Combining hybrid nanoflowers with hybridization chain reaction for highly sensitive detection of SARS-CoV-2 nucleocapsid protein.

BACKGROUND: COVID-19 (coronavirus disease 2019) pandemic has had enormous social and economic impacts so far. The nucleocapsid protein (N protein) is highly conserved and is a key antigenic marker for the diagnosis of early SARS-CoV-2 infection. RESULTS: In this study, the N protein was first captured by an aptamer (Aptamer 58) coupled to magnetic beads (MBs), which in turn were bound to another DNA sequence containing the aptamer (Aptamer 48-Initiator). After adding 5'-biotinylated hairpin DNA Amplifier 1 and Amplifier 2 with cohesive ends for complementary hybridization, the Initiator in the Aptamer 48-Initiator began to trigger the hybridization chain reaction (HCR), generating multiple biotin-labeled DNA concatamers. When incubated with synthetic streptavidin-invertase-Ca3(PO4)2 hybrid nanoflower (SICa), DNA concatamers could specifically bind to SICa through biotin-streptavidin interaction with high affinity. After adding sucrose, invertase in SICa hydrolyzed sucrose to glucose, whose concentration could be directly read with a portable glucometer, and its concentration was positively correlated with the amount of captured N protein. The method is highly sensitive with a detection limit as low as 1 pg/mL. SIGNIFICANCE: We believe this study provided a practical solution for the early detection of SARS-CoV-2 infection, and offered a new method for detecting other viruses through different target proteins.

Forsythoside a inhibits the avian infectious bronchitis virus in cell culture.

Forsythoside A is a polyphenolic constituent of the fruits of Forsythia suspensa Vahl. which is widely used as an antiinflammatory agent in traditional Chinese medicine. In the present study, the effects of forsythoside A on cell infection by avian infectious bronchitis virus were assessed. A real-time fluorescence quantitative PCR assay was used to determine mRNA content of IBV N gene. The pretreatment of cells with forsythoside A, adding forsythoside A post infection of cells, and treatment of virus with forsythoside A were analysed. The inhibitory effect of forsythoside A was confirmed by infecting primary chicken embryo kidney cells. Infected cells were inhibited by forsythoside A treatment. The data indicated that forsythoside A has the potential to prevent IBV infection in vitro. Copyright © 2010 John Wiley & Sons, Ltd.

Identification of a movement protein of the tenuivirus rice stripe virus.

Rice stripe virus (RSV) is the type member of the genus Tenuivirus. RSV has four single-stranded RNAs and causes severe disease in rice fields in different parts of China. To date, no reports have described how RSV spreads within host plants or the viral and/or host factor(s) required for tenuivirus movement. We investigated functions of six RSV-encoded proteins using trans-complementation experiments and biolistic bombardment. We demonstrate that NSvc4, encoded by RSV RNA4, supports the intercellular trafficking of a movement-deficient Potato virus X in Nicotiana benthamiana leaves. We also determined that upon biolistic bombardment or agroinfiltration, NSvc4:enhanced green fluorescent protein (eGFP) fusion proteins localize predominantly near or within the walls of onion and tobacco epidermal cells. In addition, the NSvc4:eGFP fusion protein can move from initially bombarded cells to neighboring cells in Nicotiana benthamiana leaves. Immunocytochemistry using tissue sections from RSV-infected rice leaves and an RSV NSvc4-specific antibody showed that the NSvc4 protein accumulated in walls of RSV-infected leaf cells. Gel retardation assays revealed that the NSvc4 protein interacts with single-stranded RNA in vitro, a common feature of many reported plant viral movement proteins (MPs). RSV NSvc4 failed to interact with the RSV nucleocapsid protein using yeast two-hybrid assays. Taken together, our data indicate that RSV NSvc4 is likely an MP of the virus. This is the first report describing a tenuivirus MP.

Retroviral nucleocapsid proteins display nonequivalent levels of nucleic acid chaperone activity.

Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is a nucleic acid chaperone that facilitates the remodeling of nucleic acids during various steps of the viral life cycle. Two main features of NC's chaperone activity are its abilities to aggregate and to destabilize nucleic acids. These functions are associated with NC's highly basic character and with its zinc finger domains, respectively. While the chaperone activity of HIV-1 NC has been extensively studied, less is known about the chaperone activities of other retroviral NCs. In this work, complementary experimental approaches were used to characterize and compare the chaperone activities of NC proteins from four different retroviruses: HIV-1, Moloney murine leukemia virus (MLV), Rous sarcoma virus (RSV), and human T-cell lymphotropic virus type 1 (HTLV-1). The different NCs exhibited significant differences in their overall chaperone activities, as demonstrated by gel shift annealing assays, decreasing in the order HIV-1 approximately RSV > MLV >> HTLV-1. In addition, whereas HIV-1, RSV, and MLV NCs are effective aggregating agents, HTLV-1 NC, which exhibits poor overall chaperone activity, is unable to aggregate nucleic acids. Measurements of equilibrium binding to single- and double-stranded oligonucleotides suggested that all four NC proteins have moderate duplex destabilization capabilities. Single-molecule DNA-stretching studies revealed striking differences in the kinetics of nucleic acid dissociation between the NC proteins, showing excellent correlation between nucleic acid dissociation kinetics and overall chaperone activity.

Probing nucleation, reverse annealing, and chaperone function along the reaction path of HIV-1 single-strand transfer.

Reverse transcription of the HIV-1 genome involves several nucleic acid rearrangement steps that are catalyzed (chaperoned) by the nucleocapsid protein (NC), including the annealing of the transactivation response region (TAR) RNA of the genome to the complementary sequence (TAR DNA) in minus-strand strong-stop DNA. It has been extremely challenging to obtain unambiguous mechanistic details on the annealing process at the molecular level because of the kinetic involvement of a complex and heterogeneous set of nucleic acid/protein complexes of variable structure and variable composition. Here, we investigate the in vitro annealing mechanism using a multistep single-molecule spectroscopy kinetic method. In this approach, an immobilized hairpin is exposed to a multistep programmed concentration sequence of NC, model complementary targeted-oligonucleotides, and buffer-only solutions. The sequence controllably "drags" single immobilized TAR hairpins among the kinetic stable states of the reaction mechanism; i.e., reactants, intermediates, and products. This single-molecule spectroscopy method directly probes kinetic reversibility and the chaperone (catalytic) role of NC at various stages along the reaction sequence, giving access to previously inaccessible kinetic processes and rate constants. By employing target oligonucleotides for specific TAR regions, we kinetically trap and investigate structural models for putative nucleation complexes for the annealing process. The new results lead to a more complete and detailed understanding of the ability of NC to promote nucleic acid/nucleic acid rearrangement processes. This includes information on the ability of NC to chaperone "reverse annealing" in single-strand transfer and the first observation of partially annealed, conformational substates in the annealing mechanism.

Molecular and biological comparison of two Tomato yellow ring virus (TYRV) isolates: challenging the Tospovirus species concept.

Two strains of Tomato yellow ring virus (TYRV, genus Tospovirus), one from tomato (referred to as TYRV-t) and the other from soybean and potato (denoted TYRV-s), collected from different geographical regions in Iran, were compared. Their genomic S RNA segments differed in size by 55 nucleotides. Comparison of the S RNA intergenic regions revealed the absence of a stretch of 115 nucleotides within the S RNA segment of TYRV-s and, conversely, of 56 nts in that of TYRV-t, apparently a stable genetic difference as it was also found in another isolate of TYRV-s collected from potato. Sequence comparison of the N protein ORFs revealed an identity of 92% between the N proteins of both strains, and the observed strong cross-reaction of TYRV-s in DAS-ELISA with a polyclonal antiserum directed against the TYRV-t N protein confirmed this high identity. Host range analysis revealed several differences, e.g. TYRV-s, but not TYRV-t, being able to systemically infect Nicotiana species, and TYRV-s being localised in tomato. The observed molecular and biological differences of both viruses call into question the currently used criteria for Tospovirus species demarcation.

