Interaction of chikungunya virus glycoproteins with macrophage factors controls virion production.

Despite their role as innate sentinels, macrophages can serve as cellular reservoirs of chikungunya virus (CHIKV), a highly-pathogenic arthropod-borne alphavirus that has caused large outbreaks among human populations. Here, with the use of viral chimeras and evolutionary selection analysis, we define CHIKV glycoproteins E1 and E2 as critical for virion production in THP-1 derived human macrophages. Through proteomic analysis and functional validation, we further identify signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 subunit K (eIF3k) as E1-binding host proteins with anti-CHIKV activities. We find that E1 residue V220, which has undergone positive selection, is indispensable for CHIKV production in macrophages, as its mutation attenuates E1 interaction with the host restriction factors SPCS3 and eIF3k. Finally, we show that the antiviral activity of eIF3k is translation-independent, and that CHIKV infection promotes eIF3k translocation from the nucleus to the cytoplasm, where it associates with SPCS3. These functions of CHIKV glycoproteins late in the viral life cycle provide a new example of an intracellular evolutionary arms race with host restriction factors, as well as potential targets for therapeutic intervention.

A CRISPR Activation Screen Identifies an Atypical Rho GTPase That Enhances Zika Viral Entry.

Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR-Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host-pathogen interactions.

Label-Free Proteomic Analysis of Exosomes Secreted from THP-1-Derived Macrophages Treated with IFN-α Identifies Antiviral Proteins Enriched in Exosomes.

Exosomes are extracellular vesicles that function in intercellular communication. We have previously reported that exosomes play an important role in the transmission of antiviral molecules during interferon-α (IFN-α) treatment. In this study, the protein profiles of THP-1-derived macrophages with or without interferon-α treatment and the exosomes secreted from these cells were analyzed by label-free liquid chromatography-tandem mass spectrometry quantitation technologies. A total of 1845 and 1550 protein groups were identified in the THP-1 macrophages and the corresponding exosomes, respectively. Treating the cells with IFN-α resulted in the differential abundance of 94 proteins in cells and 67 proteins in exosomes (greater than 2.0-fold), among which 23 proteins were up-regulated in both the IFN-α treated cells and corresponding exosomes, while 14 proteins were specifically up-regulated in exosomes but not in the donor cells. GO and KEGG analysis of the identified proteins suggested that IFN-α promoted the abundance of proteins involved in the "defense response to virus" and "type I interferon signaling pathway" in both exosomes and cells. Functional analysis further indicated that exosomes from IFN-α-treated cells exhibited potent antiviral activity that restored the impaired antiviral response of IFN-α in hepatitis B virus-replicating hepatocytes. These results have deepened the understanding of the exosome-mediated transfer of IFN-α-induced antiviral molecules and may provide a new basis for therapeutic strategies to control viral infection.

Exosomes Exploit the Virus Entry Machinery and Pathway To Transmit Alpha Interferon-Induced Antiviral Activity.

Alpha interferon (IFN-α) induces the transfer of resistance to hepatitis B virus (HBV) from liver nonparenchymal cells (LNPCs) to hepatocytes via exosomes. However, little is known about the entry machinery and pathway involved in the transmission of IFN-α-induced antiviral activity. In this study, we found that macrophage exosomes uniquely depend on T cell immunoglobulin and mucin receptor 1 (TIM-1), a hepatitis A virus (HAV) receptor, to enter hepatocytes for delivering IFN-α-induced anti-HBV activity. Moreover, two primary endocytic routes for virus infection, clathrin-mediated endocytosis (CME) and macropinocytosis, collaborate to permit exosome entry and anti-HBV activity transfer. Subsequently, lysobisphosphatidic acid (LBPA), an anionic lipid closely related to endosome penetration of virus, facilitates membrane fusion of exosomes in late endosomes/multivesicular bodies (LEs/MVBs) and the accompanying exosomal cargo uncoating. Together, our findings provide comprehensive insights into the transmission route of macrophage exosomes to efficiently deliver IFN-α-induced antiviral substances and highlight the similarities between the entry mechanisms of exosomes and virus.IMPORTANCE Our previous study showed that LNPC-derived exosomes could transmit IFN-α-induced antiviral activity to HBV replicating hepatocytes, but the concrete transmission mechanisms, which include exosome entry and exosomal cargo release, remain unclear. In this study, we found that virus entry machinery and pathway were also applied to exosome-mediated cell-to-cell antiviral activity transfer. Macrophage-derived exosomes distinctively exploit hepatitis A virus receptor for access to hepatocytes. Later, CME and macropinocytosis are utilized by exosomes, followed by exosome-endosome fusion for efficient transfer of IFN-α-induced anti-HBV activity. We believe that understanding the cellular entry pathway of exosomes will be beneficial to designing exosomes as efficient vehicles for antiviral therapy.