different types of shading indicate repeats of unique sequence. The open triangles preceding the poly(A) tail indicate the 19-nt that is a highly conserved sequence element (CSE), among Alphaviruses. The organization of the 3′-NTR in CHIKV revealed two copies of an incomplete RSE found in ONNV and three copies of RSE that represent the entire RRV-type RSE and repetitive sequences specific for CHIKV (slight sequence variations, below 10%, are found among the three RSE). The location of I-poly(A) in 3′-NTR of CHIKV is indicated (according to Khan et al. (2002)).

Fig. 4

Representative experiment of analysis of cells sensitivity to CHIKV. (A) Primary macrophages were exposed to 25×TCID₅₀ of 5′CHIKV-EGFP (lower panel). Infected cells were revealed by expression of green fluorescence 24 h after exposure to CHIKV. The upper panel corresponds to uninfected control primary macrophages. DAPI coloration is also shown. (B) Comparative analysis of CHIKV cytopathic effect into A549 and SH-SY5Y cell cultures. Cells were examined by phase contrast using a Leica microscope. SH-SY5Y exposed to CHIKV demonstrated syncytia formation and cell death after 48 h of culture (left, bottom panel) compared to control culture (left, upper panel). No cytopathic effect is evidenced in culture of A549 cells exposed to CHIKV.

Fig. 5

Schematic representation of the capsid protein with the main interaction domains. Adapted after Perera et al. (2001). The conserved ribosome-binding sequence (amino acids 98 to 112) is indicated. The N-terminal domain α-helix is indicated and the corresponding amino acid sequence is shown for CHIKV and related viruses. The approximate location of the RNA-binding domain (Perera et al., 2001) and E2-binding domain (Hahn et al., 1988) are indicated. The amino acid residues (H139, D-145, D161, S213), that are likely to be involved in the serine protease activity are indicated.

Fig. 6

Schematic representation of the non-structural protein nsP2, showing the location of the consensus sequences for the helicase and the proteinase, respectively. The sequence used for numbering is CHIKV sequence (Khan et al., 2002). The proteinase cleavage sites in the nsPs (nsP1/nsP2, nsP2/nsP3, and nsP3/nsP4), are shown in the lower panel (sequence of CHIKV is from Khan et al. (2002); the other sequences are from Strauss and Strauss (1994).

Fig. 7

Schematic representation of the E2 envelope glycoprotein. Adapted after Zhao et al. (1994). The amino acid sequences of the E2 cytoplasmic domain from different viruses are shown. The sequence of CHIKV is from Khan et al. (2002) (NCBI, accession no. AF369024). The Cysteine residues (C) are believed to be palmitoylated and this could serve to anchor the E2-tail at the surface of the inner membrane. The NetPhos sequence analysis (www.Cbs.dtu.dk/services/NetPhos/) predicts that threonines (T) are likely to be phosphorylated (℗) whereas the tyrosine (Y) is probably not. A stretch of highly conserved residues TP, is characteristic of an ERK2 phosphorylation site.

Fig. 8

HEK-293T cells (A and B) chronically infected with CHIKV were fixed with glutaraldehyde 48 h after infection and processed for thin-layer electron microscopy. Panel A, shows evidence of budding and the presence of an electron-dense mature particle (white arrow). Most of the particles (size ranging from 45 to 80 nm) are characterized by low density material bordered with a membrane bilayer (black arrow). The bar corresponds to 100 nm. Panel B illustrates the budding of larges particles with low density material at the cell membrane (black arrow). The bar corresponds to 100 nm. Similar experiments were performed using BHK-21 cells (C, D, and E) chronically infected with CHIKV. Panel C shows the presence of several electron-dense particles budding at the cell surface. The bar corresponds to 100 nm. Panel D, illustrated the massive production of particles from BHK-21 cells. The bar corresponds to 90 nm. Two different types of particles can be observed, viral particles of about 65 nm with an electron-dense capsid of 32 nm (white arrow) and particles of about 76 nm showing only a small electron-dense material in the middle very similar to the most frequent particles observed in CHIKV-infected HEK-293 T cells. Panel E illustrates one example of the presence of CHIKV particles within cellular vacuoles. Particles found within the endosomes appear to be electron-dense. The bar corresponds to 300 nm.

Fig. 9

Summary of the CHIKV replication cycle. The different steps of the CHIKV replication cycle are described in detail in this review.