Comparative evaluation of two severe acute respiratory syndrome (SARS) vaccine candidates in mice challenged with SARS coronavirus.

Two different severe acute respiratory syndrome (SARS) vaccine strategies were evaluated for their ability to protect against live SARS coronavirus (CoV) challenge in a murine model of infection. A whole killed (inactivated by beta-propiolactone) SARS-CoV vaccine and a combination of two adenovirus-based vectors, one expressing the nucleocapsid (N) and the other expressing the spike (S) protein (collectively designated Ad S/N), were evaluated for the induction of serum neutralizing antibodies and cellular immune responses and their ability to protect against pulmonary SARS-CoV replication. The whole killed virus (WKV) vaccine given subcutaneously to 129S6/SvEv mice was more effective than the Ad S/N vaccine administered either intranasally or intramuscularly in inhibiting SARS-CoV replication in the murine respiratory tract. This protective ability of the WKV vaccine correlated with the induction of high serum neutralizing-antibody titres, but not with cellular immune responses as measured by gamma interferon secretion by mouse splenocytes. Titres of serum neutralizing antibodies induced by the Ad S/N vaccine administered intranasally or intramuscularly were significantly lower than those induced by the WKV vaccine. However, Ad S/N administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. Among the vaccine groups, SARS-CoV-specific IgA was found only in the sera of mice immunized intranasally with Ad S/N, suggesting that mucosal immunity may play a role in protection for the intranasal Ad S/N delivery system. Finally, the sera of vaccinated mice contained antibodies to S, further suggesting a role for this protein in conferring protective immunity against SARS-CoV infection.

Multiple gene segments control the temperature sensitivity and attenuation phenotypes of ca B/Ann Arbor/1/66.

Cold-adapted (ca) B/Ann Arbor/1/66 is the influenza B virus strain master donor virus for FluMist, a live, attenuated, influenza virus vaccine licensed in 2003 in the United States. Each FluMist vaccine strain contains six gene segments of the master donor virus; these master donor gene segments control the vaccine's replication and attenuation. These gene segments also express characteristic biological traits in model systems. Unlike most virulent wild-type (wt) influenza B viruses, ca B/Ann Arbor/1/66 is temperature sensitive (ts) at 37 degrees C and attenuated (att) in the ferret model. In order to define the minimal genetic components of these phenotypes, the amino acid sequences of the internal genes of ca B/Ann Arbor/1/66 were aligned to those of other influenza B viruses. These analyses revealed eight unique amino acids in three proteins: two in the polymerase subunit PA, two in the M1 matrix protein, and four in the nucleoprotein (NP). Using reverse genetics, these eight wt amino acids were engineered into a plasmid-derived recombinant of ca B/Ann Arbor/1/66, and these changes reverted both the ts and the att phenotypes. A detailed mutational analysis revealed that a combination of two sites in NP (A114 and H410) and one in PA (M431) controlled expression of ts, whereas these same changes plus two additional residues in M1 (Q159 and V183) controlled the att phenotype. Transferring this genetic signature to the divergent wt B/Yamanashi/166/98 strain conferred both the ts and the att phenotypes on the recombinant, demonstrating that this small, complex, genetic signature encoded the essential elements for these traits.

Purification and serological analyses of tospoviral nucleocapsid proteins expressed by Zucchini yellow mosaic virus vector in squash.

