The RNA-dependent RNA polymerase nsP4 is an integral part of the alphavirus replication complex. To define the role of nsP4 in viral RNA replication and for a structure-function analysis, we expressed Sindbis virus nsP4 in Escherichia coli. The core catalytic domain of nsP4 (⌬97nsP4, a deletion of the N-terminal 97 amino acids), which consists of the predicted polymerase domain containing the GDD amino acid motif required for viral RNA synthesis, was stable against proteolytic degradation during expression. Therefore, the recombinant core domain and selected mutants were expressed and purified to homogeneity. We determined that ⌬97nsP4 possesses terminal adenylyltransferase (TATase) activity, as it specifically catalyzed the addition of adenine to the 3 end of an acceptor RNA in the presence of divalent cations. Furthermore, ⌬97nsP4 is unable to transfer other nucleotides (UTP, CTP, GTP, and dATP) to the acceptor RNA in the absence or presence of other nucleotides. ⌬97nsP4 possessing a GDD-to-GAA mutation completely inactivates the enzymatic activity. However, a GDD-to-SNN mutation did not inactivate the enzyme but reduced its activity to ϳ45% of that of the wild type in the presence of Mg 2؉ . Investigation of the TATase of the GDD-to-SNN mutant revealed that it had TATase equivalent to that of the wild type in the presence of Mn 2؉ . Identification of ⌬97nsP4 TATase activity suggests a novel function of the alphavirus RNA-dependent RNA polymerase in the maintenance and repair of the poly(A) tail, an element required for replication of the viral genome.Sindbis virus (SINV), the type species of the Alphavirus genus, is a plus-sense, single-stranded RNA virus. Its 11.7-kb genome is capped at the 5Ј end and polyadenylated at the 3Ј end (51). The 5Ј-terminal two-thirds of the genomic RNA encodes four nonstructural or replicase proteins. These nonstructural proteins are directly synthesized from the genomic RNA as two overlapping polyproteins, P123 and P1234. P1234 is produced by readthrough of a UGA termination codon present between the sequences encoding nsP3 and nsP4, which occurs 5 to 20% of the time during translation (30,50,51). The protease domain of nsP2 processes the polyproteins into various intermediates and individual nonstructural proteins (20). Nonstructural proteins together with an unknown host factor(s) constitute the alphavirus replication complex (51). The RNA-dependent RNA polymerase (RdRp), nsP4, which contains the signature GDD motif of viral RNA polymerases (24,25), is activated by cleavage from nascent P1234 to form an initial P123/nsP4 replication complex (47). This complex uses the genomic RNA as a template for the synthesis of minusstrand RNA. P123 is further processed at the 1/2 junction to produce an nsP1/P23/nsP4 complex, capable of synthesizing both minus-strand and 49S genomic RNAs. Finally, polyprotein P23 is cleaved at the 2/3 junction to produce a replication complex consisting of fully processed nonstructural proteins, which is responsible for the synthesis of 26S subgenomic mRNA ...

The rapidly spreading, highly contagious and pathogenic SARS-coronavirus 2 (SARS-CoV-2) associated Coronavirus Disease 2019 (COVID-19) has been declared as a pandemic by the World Health Organization (WHO). The novel 2019 SARS-CoV-2 enters the host cell by binding of the viral surface spike glycoprotein (S-protein) to cellular angiotensin converting enzyme 2 (ACE2) receptor. The virus specific molecular interaction with the host cell represents a promising therapeutic target for identifying SARS-CoV-2 antiviral drugs. The repurposing of drugs can provide a rapid and potential cure toward exponentially expanding COVID-19. Thereto, high throughput virtual screening approach was used to investigate FDA approved LOPAC library drugs against both the receptor binding domain of spike protein (S-RBD) and ACE2 host cell receptor. Primary screening identified a few promising molecules for both the targets, which were further analyzed in details by their binding energy, binding modes through molecular docking, dynamics and simulations. Evidently, GR 127935 hydrochloride hydrate, GNF-5, RS504393, TNP, and eptifibatide acetate were found binding to virus binding motifs of ACE2 receptor. Additionally, KT203, BMS195614, KT185, RS504393, and GSK1838705A were identified to bind at the receptor binding site on the viral S-protein. These identified molecules may effectively assist in controlling the rapid spread of SARS-CoV-2 by not only potentially inhibiting the virus at entry step but are also hypothesized to act as anti-inflammatory agents, which could impart relief in lung inflammation. Timely identification and determination of an effective drug to combat and tranquilize the COVID-19 global crisis is the utmost need of hour. Further, prompt in vivo testing to validate the anti-SARS-CoV-2 inhibition efficiency by these molecules could save lives is justified.

