African swine fever virus (ASFV), a devastating pathogen to the worldwide swine industry, mainly targets macrophage/monocyte lineage, but how the virus enters host cells has remained unclear. Here, we report that ASFV utilizes apoptotic bodies (ApoBDs) for infection and cell–cell transmission. We show that ASFV induces cell apoptosis of primary porcine alveolar macrophages (PAMs) at the late stage of infection to productively shed ApoBDs that are subsequently swallowed by neighboring PAMs to initiate a secondary infection as evidenced by electron microscopy and live-cell imaging. Interestingly, the virions loaded within ApoBDs are exclusively single-enveloped particles that are devoid of the outer layer of membrane and represent a predominant form produced during late infection. The in vitro purified ApoBD vesicles are capable of mediating virus infection of naive PAMs, but the transmission can be significantly inhibited by blocking the “eat-me” signal phosphatidyserine on the surface of ApoBDs via Annexin V or the efferocytosis receptor TIM4 on the recipient PAMs via anti-TIM4 antibody, whereas overexpression of TIM4 enhances virus infection. The same treatment however did not affect the infection by intracellular viruses. Importantly, the swine sera to ASFV exert no effect on the ApoBD-mediated transmission but can partially act on the virions lacking the outer layer of membrane. Thus, ASFV has evolved to hijack a normal cellular pathway for cell–cell spread to evade host responses.

Protein kinase R (PKR) is a critical host restriction factor against invading viral pathogens. However, this molecule is inactivated in the cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), an economically devastating pathogen to the world swine industry. Here, we report that this event is to suppress cellular inflammation and is mediated by the viral replicase protein nsp1β. We show that nsp1β is a stress-responsive protein, enters virus-induced stress granules (SGs) during infection, and repurposes SGs into a proviral platform, where it co-opts the SG core component G3BP1 to interact with PKR in a regulated manner. RNA interference silencing of G3BP1 or mutation of specific nsp1β residues (VS19GG) can abolish the antagonization of PKR activation. The viral mutant carrying the corresponding mutations induces elevated level of PKR phosphorylation and pronounced production of inflammatory cytokines (e.g., tumor necrosis factor-α, interleukin [IL]-6, and IL-8), whereas small-interfering RNA knockdown of PKR or treatment with C16, a PKR inhibitor, blocks this effect. Thus, PRRSV has evolved a unique strategy to evade PKR restriction to suppress host inflammatory responses.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been a major threat to the world swine industry. In the field, rapid genetic variations (e.g., deletion, mutation, recombination, etc.) within the nsp2 region present an intriguing conundrum to PRRSV biology and pathogenesis.

The papain-like cysteine protease 2 (PLP2) within the N-terminus of the porcine reproductive and respiratory syndrome virus (PRRSV) nsp2 replicase protein specifies a deubiquitinating enzyme (DUB), but its biochemical properties and the role in infection have remained poorly defined. By using in vitro assays, we found that the purified PLP2 could efficiently cleave K63 and K48 linked polyubiquitin chains Ub3-7 in vitro although displaying a differential activity in converting the respective ubiquitin dimers to monomer. The subsequent mutagenesis analyses revealed that the requirement for PLP2 DUB activity surprisingly resembled that for cis-cleavage activity, as several mutations (e.g., D91R, D85R, etc.) that largely ablated the DUB function also blocked the cis- but not trans-proteolytic cleavage of nsp2/3 polyprotein. Moreover, the analyses identified key mutations that could differentiate DUB from PLP2 cis- and trans-cleavage activities. Further reverse genetics analyses revealed the following findings: (i) mutations that largely blocked the DUB activity were all lethal to the virus, (ii) a point mutation T88G that selectively blocked the cis-cleavage activity of PLP2 did not affect viral viability in cell culture, and (iii) an E90Q mutation that did not affect either of the PLP2 activities led to rescue of WT-like virus but displayed significantly reduced ability to induce TNF-α production. Our findings support the possibility that the PLP2 DUB activity, but not cis-cleavage activity, is essential for PRRSV replication. The data also establish a strong link of nsp2 to pro-inflammatory cytokine induction during infection that operates in a manner independent of PLP2 DUB activity.

As a member of nucleocytoplasmic large DNA viruses, African swine fever virus (ASFV) has been proposed to include an intriguing nuclear stage for DNA replication. In this report, we revisited this early event by using the ASFV type II strain HN09 as a model organism. Our studies began with dissecting the early stages of the ASFV life cycle via analyses by viral growth kinetics, electron microscopy, and quantitative PCR (qPCR). We showed that the single replication cycle lasted about 9 h, with the initial early mRNA transcription taking place around 0.5 h upon virus incubation with host cells, a timing that is much earlier than previously found; the viral DNA replication occurred around 4–5 hours post-infection (hpi), coinciding with the formation of early viral factory (VF), followed by virion morphogenesis at 6–9 hpi. The time-course tracking of viral genomic and newly synthesized DNA via EdU (5-ethynyl-2-deoxyuridine) labeling and DNAscope in situ hybridization combined with 3D reconstruction revealed that the viral DNA remained all the time in the cytoplasm. Further transcriptional studies by RNAscope targeting three early mRNAs ( CP204L , F334L , and H359L ) and EU labeling of nascent viral RNAs revealed similar results regarding their localization. In line with these observations, disruption of the nucleocytoplasmic shuttling with either small interfering RNA (siRNA) or chemical inhibitor did not affect ASFV replication. In contrast, the replication of porcine pseudorabies virus (PRV) was significantly reduced. Thus, we provide strong evidence to suggest that ASFV DNA replication does not involve a nuclear stage. Our findings provide great insight into the replication biology of ASFV. IMPORTANCE: African swine fever virus (ASFV) represents a devastating threat to the global swine industry. This virus has a large genomic size of 170 to 200 kb with a complex virion structure, but how this virus coordinates transcription/replication cascades has remained poorly defined. By using modern techniques, including EdU (5-ethynyl-2-deoxyuridine) and EU labeling, DNAscope and RNAscope, 3D reconstruction, and RNA interference (RNAi), we provide compelling evidence to show that the ASFV life cycle does not involve a nuclear stage, with both viral transcription and DNA replication confined to the cell cytoplasm. Our findings provide important insight into ASFV replication biology and into seeking targets for antiviral drug development.

The autophagy–lysosome axis is an evolutionarily conserved intracellular degradation pathway which constitutes an important component of host innate immunity against microbial infections. Here, we show that African swine fever virus (ASFV), one of most devastating pathogens to the worldwide swine industry, can reshape the autophagy–lysosome axis by recruiting the critical lysosome membrane proteins (LAMP1 and LAMP2) to viral factories while inhibiting autophagic induction in macrophages. The screening of viral membrane proteins led to the identification of several ASFV membrane proteins, exemplified by viral protein pEP153R, that could significantly alter the subcellular localization of LAMP1/2 when expressed alone in transfected cells. Further analysis showed that pEP153R was also a component of viral factories and could induce endoplasmic reticulum (ER) retention of LAMP1/2, leading to the inhibition of the fusion of autophagosomes with lysosomes. Interestingly, the ASFV mutant lacking EP153R could still actively recruit LAMP into viral factories (VFs) and inhibit autophagic flux, indicating the existence of a functional redundancy of other viral proteins in the absence of pEP153R and highlighting the complexity of ASFV replication biology. Taken together, our results reveal novel information about the interplay of ASFV with the autophagy–lysosome axis and a previously unrecognized function of ASFV protein pEP153R in regulating the cellular autophagic process.