A plant viral vector engineered from an in vivo infectious clone of Zucchini yellow mosaic virus (ZYMV) was used to express the nucleocapsid proteins (NPs) of tospoviruses in planta. The open reading frames (ORFs) of NPs of different serogroups of tospoviruses, including Tomato spotted wilt virus, Impatiens necrotic spot virus, Watermelon silver mottle virus, Peanut bud necrosis virus, and Watermelon bud necrosis virus (WBNV), were in frame inserted in between the P1 and HC-Pro genes of the ZYMV vector. Six histidine residues and an NIa protease cleavage site were added at the C-terminal region of the inserts to facilitate purification and process of free form of the expressed NPs, respectively. Approximately 1.2-2.5 mg/NPs 100 g tissues were purified from leaf extracts of zucchini squash. The expressed WBNV NP was used as an immunogen for the production of highly specific polyclonal antisera and monoclonal antibodies. The procedure provides a convenient and fast way for production of large quantities of pure NPs of tospoviruses in planta. The system also has a potential for production of any proteins of interest in cucurbits.

Complementary function of the two catalytic domains of APOBEC3G.

The HIV-1 viral accessory protein Vif prevents the encapsidation of the antiviral cellular cytidine deaminases APOBEC3F and APOBEC3G by inducing their proteasomal degradation. In the absence of Vif, APOBEC3G is encapsidated and blocks virus replication by deaminating cytosines of the viral cDNA. APOBEC3G encapsidation has been recently shown to depend on the viral nucleocapsid protein; however, the role of RNA remains unclear. Using APOBEC3G deletion and point mutants, we mapped the encapsidation determinant to the Zn(2+) coordination residues of the N-terminal catalytic domain (CD1). Notably, these residues were also required for RNA binding. Mutations in the two aromatic residues of CD1 but not CD2, which are conserved in cytidine deaminase core domains and are required for RNA binding, prevented encapsidation into HIV-1, HTLV-I and MLV. The Zn(2+) coordination residues of the C-terminal catalytic domain (CD2) were not required for encapsidation but were essential for cytidine deaminase activity and the antiviral effect. These findings suggest a model in which CD1 mediates encapsidation and RNA binding while CD2 mediates cytidine deaminase activity. Interestingly, HTLV-I was relatively resistant to the antiviral effects of encapsidated APOBEC3G.

Severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice.

Severe acute respiratory syndrome coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. Thus, vaccination against SARS-CoV may represent an effective approach towards controlling SARS. The nucleocapsid (N) protein is thought to play a role in induction of cell-mediated immunity to SARS-CoV and thus it is important to characterize this protein. In the present study, an E1/partially E3-deleted, replication-defective human adenovirus 5 (Ad5) vector (Ad5-N-V) expressing the SARS-CoV N protein was constructed. The N protein, expressed in vitro by Ad5-N-V, was of the expected molecular mass of 50 kDa and was phosphorylated. Vaccination of C57BL/6 mice with Ad5-N-V generated potent SARS-CoV-specific humoral and T cell-mediated immune responses. These results show that Ad5-N-V may potentially be used as a SARS-CoV vaccine.

Zinc finger-dependent HIV-1 nucleocapsid protein-TAR RNA interactions.

In the minus-strand transfer step of HIV-1 reverse transcription, the nucleocapsid protein (NC) promotes annealing of the 3' 'R' (repeat) region of the RNA genome to its complementary sequence located in the newly synthesized minus-strand strong-stop DNA. The R region contains the highly stable transactivation response (TAR) RNA hairpin. To gain insights into the molecular details of TAR RNA-NC interactions, we carried out hydroxyl radical footprinting, as well as gel-shift and fluorescence anisotropy binding assays using wild-type and mutant forms of NC. Our results support the conclusion that NC variants with mutations in their zinc finger domains have dramatically altered TAR RNA binding interactions relative to wild-type NC. These data demonstrate that a specific zinc finger architecture is required for optimal TAR RNA binding, and help to explain the requirement for the zinc finger motifs of NC in its role as a nucleic acid chaperone in minus-strand transfer.

Differing roles of the N- and C-terminal zinc fingers in human immunodeficiency virus nucleocapsid protein-enhanced nucleic acid annealing.