The sudden emergence of a highly transmissible and pathogenic coronavirus SARS-CoV-2 in December 2019 from China and its rapid global spread has posed an international health emergency. The rapid development of an effective vaccine is imperative to control the spread of SARS-CoV-2. A number of concurrent efforts to find an effective therapeutic agent or vaccine for COVID-19 (coronavirus disease 2019) are being undertaken globally. Oral and nasal mucosal surfaces serve as the primary portal of entry for pathogens like coronaviruses in the human body. As evidenced by studies on similar coronaviruses (SARS-CoV and MERS-CoV), mucosal vaccination can provide a safe and effective means for the induction of long-lasting systemic and mucosal immunity to confer protection against SARS-CoV-2. This article summarizes the approaches to an effective mucosal vaccine formulation which can be a rewarding approach to combat the unprecedented threat posed by this emerging global pandemic.

The rapidly spreading, highly contagious and pathogenic SARS-coronavirus 2 (SARS-CoV-2) associated Coronavirus Disease 2019 (COVID-19) has been declared as a pandemic by the World Health Organization (WHO). The novel 2019 SARS-CoV-2 enters the host cell by binding of the viral surface spike glycoprotein (S-protein) to angiotensin converting enzyme 2 (ACE2). The virus specific molecular interaction with the host cell represents a promising therapeutic target for identifying SARS-CoV-2 antiviral drugs. The repurposing of drugs can provide a rapid and potential cure towards exponentially expending COVID-19. Thereto, high-throughput virtual screening approach was used to investigate FDA approved LOPAC library drugs against both the S-protein and ACE2 host cell receptor. Primary screening identified a few promising drugs for both the targets, which were further analyzed in details by their binding energy, binding modes through molecular docking, dynamics and simulations. Evidently, Eptifibatide acetate, TNP, GNF5, GR 127935 hydrochloride hydrate and RS504393 were found binding to virus binding motifs of ACE2 receptor. Additionally, KT185, KT203 GSK1838705A, BMS195614, and RS504393 were identified to bind at the receptor binding site on the viral S-protein. These identified drug molecules may effectively assist in controlling the rapid spread of SARS-COV-2 by not only potentially inhibiting the virus at entry step but also as anti-inflammatory agents which could impart relief in lung injuries. Timely identification and determination of an effective drug to combat and tranquilize the COVID-19 global crisis is the utmost need of hour. Further, prompt in vivo testing to validate the anti-SARS-COV-2 inhibition by these drugs could save lives is justified.

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The plant genome contains a large number of sequences that encode catalytically inactive chitinases referred to as chitinase-like proteins (CLPs). Although CLPs share high sequence and structural homology with chitinases of glycosyl hydrolase 18 (TIM barrel domain) and 19 families, they may lack the binding/catalytic activity. Molecular genetic analysis revealed that gene duplication events followed by mutation in the existing chitinase gene have resulted in the loss of activity. The evidences show that adaptive functional diversification of the CLPs has been achieved through alterations in the flexible regions than in the rigid structural elements. The CLPs plays an important role in the defense response against pathogenic attack, biotic and abiotic stress. They are also involved in the growth and developmental processes of plants. Since the physiological roles of CLPs are similar to chitinase, such mutations have led to plurifunctional enzymes. The biochemical and structural characterization of the CLPs is essential for understanding their roles and to develop potential utility in biotechnological industries. This review sheds light on the structure-function evolution of CLPs from chitinases.

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