Development and in vivo evaluation of MGF100-1R deletion mutant in an African swine fever virus Chinese strain.

African swine fever virus (ASFV) is a large nucleoplasmic DNA virus, in which the genome is around 170-198 kilobases (kb). More than 50 % genes have unknown functions. Here, MGF100-1R gene is chosen to study the primary function and sublocalization. The gene was located at the left variable region of the ASFV genome that belongs to MGF100 families. It located at the cytoplasm without cytotoxic activities. However, it related to induce the transcriptional levels of pro-inflammatory cytokines. A deletion mutant of MGF100-1R gene was constructed based on ASFV Chinese strain GZ201801. The recombinant deletion mutant (ASFV△MGF100-1R) was demonstrated in vitro that the gene is non-essential for virus replication with a similar replication kinetics in bone marrow-derived macrophages (BMDMs) cell cultures when compared to parental virus. In vivo evaluation, ASFV△MGF100-1R was inoculated intramuscularly and led to a similar pathogenesis that caused by the parental ASFV GZ201801, confirming that deletion of MGF100-1R gene from the ASFV genome does not impact virulence.

A novel function of African Swine Fever Virus pE66L in inhibition of host translation by the PKR/eIF2α pathway.

African swine fever virus (ASFV) is one of the most contagious and lethal viruses infecting pigs. This virus is endemic in many countries and has very recently spread to China, but no licensed vaccines or treatments are currently available. Despite extensive research, the basic question of how ASFV-encoded proteins inhibit host translation remains. Here, we examined how ASFV interfered with host translation and optimized viral gene expression. We found that 14 ASFV proteins inhibited Renilla luciferase (Rluc) activity greater than 5-fold, and the protein with the strongest inhibitory effect was pE66L, which was not previously reported. Combined with bioinformatical analysis and biochemical experiment, we determined that the transmembrane (TM) domain (amino acids 13-34) of pE66L was required for the inhibition of host gene expression. Notably, we constructed a recombinant plasmid with the TM domain linked to enhanced green fluorescent protein (EGFP) and further demonstrated that this domain broadly inhibited protein synthesis. Confocal and biochemical analyses indicated that the TM domain might help proteins locate to the endoplasmic reticulum (ER) to suppress translation though the PKR/eIF2α pathway. Deletion of the E66L gene had little effect on virus replication in macrophages, but significantly recovered host gene expression. Taken together, our findings complement studies on the host translation of ASFV proteins and suggest that ASFV pE66L induces host translation shutoff, which is dependent on activation of the PKR/eIF2α pathway.Importance African swine fever virus (ASFV) is a member of the nucleocytoplasmic large DNA virus superfamily that predominantly replicates in the cytoplasm of infected cells. The ASFV double-stranded DNA genome varies in length from approximately 170 to 193 kbp depending on the isolate and contains between 150 and 167 open reading frames (ORFs), of which half the encoded proteins have not been explored. Our study showed that 14 proteins had an obvious inhibitory effect on Renilla luciferase (Rluc) gene synthesis, with pE66L showing the most significant effect. Furthermore, the transmembrane (TM) domain of pE66L broadly inhibited host protein synthesis in a PKR/eIF2a pathway-dependent manner. Loss of pE66L during ASFV infection had little effect on virus replication, but significantly recovered host protein synthetic. Based on the above results, our findings expand our view of ASFV in determining the fate of host-pathogen interactions.