Experimental systems for the study of KSHV infection

KSHV field has for long suffered from the absence of any physiologically relevant animal model that could be successfully infected by the virus, as no natural host other than human had been reported.

Some studies have been performed in mouse with the goal of generating murine models. Two endothelial cell lines stably carrying the KSHV genome have been generated, the first by infection of telomerase-immortalised HUVECs (TIVE-LTC) and the second by transfection of a KSHV bacterial artificial chromosome (BAC36 - described below) into mouse bone marrow endothelial-lineage cells (mECK36), and have succeeded to generate KSHV-infected tumours when injected into nude mice. It is worth noting that both systems suggest that KSHV tumour formation requires both latent and lytic viral gene expression, in contrasts with classic viral-induced tumours such as EBV-driven lymphoma or human papillomavirus (HPV)-associated cervical cancer, respectively in which lytic or abortive infection plays little, if any, role (An et al., 2006; Mesri et al., 2010; Mutlu et al., 2007). The mECK36 system is of particular interest, as the cell line induces lesions resembling KS in mice, with characteristic viral and host transcriptome. It demonstrates the de novo tumourigenicity of KSHV infection in normal mouse cells, showing that the virus readily provides a survival advantage to cells in vivo (Mutlu et al., 2007). Other attempts, aiming to establish a more physiologically relevant in vivo model of KSHV infection of human cells, have used different types of humanised immunodeficient mice injected intravenously or directly into the implant with purified virus.

These models resulted especially into infection of B-cells, mimicking KSHV natural tropism, with an early phase of lytic replication accompanied and followed by sustained latency, but failed to generate KS-like tumours; no spread to mouse tissue was observed, and no disease state developed in infected animals (Dittmer et al., 1999; Parsons, 2006). Finally, Jones et al.

have recently developed a new interesting mouse model, which pathological and viral features resemble those in KS tumours. Indeed, they succeeded in transforming primary rat embryonic metanephric mesenchymal precursor cells by infecting them with KSHV. In effect, the cells, which are multipotent cells, were reported as being reprogrammed following infection as expressing, in addition to mesenchymal markers, hallmark vascular endothelial and lymphatic endothelial markers. Then, when introduced into nude mice, transformed cells efficiently induced tumours. The resulting tumours often manifested as reddish lesions and dissemination to visceral organs, and consisted of proliferating spindle cells with vast inflammatory infiltrates and neoangiogenesis. KSHV infection of tumourous cells was characterised at the latent stage, with some cells undergoing spontaneous lytic infection (Jones et al., 2012).

Recently in 2009, Chang et al. reported a successful zoonotic transmission of KSHV into the common marmosets Callithrix jacchus, a New World primate. Viral infection was established both through intravenous injection and oral inoculation. Although a lower efficiency was

tumour, with infiltration of leukocytes by spindle cells expressing both latent and lytic viral proteins. This study thus provides the first animal model, which significantly recapitulates the important aspects of KSHV infection in humans, and will permit a better comprehension of the disease’s physiology (Chang et al., 2009).

5.4.2. Cell culture systems

The first successful attempts of cultivation of KSHV-infected cells were performed with PEL cell lines explanted from AIDS-patients; many such cell lines have now been established from EBV-positive and EBV-negative PEL, and they tend to grow readily in cell culture. Although the majority of PEL cells harbour latent KSHV genomes, from which only a small fraction of viral genes are expressed without any viral production, in most experimental culture, 1% to 5% of cells spontaneously display lytic replication, in which the whole genome is expressed and results in the release of infectious virions. The titers of virus particles thus obtained in the culture supernatant is generally low, but can strongly be increased by chemical treatments with drugs such as 12-O-tetradecanoylphorbol-13-acetate (TPA or PMA) phorbol ester, or the histone deacetylase inhibitors sodium butyrate or valproic acid. Following such treatments, 15% to 30% of the cells are switched into lytic replication, and virus stock obtained after purification from induced PEL cells can be used to establish de novo infection in other cell lines or animals. PEL-derived virus stocks have thus allowed the in vitro screening of multiple different cell lines for susceptibility to KSHV infection, and have shown that most de novo infection in vitro results in immediate entry into latency and no lytic growth (reviewed in (Ganem, 2007)).

Although fibroblasts and epithelial cells have not been reliably observed to be infected in vivo, these cell types are readily susceptible to KSHV infection in vitro but display no phenotypic changes on latent infection. By contrast, primary endothelial cell lines, which are also susceptible to in vitro infection, undergo important morphological changes upon KSHV latent infection, such as cell elongation, and rearrangement of their actin cytoskeleton, thus resembling the spindle cells of KS. Early after infection, a substantial amount of lytic infection is observed, and efficient spread of the virus occurs through cells on the dish, in contrast to most other in vitro culture systems, where horizontal transmission has been reported to be relatively poor. Within few days, the viral spread subsides, resulting in a fully latent culture. Importantly, endothelial cells lines are not immortalised by the infection. B-

cells lines, despite their willing susceptibility to infection in vivo, are the most refractory to infection in vitro (reviewed in (Ganem, 2007)).

5.4.3. Genetic analysis

As opposed to the other herpesvirus subfamilies, gammaherpesvirinae are viruses very difficult to genetically engineer by homologous recombination. The critical step seems to be an inefficient rescue of the recombinant viral genome, which may reflect an inefficient virion infectivity and spread. The basis for this inefficiency is unknown, so no rational solution exists to solve the problem. However, a few teams have succeeded with great efforts in the construction of mutant KSHV, by persisting in laborious screening and amplification of many clones. Two successful general strategies have been employed so far (Ganem, 2007).

In 2001, Vieira et al. performed homologous recombination directly by transfection in PEL cells to generate a recombinant KSHV expressing a selectable marker and a green fluorescent protein (GFP) cassette (Vieira et al., 2001). In 2002, Gao’s laboratory has achieved the cloning of KSHV genome from infected BCBL-1 cells by inserting a BAC into the viral genome by homologous recombination. The recombinant BAC plasmid, named BAC36, was then transfected into HEK293 epithelial cell line, and infectious virions produced by induction with TPA. The KSHV-BAC was then showed to efficiently serve for KSHV genetic analysis as the starting point for additional rounds of homologous recombination in Escherichia coli, with the subsequent successful generation of a KSHV deletion mutant (Zhou et al., 2002). However, recent studies have shown that BAC36 contains a 9 kb duplication of the KSHV genome, which is located within the unstable TR region at the extremities of the viral genome. This duplication includes two viral ORFs, between which is actually located the BAC vector backbone of BAC36 that was effectively initially designed to integrate between the latter ORFs, but within their original location in the central region of the KSHV genome.

This duplication, in addition of this TR-localisation of the BAC vector backbone could explain why BAC36 has frequently been observed to undergo homologous recombination events, which result in large deletions of the KSHV genome. To solve this issue, a new KSHV-BAC, BAC16, which shows minimal sequence variations compared to other KSHV strains, was then recently successfully generated from another PEL cell line, and thus can efficiently serve for knockout and recombinants studies on KSHV (Brulois et al., 2012).