Site-directed mutagenesis of the VP2 gene of Chicken anemia virus affects virus replication, cytopathology and host-cell MHC class I expression

Abstract

Chicken anemia virus (CAV) is an immunosuppressive pathogen of chickens. To further examine the role of viral protein 2 (VP2), which possesses dual-specificity protein phosphatase (DSP) activity, in viral cytopathogenicity and its influence on viral growth and virulence, an infectious genomic clone of CAV was subjected to site-directed mutagenesis. Substitution mutations C87R, R101G, K102D and H103Y were introduced into the DSP catalytic motif and R129G, Q131P, R/K/K150/151/152G/A/A, D/E161/162G/G, L163P, D169G and E186G into a region predicted to have a high degree of secondary structure. All mutant constructs were infectious, but their growth curves differed. The growth curve for mutant virus R/K/K150/151/152G/A/A was similar to that for wild-type virus, a second cluster of mutant viruses had an extended latent period and a third cluster of mutant viruses had extended latent and eclipse periods. All mutants had a reduced cytopathogenic effect in infected cells and VP3 was restricted to the cytoplasm. Mutation of the second basic residue (K102D) in the atypical DSP signature motif resulted in a marked reduction in virus replication efficiency, whereas mutation of the first basic residue (R101G) attenuated cytopathogenicity, but did not reduce replication efficiency. Expression of major histocompatibility complex (MHC) class I was markedly downregulated in cells infected with wild-type CAV, but not in those infected with mutants. This study further demonstrates the significance of VP2 in CAV replication and shows that specific mutations introduced into the gene encoding this protein can reduce virus replication, cytopathogenicity and downregulation of MHC I in infected cells.

Avian Immunosuppressive Diseases and Immunoevasion

“…In contrast to the effect on CTL, natural killer (NK) cells were not affected by CIAV infection [81]. Based on their studies of CIAV infection in MSB-1 cells, Peters et al [82] suggested that VP2 may also play a role in immunosuppression through down-regulation of major histocompatibility complex (MHC) class I antigens. The importance of this observation for immunosuppression is difficult to evaluate because the assumption is that CIAV-infected cells will become apoptotic.…”

Avian Immunosuppressive Diseases and Immune Evasion

“…In contrast to the effect on CTL, natural killer (NK) cells were not affected by CIAV infection (Markowski-Grimsrud and . Based on the studies in MSB-1 cells, Peters et al (2006) recently suggested that VP2 may also play a role in immunosuppression through downregulation of major histocompatibility complex (MHC) class I antigens. The importance of this observation for immunosuppression is difficult to evaluate because the assumption is that CIAV-infected cells will become apoptotic.…”

Viral Manipulation of Cell Death

“…The CAV genome is circular and ~2.4Kb in length, encoding three distinct viral proteins from a single transcribed mRNA containing 3 overlapping ORF's. VP3, renamed apoptin for its ability to promote apoptosis in transformed and tumorigenic cell lines, is the primary cause of the CAV's deleterious effects [98][99][100]. VP2, a dual-specificity protein phosphatase, is essential for efficient viral replication and has mild apoptotic effects [99].…”

“…Apoptin has been shown to induce apoptosis in a variety of tumorigenic cell lines, but normal cells are spared from death. In non-transformed cells, threonine 108 is not phosphorylated and apoptin segregates to the cytoplasm, preventing induction of apoptosis [100]. Tumorigenic or transformed cells, however, target apoptin to the nucleus [102], where it initiates an unknown series of events leading to apoptosis.…”