Deyin Guo

Towards a protein interaction map of potyviruses: protein interaction matrixes of two potyviruses based on the yeast two-hybrid system.

A map for the interactions of the major proteins from Potato virus A (PVA) and Pea seed-borne mosaic virus (PSbMV) (members of the genus POTYVIRUS:, family POTYVIRIDAE:) was generated using the yeast two-hybrid system (YTHS). Interactions were readily detected with five PVA protein combinations (HC-HC, HC-CI, VPg-VPg, NIa-NIb and CP-CP) and weak but reproducible interactions were detected for seven additional combinations (P1-CI, P3-NIb, NIaPro-NIb, VPg-NIa, VPg-NIaPro, NIaPro-NIa and NIa-NIa). In PSbMV, readily detectable interactions were found in five protein combinations (HC-HC, VPg-VPg, VPg-NIa, NIa-NIa and NIa-NIb) and weaker but reproducible interactions were detected for three additional combinations (P3-NIa, NIa-NIaPro and CP-CP). The self-interactions of HC, VPg, NIa and CP and the interactions of VPg-NIa, NIa-NIaPro and NIa-NIb were, therefore, common for the two potyviruses. The multiple protein interactions revealed in this study shed light on the co-ordinated functions of potyviral proteins involved in virus movement and replication.

Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase

The N7-methylguanosine (m7G) cap is the defining structural feature of eukaryotic mRNAs. Most eukaryotic viruses that replicate in the cytoplasm, including coronaviruses, have evolved strategies to cap their RNAs. In this report, we used a yeast genetic system to functionally screen for the cap-forming enzymes encoded by severe acute respiratory syndrome (SARS) coronavirus and identified the nonstructural protein (nsp) 14 of SARS coronavirus as a (guanine-N7)-methyltransferase (N7-MTase) in vivo in yeast cells and in vitro using purified enzymes and RNA substrates. Interestingly, coronavirus nsp14 was previously characterized as a 3′-to-5′ exoribonuclease, and by mutational analysis, we mapped the N7-MTase domain to the carboxy-terminal part of nsp14 that shows features conserved with cellular N7-MTase in structure-based sequence alignment. The exoribonuclease active site was dispensable but the exoribonuclease domain was required for N7-MTase activity. Such combination of the 2 functional domains in coronavirus nsp14 suggests that it may represent a novel form of RNA-processing enzymes. Mutational analysis in a replicon system showed that the N7-MTase activity was important for SARS virus replication/transcription and can thus be used as an attractive drug target to develop antivirals for control of coronaviruses including the deadly SARS virus. Furthermore, the observation that the N7-MTase of RNA life could function in lieu of that in DNA life provides interesting evolutionary insight and practical possibilities in antiviral drug screening.

Hantavirus nucleocapsid protein interacts with the Fas-mediated apoptosis enhancer Daxx.

Hantaviruses cause two severe diseases, haemorrhagic fever with renal syndrome in Eurasia and hantavirus pulmonary syndrome in the Americas. To understand more about the molecular mechanisms that lead to these diseases, the associations of Puumala virus nucleocapsid protein (PUUV-N) with cellular proteins were studied by yeast two-hybrid screening. Daxx, known as an apoptosis enhancer, was identified from a HeLa cDNA library and its interaction with PUUV-N was confirmed by GST pull-down assay, co-immunoprecipitation and co-localization studies. Furthermore, domains of interaction were mapped to the carboxyl-terminal region of 142 amino acids in Daxx and the carboxyl-terminal 57 residues in PUUV-N, respectively. In pepscan assays, the binding sites of Daxx to PUUV-N were mapped further to two lysine-rich regions, of which one overlaps the sequence of the predicted nuclear localization signal of Daxx. These data suggest a direct link between host cell machinery and a hantavirus structural component.

Genome-Wide Analysis of Protein-Protein Interactions and Involvement of Viral Proteins in SARS-CoV Replication

Analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV) and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp) 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.

Single mutation at the amino acid position 627 of PB2 that leads to increased virulence of an H5N1 avian influenza virus during adaptation in mice can be compensated by multiple mutations at other sites of PB2

To understand the adaptation of H5N1 influenza viruses to mammals, a non-pathogenic influenza H5N1 virus (HN021) in mice was passaged for 15 times in mammalian host. Animal experimental results indicated that the mouse-adapted (MA) variants became highly pathogenic in mice after the passages. Sequence analysis showed that there was one amino acid substitution in PB2 protein of MA mutants after first passage (MA1), three amino acid substitutions in PB2 protein of MA5 and one amino acid in M1 protein, seven amino acids in HA protein and seven amino acids in PB2 protein of MA15, respectively. Animal experiments and growth assays with reassortant viruses produced by reverse genetics showed that mutations in PB2 alone contributed to the increase in virulence of HN021 in mice. Polymerase activity assays showed that the mutations in PB2 enhanced ribonucleoprotein complex polymerase activity in mammalian cells. Interestingly, one reverse mutation (K627E) took place at the amino acid position 627 of PB2 during passages of MA5 to MA15, indicating that a lysine at position 627 of PB2 is not absolutely needed for virulence and adaptation in mice by H5N1 virus. Taken together, the results suggest that mutations at multiple sites of PB2 contributed to the virulence and adaptation in mice, and the E627K mutation of PB2 is not an indispensable determinant in PB2 for mammalian adaptation by H5N1 avian influenza virus.

