Possible functional co-operation of palindromes hr3 and hr4 in the genome of Cydia pomonella granulovirus affects viral replication capacity

After previous studies had shown that natural transposon insertion between the two homologous regions hr3 and hr4 of the genome of the Mexican (M) strain of Cydia pomonella granulovirus (CpGV-M) resulted in a loss of viral competitiveness, the function of these homologous regions was investigated. A CpGV-based bacmid (CpBAC) was constructed and mutants with deleted hr3 and hr4 palindromes (CpBAChr3/hr4KO) and a construct (CpBAChr3-kan-hr4) with physically separated hr3 and hr4 repeats were generated to investigate their involvement in in vivo replication. Based on median lethal concentration (LC50) and median survival time (ST50) of the mutant viruses vCpBAChr3/hr4KO and vCpBAChr3-kan-hr4 it was found that the infectivity of both mutants for codling moth Cydia pomonella L. (Lep.: Tortricidae) larvae was not influenced compared with the parental virus vCpBAC. Co-infection experiments with vCpBAChr3-kan-hr4 and vCpBAC using different virus ratios revealed that vCpBAChr3-kan-hr4 was efficiently out-competed by vCpBAC during in vivo replication. These findings suggested that the separation of hr3 and hr4 resulted in a replication disadvantage of the mutant similar to the observation made in previous co-infection experiments using the transposon-carrying mutant CpGV-MCp5 and WT CpGV-M. It was concluded that the palindromes hr3 and hr4 may play a non-essential but co-functional role in the replication of CpGV-M.