Junsong Zhang

Cellular microRNAs up-regulate transcription via interaction with promoter TATA-box motifs

The TATA box represents one of the most prevalent core promoters where the pre-initiation complexes (PICs) for gene transcription are assembled. This assembly is crucial for transcription initiation and well regulated. Here we show that some cellular microRNAs (miRNAs) are associated with RNA polymerase II (Pol II) and TATA box-binding protein (TBP) in human peripheral blood mononuclear cells (PBMCs). Among them, let-7i sequence specifically binds to the TATA-box motif of interleukin-2 (IL-2) gene and elevates IL-2 mRNA and protein production in CD4(+) T-lymphocytes in vitro and in vivo. Through direct interaction with the TATA-box motif, let-7i facilitates the PIC assembly and transcription initiation of IL-2 promoter. Several other cellular miRNAs, such as mir-138, mir-92a or mir-181d, also enhance the promoter activities via binding to the TATA-box motifs of insulin, calcitonin or c-myc, respectively. In agreement with the finding that an HIV-1-encoded miRNA could enhance viral replication through targeting the viral promoter TATA-box motif, our data demonstrate that the interaction with core transcription machinery is a novel mechanism for miRNAs to regulate gene expression.

Glycopeptide Antibiotics Potently Inhibit Cathepsin L in the Late Endosome/Lysosome and Block the Entry of Ebola Virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)

Ebola virus infection can cause severe hemorrhagic fever with a high mortality in humans. The outbreaks of Ebola viruses in 2014 represented the most serious Ebola epidemics in history and greatly threatened public health worldwide. The development of additional effective anti-Ebola therapeutic agents is therefore quite urgent. In this study, via high throughput screening of Food and Drug Administration-approved drugs, we identified that teicoplanin, a glycopeptide antibiotic, potently prevents the entry of Ebola envelope pseudotyped viruses into the cytoplasm. Furthermore, teicoplanin also has an inhibitory effect on transcription- and replication-competent virus-like particles, with an IC50 as low as 330 nm. Comparative analysis further demonstrated that teicoplanin is able to block the entry of Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) envelope pseudotyped viruses as well. Teicoplanin derivatives such as dalbavancin, oritavancin, and telavancin can also inhibit the entry of Ebola, MERS, and SARS viruses. Mechanistic studies showed that teicoplanin blocks Ebola virus entry by specifically inhibiting the activity of cathepsin L, opening a novel avenue for the development of additional glycopeptides as potential inhibitors of cathepsin L-dependent viruses. Notably, given that teicoplanin has routinely been used in the clinic with low toxicity, our work provides a promising prospect for the prophylaxis and treatment of Ebola, MERS, and SARS virus infection.

Host Protein Moloney Leukemia Virus 10 (MOV10) Acts as a Restriction Factor of Influenza A Virus by Inhibiting the Nuclear Import of the Viral Nucleoprotein

ABSTRACT The viral ribonucleoprotein (vRNP) complex of influenza A viruses (IAVs) contains an RNA-dependent RNA polymerase complex (RdRp) and nucleoprotein (NP) and is the functional unit for viral RNA transcription and replication. The vRNP complex is an important determinant of virus pathogenicity and host adaptation, implying that its function can be affected by host factors. In our study, we identified host protein Moloney leukemia virus 10 (MOV10) as an inhibitor of IAV replication, since depletion of MOV10 resulted in a significant increase in virus yield. MOV10 inhibited the polymerase activity in a minigenome system through RNA-mediated interaction with the NP subunit of vRNP complex. Importantly, we found that the interaction between MOV10 and NP prevented the binding of NP to importin-α, resulting in the retention of NP in the cytoplasm. Both the binding of MOV10 to NP and its inhibitory effect on polymerase activity were independent of its helicase activity. These results suggest that MOV10 acts as an anti-influenza virus factor through specifically inhibiting the nuclear transportation of NP and subsequently inhibiting the function of the vRNP complex. IMPORTANCE The interaction between the influenza virus vRNP complex and host factors is a major determinant of viral tropism and pathogenicity. Our study identified MOV10 as a novel host restriction factor for the influenza virus life cycle since it inhibited the viral growth rate. Conversely, importin-α has been shown as a determinant for influenza tropism and a positive regulator for viral polymerase activity in mammalian cells but not in avian cells. MOV10 disrupted the interaction between NP and importin-α, suggesting that MOV10 could also be an important host factor for influenza virus transmission and pathogenicity. Importantly, as an interferon (IFN)-inducible protein, MOV10 exerted a novel mechanism for IFNs to inhibit the replication of influenza viruses. Furthermore, our study potentially provides a new drug design strategy, the use of molecules that mimic the antiviral mechanism of MOV10.

RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle.

Development of an Attenuated Tat Protein as a Highly-effective Agent to Specifically Activate HIV-1 Latency

Although combined antiretroviral therapy (cART) successfully decreases plasma viremia to undetectable levels, the complete eradication of human immunodeficiency virus type 1 (HIV-1) remains impractical because of the existence of a viral reservoir, mainly in resting memory CD4+ T cells. Various cytokines, protein kinase C activators, and histone deacetylase inhibitors (HDACi) have been used as latency-reversing agents (LRAs), but their unacceptable side effects or low efficiencies limit their clinical use. Here, by a mutation accumulation strategy, we generated an attenuated HIV-1 Tat protein named Tat-R5M4, which has significantly reduced cytotoxicity and immunogenicity, yet retaining potent transactivation and membrane-penetration activity. Combined with HDACi, Tat-R5M4 activates highly genetically diverse and replication-competent viruses from resting CD4+ T lymphocytes isolated from HIV-1-infected individuals receiving suppressive cART. Thus, Tat-R5M4 has promising potential as a safe, efficient, and specific LRA in HIV-1 treatment.

