Deletion of the H240R Gene of African Swine Fever Virus Decreases Infectious Progeny Virus Production Due to Aberrant Virion Morphogenesis and Enhances Inflammatory Cytokine Expression in Porcine Macrophages

African swine fever is a lethal hemorrhagic disease of global concern that is caused by African swine fever virus (ASFV). Despite extensive research, there exist relevant gaps in knowledge of the fundamental biology of the viral life cycle. ABSTRACT African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus that causes African swine fever, a lethal hemorrhagic disease that currently threatens the pig industry. Recent studies have identified the viral structural proteins of infectious ASFV particles. However, the functional roles of several ASFV structural proteins remain largely unknown. Here, we characterized the function of the ASFV structural protein H240R (pH240R) in virus morphogenesis. pH240R was identified as a capsid protein by using immunoelectron microscopy and interacted with the major capsid protein p72 by pulldown assays. Using a recombinant ASFV, ASFV-ΔH240R, with the H240R gene deleted from the wild-type ASFV (ASFV-WT) genome, we revealed that the infectious progeny virus titers were reduced by approximately 2.0 logs compared with those of ASFV-WT. Furthermore, we demonstrated that the growth defect was due to the generation of noninfectious particles with a higher particle-to-infectious titer ratio in ASFV-ΔH240R-infected primary porcine alveolar macrophages (PAMs) than in those infected with ASFV-WT. Importantly, we found that pH240R did not affect virus-cell binding, endocytosis, or egress but did affect ASFV assembly; noninfectious virions containing large aberrant tubular and bilobulate structures comprised nearly 98% of all virions observed in ASFV-ΔH240R-infected PAMs by electron microscopy. Notably, we demonstrated that ASFV-ΔH240R infection induced high-level expression of inflammatory cytokines in PAMs. Collectively, we show for the first time that pH240R is essential for ASFV icosahedral capsid formation and infectious particle production. Also, these results highlight the importance of pH240R in ASFV morphogenesis and provide a novel target for the development of ASF vaccines and antivirals. IMPORTANCE African swine fever is a lethal hemorrhagic disease of global concern that is caused by African swine fever virus (ASFV). Despite extensive research, there exist relevant gaps in knowledge of the fundamental biology of the viral life cycle. In this study, we identified pH240R as a capsid protein that interacts with the major capsid protein p72. Furthermore, we showed that pH240R was required for the efficient production of infectious progeny virions as indicated by the H240R-deleted ASFV mutant (ASFV-ΔH240R). More specifically, pH240R directs the morphogenesis of ASFV toward the icosahedral capsid in the process of assembly. In addition, ASFV-ΔH240R infection induced high-level expression of inflammatory cytokines in primary porcine alveolar macrophages. Our results elucidate the role of pH240R in the process of ASFV assembly, which may instruct future research on effective vaccines or antiviral strategies.