Generation and characterization of a Cowpox virus mutant lacking host range factor CP77

Abstract

Cowpox virus (CPXV) host range factor CP77 was identified to be required for virus replication in Chinese hamster ovary (CHO) cells, but the underlying molecular mechanism by which CP77 modulates host range has remained unclear. Therefore, a CPXVΔCP77 deletion mutant was constructed by applying bacterial artificial chromosome (BAC) technology. Integrity of BAC-derived viral DNA was confirmed by whole genome sequencing. In vitro growth characteristics of CPXV wild type (WT), BAC-derived vCPXV WT and vCPXVΔCP77 were virtually indistinguishable in HEK293T cells, whereas in CHO-K1 cells replication of virus lacking CP77 was unambiguously attenuated. This block of viral replication was confirmed by lack of late viral protein expression. The replication defect of various Orthopoxviruses lacking CP77 in CHO cells could be restored by recombinant expression of CP77. Thus, for the first time, the described CP77-dependent host range effect in CHO cells was shown in the background of CPXV as well as Camelpox virus.

To further characterize the mutant virus, cells of several different species were comparably infected with vCPXV WT and vCPXVΔCP77, respectively. Interestingly, except for CHO-K1 cells, vCPXV WT and vCPXVΔCP77 showed no significant difference in terms of morphology of cytopathic effects, expression of a late transcribed virus-encoded green fluorescent protein and virus reproduction, even in other hamster-derived cells. Additionally, in ovo inoculation with either virus revealed the same red-pock phenotype on chicken egg chorioallantoic membranes.

Since the data presented indicate a CP77-dependent host range effect only for CHO cells, we conclude that the protein might mediate additional functions not identified yet. The vCPXVΔCP77 deletion mutant generated can now be applied as a useful tool to investigate the function of the putative host range protein CP77.

Introduction

An increasing natural emergence of Orthopoxviruses (OPV) in the human population through zoonotic infections is being observed. Outbreaks associated with Vaccinia virus (VACV) in Brazil, Buffalopox virus in India, Monkeypox virus (MPXV) in Africa and the USA and Cowpox virus (CPXV) in Europe (reviewed in Essbauer et al., 2010) cause concern regarding the unvaccinated population and highlight the importance of understanding details of virus–host interaction for a reliable risk assessment.

The genus OPV comprises virus species that can either show a narrow host specificity, like Variola virus (VARV) that exclusively infected humans, or that can infect a broad spectrum of vertebrate species, like MPXV and CPXV. Although complete genomic information is known for more than 100 OPV, so far this varying host range is not understood at the molecular level. Among the published OPV sequences, CPXV have the largest genome and most complete gene repertoire (Gubser et al., 2004, Shchelkunov et al., 1998).

CPXV are of particular interest due to the increasing number of CPXV infections in humans as well as in animals. While no infections of cows have been reported in Germany for decades, rodents are supposed to transmit virus to cats which represent the most common source of human infections (Baxby and Bennett, 1997, Bennett and Begon, 1997). Virus isolates derived from clinical cases are usually unique in sequence and can be distinct in their repertoire of genes responsible for virus–host interaction.

One of the CPXV proteins known to mediate host range is a 77 kDa protein named CP77 or CHOhr which was discovered to permit CPXV replication in CHO cells (Spehner et al., 1988). The protein encoded by the CPXV025 gene (GenBank ID: AAM13472.1) is expressed early in the viral replication cycle and consists of 668 amino acids containing ankyrin repeats. It is well conserved among CPXV, sharing 95% protein identity among 34 CPXV sequences analyzed (partly unpublished), but not well conserved among the OPV. For example, the orthologous gene is deleted in Camelpox virus (CMLV) and truncated in Ectromelia virus (ECTV) and VACV strain Western Reserve, while VARV codes for a shorter 451 amino acid protein with 87% sequence identity with unknown function (http://poxvirus.org). After infection of CHO cells with VACV, a block of viral replication at the stage of intermediate gene translation was observed (Ramsey-Ewing and Moss, 1995). This block of replication is accompanied by a complete shut-off of viral and cellular protein synthesis (Drillien et al., 1978, Hsiao et al., 2004), while viral DNA replication was shown to proceed unhampered (Ramsey-Ewing and Moss, 1995). The host range defect described for VACV and ECTV (both lacking functional CP77 expression) in CHO cells could be complemented by in trans expression of CP77 (Chen et al., 1992, Ramsey-Ewing and Moss, 1995, Spehner et al., 1988). Interestingly, although sharing little amino acid sequence homology, CP77 provides host range functions equivalent to C7L and K1L in some cell lines. In human and porcine cells the host range defect of VACV deleted in C7L and K1L was shown to be restored by expression of CP77 (Perkus et al., 1990, Ramsey-Ewing and Moss, 1996). Similarly, replication of K1L-deleted VACV is permitted by CP77 in RK-13 cells (Chung et al., 1997, Ramsey-Ewing and Moss, 1996). On the other hand, C7L and K1L cannot complement the lack of CP77 for VACV replication in CHO cells.

A potential role of CP77 in NF-κB, protein kinase R and apoptosis signaling as well as its function in regulating viral DNA architecture is discussed in the literature (Chang et al., 2009, Hsiao et al., 2004, Hsiao et al., 2006, Ink et al., 1995, Ramsey-Ewing and Moss, 1998). However, the precise molecular mechanisms enabling CP77 to expand host range could not be solved yet and need to be investigated intensively in the future. Therefore, in the present study a CPXVΔCP77 deletion mutant was constructed by applying bacterial artificial chromosome (BAC) technology.

BACs have first been described by Shizuya et al. (1992) as vectors based on minimal E. coli F-plasmids, providing a high cloning capacity to allow maintenance mapping and sequencing of large genomes. One of the most useful advantages of the BAC system is the possibility of scarless modification of the cloned DNA, like the introduction of frame-shift mutations, point mutations, deletions or insertions of longer sequences. The first full-length virus cloned as a BAC was the mouse cytomegalovirus (Messerle et al., 1997). Since then, several viruses have been cloned and manipulated as BACs, providing the basis for studies of viral gene function and pathogenesis as well as for the development of vaccines and gene therapy approaches (Almazan et al., 2000, Domi and Moss, 2002, Schleiss et al., 2006, Warden et al., 2011).

In the present study a CPXV BAC clone was used for generating a vCPXVΔCP77 deletion mutant which was biologically characterized to elucidate the potential host range function of CP77.