Complex reassortment of polymerase genes in Asian influenza A virus H7 and H9 subtypes

The epidemic first caused by a novel H7N9 avian influenza A virus (IAV) has emerged in China recently. Meanwhile, a novel H7N7 IAV with the ability to infect mammals was also found in China. Both IAVs of H7 subtype possess internal genes originating from H9N2. As internal polymerase genes play a key role for interspecies transmission of IAVs, it is important to trace the reassortment history of polymerase genes in the IAVs of H7 and H9 subtypes. Here, by comprehensive phylogenetic analyses of Asian H7 and H9 polymerases, we showed a significant incongruence among the tree topologies of polymerase genes PA, PB1 and PB2, which suggested frequent intra-subtype reassortments in the IAVs of H9N2. Moreover, the PA gene of H1N1pdm09 clustered with that of H9N2 located at the basal position of clade A, including most strains isolated from mammals and the recent novel H7N9 in the phylogenetic tree of PA. This finding indicated that the H1N1pdm09-like PA gene may play an important role in the human H7N9 epidemic. Results also showed that the earlier strains of H7 subtype were divided into several clusters dispersed within the strains of H9N2, implying multiple direct and/or indirect reassortments may occur between H7 and H9 polymerase genes. Furthermore, the most recent reassortments occurred multiply on the polymerase genes of the newly emerging H7N9 isolated from human in South China, evolving E627K mutation in PB2 independently. These results suggest that the reassortment history of polymerase genes in Asian IAVs of H7 and H9 subtypes is complex and timely evolutionary analyses on the novel H7N9 with newly adapted polymerase are necessary for preventing a potential outbreak in South China.

MicroRNA Expression Profile of Whole Blood Is Altered in Adenovirus-Infected Pneumonia Children

Human adenovirus (Adv) infection is responsible for most community-acquired pneumonia in infants and children, which results in significant morbidity and mortality in children every year. MicroRNAs (miRNAs) are associated with viral replication and host immune response. Knowing the miRNA expression profile will help understand the role of miRNAs in modulating the host response to adenovirus infection and possibly improve the diagnosis of adenovirus-infected pneumonia. In our study, total RNA extracted from whole blood of adenovirus-infected pneumonia children and healthy controls were analyzed by small RNA deep sequencing. Expression profiles of whole blood microRNAs were altered and distinctly different in adenovirus-infected children. The top 3 upregulated miRNA (hsa-miR-127-3p, hsa-miR-493-5p, and hsa-miR-409-3p) were identified in adenovirus-infected children and provided a clear distinction between infected and healthy individuals. Potential host target genes were predicated and validated by qRT-PCR to study the impact of microRNAs on the host genes. Most of the target genes were involved in the MAPK signaling pathway and innate immune response. These highly upregulated microRNAs may have crucial roles in Adv pathogenesis and are potential biomarkers for adenovirus-infected pneumonia.

Identification of a novel compound targeting the nuclear export of influenza A virus nucleoprotein

Although antiviral drugs are available for the treatment of influenza infection, it is an urgent requirement to develop new antiviral drugs regarding the emergence of drug‐resistant viruses. The nucleoprotein (NP) is conserved among all influenza A viruses (IAVs) and has no cellular equivalent. Therefore, NP is an ideal target for the development of new IAV inhibitors. In this study, we identified a novel anti‐influenza compound, ZBMD‐1, from a library of 20,000 compounds using cell‐based influenza A infection assays. We found that ZBMD‐1 inhibited the replication of H1N1 and H3N2 influenza A virus strains in vitro, with an IC50 ranging from 0.41–1.14 μM. Furthermore, ZBMD‐1 inhibited the polymerase activity and specifically impaired the nuclear export of NP. Further investigation indicated that ZBMD‐1 binds to the nuclear export signal 3 (NES3) domain and the dimer interface of the NP pocket. ZBMD‐1 also protected mice that were challenged with lethal doses of A/PR/8/1934 (H1N1) virus, effectively relieving lung histopathology changes, as well as strongly inhibiting the expression of pro‐inflammatory cytokines/chemokines, without inducing toxicity effects in mice. These results suggest that ZBMD‐1 is a promising anti‐influenza compound which can be further investigated as a useful strategy against IAVs in the future.

Characteristic amino acid changes of influenza A(H1N1)pdm09 virus PA protein enhance A(H7N9) viral polymerase activity

Human coinfection with a novel H7N9 influenza virus and the 2009 pandemic A(H1N1) influenza virus, H1N1pdm09, has recently been reported in China. Because reassortment can occur during coinfection, it is necessary to clarify the effects of gene reassortment between these two viruses. Among the viral ribonucleoprotein complex (vRNP) genes, only the PA gene of H1N1pdm09 enhances the avian influenza viral polymerase activity. Based on a phylogenetic analysis, we show a special evolutionary feature of the H1N1pdm09 PA gene, which clustered with those of the novel H7N9 virus and related H9N2 viruses, rather than in the outgroup as the H1N1pdm09 genes do on the phylogenetic trees of other vRNP genes. Using a minigenome system of the novel H7N9 virus, we further demonstrate that replacement of its PA gene significantly enhanced its polymerase activity, whereas replacement of the other vRNP genes reduced its polymerase activity. We also show that the residues of PA evolutionarily conserved between H1N1pdm09 and the novel H7N9 virus are associated with attenuated or neutral polymerase activity. The mutations associated with the increased activity of the novel H7N9 polymerase are characteristic of the H1N1pdm09 gene, and are located almost adjacent to the surface of the PA protein. Our results suggest that the novel H7N9 virus has more effective PB1, PB2, and NP genes than H1N1pdm09, and that H1N1pdm09-like PA mutations enhance the novel H7N9 polymerase function.