The replication process of human immunodeficiency virus requires a number of nucleic acid annealing steps facilitated by the hybridization and helix-destabilizing activities of human immunodeficiency virus nucleocapsid (NC) protein. NC contains two CCHC zinc finger motifs numbered 1 and 2 from the N terminus. The amino acids surrounding the CCHC residues differ between the two zinc fingers. Assays were preformed to investigate the activities of the fingers by determining the effect of mutant and wild-type proteins on annealing of 42-nucleotide RNA and DNA complements. The mutants 1.1 NC and 2.2 NC had duplications of the N- and C-terminal zinc fingers in positions 1 and 2. The mutant 2.1 NC had the native zinc fingers with their positions switched. Annealing assays were completed with unstructured and highly structured oligonucleotide complements. 2.2 NC had a near wild-type level of annealing of unstructured nucleic acids, whereas it was completely unable to stimulate annealing of highly structured nucleic acids. In contrast, 1.1 NC was able to stimulate annealing of both unstructured and structured substrates, but to a lesser degree than the wild-type protein. Results suggest that finger 1 has a greater role in unfolding of strong secondary structures, whereas finger 2 serves an accessory role that leads to a further increase in the rate of annealing.

Destabilization of the HIV-1 complementary sequence of TAR by the nucleocapsid protein through activation of conformational fluctuations.

The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are critical for the two obligatory strand transfer reactions required for the synthesis of a complete proviral DNA by reverse transcriptase. The first DNA strand transfer relies on the destabilization by NCp7 of double-stranded segments of the transactivation response region (TAR) sequence at the 3' end of the genomic RNA and the complementary sequence cTAR at the 3' terminus of minus strong-stop DNA, the early product of reverse transcription. In order to determine the dynamics of NCp7-mediated nucleic acid destabilization, we investigated by time-resolved fluorescence spectroscopy and two photon fluorescence correlation spectroscopy, the interaction of a doubly labeled cTAR sequence with NC(12-55) containing NCp7 CCHC zinc fingers and flanking basic amino acid residues. From the chemical rates and the activation energy associated with the conformational fluctuations observed in the absence of NC, it is concluded that such fluctuations are associated with the opening and closing of the double-stranded terminal segments of cTAR. The destabilizing activity of NC(12-55) occurs mainly through a major increase of the opening rate constant of cTAR. Moreover, NC appears to augment the number of pathways between the open and closed states of cTAR, suggesting that it initiates melting of base-pairs at different locations within the terminal segments of cTAR. This activity of NC on the dynamics of cTAR secondary structure is thought to be critical for the formation of the cTAR-TAR complex, which is essential for the specificity and extent of proviral DNA synthesis by reverse transcriptase.

Zinc finger structures in the human immunodeficiency virus type 1 nucleocapsid protein facilitate efficient minus- and plus-strand transfer.

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) has two zinc fingers, each containing the invariant metal ion binding residues CCHC. Recent reports indicate that mutations in the CCHC motifs are deleterious for reverse transcription in vivo. To identify reverse transcriptase (RT) reactions affected by such changes, we have probed zinc finger functions in NC-dependent RT-catalyzed HIV-1 minus- and plus-strand transfer model systems. Our approach was to examine the activities of wild-type NC and a mutant in which all six cysteine residues were replaced by serine (SSHS NC); this mutation severely disrupts zinc coordination. We find that the zinc fingers contribute to the role of NC in complete tRNA primer removal from minus-strand DNA during plus-strand transfer. Annealing of the primer binding site sequences in plus-strand strong-stop DNA [(+) SSDNA] to its complement in minus-strand acceptor DNA is not dependent on NC zinc fingers. In contrast, the rate of annealing of the complementary R regions in (-) SSDNA and 3' viral RNA during minus-strand transfer is approximately eightfold lower when SSHS NC is used in place of wild-type NC. Moreover, unlike wild-type NC, SSHS NC has only a small stimulatory effect on minus-strand transfer and is essentially unable to block TAR-induced self-priming from (-) SSDNA. Our results strongly suggest that NC zinc finger structures are needed to unfold highly structured RNA and DNA strand transfer intermediates. Thus, it appears that in these cases, zinc finger interactions are important components of NC nucleic acid chaperone activity.