Inhibition of hepatitis B virus by the CRISPR/Cas9 system via targeting the conserved regions of the viral genome.

Hepatitis B virus (HBV) remains a global health threat as chronic HBV infection may lead to liver cirrhosis or cancer. Current antiviral therapies with nucleoside analogues can inhibit the replication of HBV, but do not disrupt the already existing HBV covalently closed circular DNA. The newly developed CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a powerful tool to target cellular genome DNA for gene editing. In order to investigate the possibility of using the CRISPR/Cas9 system to disrupt the HBV DNA templates, we designed eight guide RNAs (gRNAs) that targeted the conserved regions of different HBV genotypes, which could significantly inhibit HBV replication both in vitro and in vivo. Moreover, the HBV-specific gRNA/Cas9 system could inhibit the replication of HBV of different genotypes in cells, and the viral DNA was significantly reduced by a single gRNA/Cas9 system and cleared by a combination of different gRNA/Cas9 systems.

Biochemical and Structural Insights into the Mechanisms of SARS Coronavirus RNA Ribose 2′-O-Methylation by nsp16/nsp10 Protein Complex

The 5′-cap structure is a distinct feature of eukaryotic mRNAs, and eukaryotic viruses generally modify the 5′-end of viral RNAs to mimic cellular mRNA structure, which is important for RNA stability, protein translation and viral immune escape. SARS coronavirus (SARS-CoV) encodes two S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTase) which sequentially methylate the RNA cap at guanosine-N7 and ribose 2′-O positions, catalyzed by nsp14 N7-MTase and nsp16 2′-O-MTase, respectively. A unique feature for SARS-CoV is that nsp16 requires non-structural protein nsp10 as a stimulatory factor to execute its MTase activity. Here we report the biochemical characterization of SARS-CoV 2′-O-MTase and the crystal structure of nsp16/nsp10 complex bound with methyl donor SAM. We found that SARS-CoV nsp16 MTase methylated m7GpppA-RNA but not m7GpppG-RNA, which is in contrast with nsp14 MTase that functions in a sequence-independent manner. We demonstrated that nsp10 is required for nsp16 to bind both m7GpppA-RNA substrate and SAM cofactor. Structural analysis revealed that nsp16 possesses the canonical scaffold of MTase and associates with nsp10 at 1∶1 ratio. The structure of the nsp16/nsp10 interaction interface shows that nsp10 may stabilize the SAM-binding pocket and extend the substrate RNA-binding groove of nsp16, consistent with the findings in biochemical assays. These results suggest that nsp16/nsp10 interface may represent a better drug target than the viral MTase active site for developing highly specific anti-coronavirus drugs.

SELF-ASSOCIATION AND MAPPING OF INTERACTION DOMAINS OF HELPER COMPONENT-PROTEINASE OF POTATO A POTYVIRUS

Potyviral helper component-proteinase (HC-Pro) is a multifunctional protein involved in aphid transmission, long-distance movement, polyprotein processing, genome amplification and symptom expression. It has been proposed that the active form of HC-Pro is a dimer and that coat protein (CP)-HC-Pro interaction is required for aphid transmission. To test these proposed interactions between CP and HC-Pro of potato A potyvirus (PVA), the yeast two-hybrid system was used. HC-Pro was shown to interact with itself in vivo in yeast cells, as did CP. Taken together with previous observations, we conclude that the functional HC-Pro is a homodimer. Deletion analysis showed that a 24 aa domain in the N-terminal half and the C-terminal proteinase part of HC-Pro were required for the interaction between HC-Pro molecules. No interactions were found between HC-Pro and CP using the genes of aphid-transmissible as well as aphid non-transmissible strains of PVA.

Knockdown of Cellular RNA Helicase DDX3 by Short Hairpin RNAs Suppresses HIV-1 Viral Replication Without Inducing Apoptosis

The targeting of a cellular co-factor, rather than the HIV-1-specific RNAs, by small interfering RNAs holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi against this virus. The DEAD-box RNA helicase DDX3 is an essential Rev co-factor in the CRM1-Rev-RRE complex that promotes the export of unspliced and single-spliced HIV-1 RNAs from the nucleus to cytoplasm. In this report, human DDX3 was targeted by specific short hairpin RNAs, and the down-regulation of cells endogenous DDX3 suppressed the nuclear export of unspliced HIV-1 RNAs but did not affect the cell viability. We further showed that the knockdown of cellular DDX3 could effectively inhibit the replication of HIV-1. Therefore, the current results suggest that the RNA helicase DDX3 may become a potential target by RNAi for future genetic therapy of HIV/AIDS.

Two Potato Proteins, Including a Novel RING Finger Protein (HIP1), Interact with the Potyviral Multifunctional Protein HCpro

Potyviral helper-component proteinase (HCpro) is a multifunctional protein exerting its cellular functions in interaction with putative host proteins. In this study, cellular protein partners of the HCpro encoded by Potato virus A (PVA) (genus Potyvirus) were screened in a potato leaf cDNA library using a yeast two-hybrid system. Two cellular proteins were obtained that interact specifically with PVA HCpro in yeast and in the two in vitro binding assays used. Both proteins are encoded by single-copy genes in the potato genome. Analysis of the deduced amino acid sequences revealed that one (HIP1) of the two HCpro interactors is a novel RING finger protein. The sequence of the other protein (HIP2) showed no resemblance to the protein sequences available from databanks and has known biological functions.