RNA Replication In Cells Research Articles

Production of single-cycle infectious SARS-CoV-2 through a trans-complemented replicon.

Single‐cycle infectious virus can elicit close‐to‐natural immune response and memory. One approach to generate single‐cycle severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) is through deletion of structural genes such as spike (S) and nucleocapsid (N). Transcomplementation of the resulting ΔS or ΔN virus through enforced expression of S or N protein in the cells gives rise to a live but unproductive virus. In this study, ΔS and ΔN BAC clones were constructed and their live virions were rescued by transient expression of S and N proteins from the ancestral and the Omicron strains. ΔS and ΔN virions were visualized by transmission electron microscopy. Virion production of ΔS was more efficient than that of ΔN. The coated S protein from ΔS was delivered to infected cells in which the expression of N protein was also robust. In contrast, expression of neither S nor N was detected in ΔN‐infected cells. ΔS underwent viral RNA replication, induced type I interferon (IFN) response, but did not form plaques. Despite RNA replication in cells, ΔS infection did not produce viral progeny in culture supernatant. Interestingly, viral RNA replication was not further enhanced upon overexpression of S protein. Taken together, our work provides a versatile platform for development of single‐cycle vaccines for SARS‐CoV‐2.

Significant Inactivation of SARS-CoV-2 In Vitro by a Green Tea Catechin, a Catechin-Derivative, and Black Tea Galloylated Theaflavins.

Potential effects of tea and its constituents on SARS-CoV-2 infection were assessed in vitro. Infectivity of SARS-CoV-2 was decreased to 1/100 to undetectable levels after a treatment with black tea, green tea, roasted green tea, or oolong tea for 1 min. An addition of (−) epigallocatechin gallate (EGCG) significantly inactivated SARS-CoV-2, while the same concentration of theasinensin A (TSA) and galloylated theaflavins including theaflavin 3,3′-di-O-gallate (TFDG) had more remarkable anti-viral activities. EGCG, TSA, and TFDG at 1 mM, 40 µM, and 60 µM, respectively, which are comparable to the concentrations of these compounds in tea beverages, significantly reduced infectivity of the virus, viral RNA replication in cells, and secondary virus production from the cells. EGCG, TSA, and TFDG significantly inhibited interaction between recombinant ACE2 and RBD of S protein. These results suggest potential usefulness of tea in prevention of person-to-person transmission of the novel coronavirus.

Determination of the vRNA and cRNA promoter activity by M segment-specific non-coding nucleotides of influenza A virus

ABSTRACT Eight-segmented, negative-sense, single-stranded genomic RNAs of influenza A virus are terminated with 5′ and 3′ untranslated regions (UTRs). All segments have highly conserved extremities of 13 and 12 nucleotides at the 5′ and 3′ UTRs, respectively, constructing the viral RNA (vRNA) promoter. Adjacent to the duplex stem of 3 base pairs (bps) between the two conserved strands, additional 1–4 bps are existing in a segment-specific manner. We investigated the roles of the matrix (M) segment-specific base pair between the 14th nucleotide uridine (U14′) of the 5′ UTR and the 13th nucleotide adenosine (A13) of the 3′ UTR by preparing possible vRNA promoters, named vXY, as well as cRNA promoters, named cYX. We analysed their RNA-dependent RNA replication efficiency using the minigenome replicon system and an enzyme assay system in vitro with synthetic RNA promoters. Notably, in contrast to vAC(s) that is a synthetic vRNA promoter with A14′ and C13, base-pair disruption at the complementary RNA (cRNA) promoter in cAC(s), which has A13′ and C14, not only reduced viral RNA replication in cells but also impaired de novo initiation of unprimed vRNA synthesis. Reverse genetics experiments confirmatively exhibited that this breakage in the cRNA promoter affected the rescue of infectious virus. The present study suggests that the first segment-specific base pair plays an essential role in generating infectious viruses by regulating the promoter activity of cRNA rather than vRNA. It could provide insights into the role of the segment-specific nucleotides in viral genome replication for sustainable infection.

A Chikungunya Virus trans-Replicase System Reveals the Importance of Delayed Nonstructural Polyprotein Processing for Efficient Replication Complex Formation in Mosquito Cells.

Chikungunya virus (CHIKV) is a medically important alphavirus that is transmitted by Aedes aegypti and Aedes albopictus mosquitoes. The viral replicase complex consists of four nonstructural proteins (nsPs) expressed as a polyprotein precursor and encompasses all enzymatic activities required for viral RNA replication. nsPs interact with host components of which most are still poorly understood, especially in mosquitos. A CHIKV trans-replicase system that allows the uncoupling of RNA replication and nsP expression was adapted to mosquito cells and subsequently used for analysis of universal and host-specific effects of 17 different nonstructural polyprotein (ns-polyprotein) mutations. It was found that mutations blocking nsP enzymatic activities as well as insertions of enhanced green fluorescent protein (EGFP) into different nsPs had similar effects on trans-replicase activity regardless of the host (i.e., mammalian or mosquito). Mutations that slow down or accelerate ns-polyprotein processing generally had no effect or reduced trans-replicase activity in mammalian cells, while in mosquito cells most of them increased trans-replicase activity prominently. Increased RNA replication in mosquito cells was counteracted by an antiviral RNA interference (RNAi) response. Substitution of the W258 residue in the membrane binding peptide of nsP1 resulted in a temperature-sensitive defect, in the context of both the trans-replicase and infectious CHIKV. The defect was compensated for by secondary mutations selected during passaging of mutant CHIKV. These findings demonstrate the value of alphavirus trans-replicase systems for studies of viral RNA replication and virus-host interactions.IMPORTANCE Chikungunya virus is an important mosquito-transmitted human pathogen. This virus actively replicates in mosquitoes, but the underlying molecular mechanisms and interactions of viral and host components are poorly understood. This is partly due to the lack of reliable systems for functional analysis of viral nonstructural polyproteins (ns-polyproteins) and nonstructural proteins (nsPs) in mosquito cells. Adaption of a CHIKV trans-replicase system allowed study of the effects of mutations in the ns-polyprotein on RNA replication in cells derived from mammalian and mosquito hosts. We found that a slowdown of ns-polyprotein processing facilitates replication complex formation and/or functioning in mosquito cells and that this process is antagonized by the natural RNAi defense system present in mosquito cells. The mosquito-adapted CHIKV trans-replicase system represents a valuable tool to study alphavirus-mosquito interactions at the molecular level and to develop advanced antiviral strategies.

A Human Proteome Microarray Identifies that the Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) Recognizes the 5′ Terminal Sequence of the Hepatitis C Virus RNA

Stem-loop I (SL1) located in the 5' untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of ∼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.

Active RNA Replication of Hepatitis C Virus Downregulates CD81 Expression

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

Control of Innate Immune Signaling and Membrane Targeting by the Hepatitis C Virus NS3/4A Protease Are Governed by the NS3 Helix α 0

Hepatitis C virus (HCV) infection is sensed in the host cell by the cytosolic pathogen recognition receptor RIG-I. RIG-I signaling is propagated through its signaling adaptor protein MAVS to drive activation of innate immunity. However, HCV blocks RIG-I signaling through viral NS3/4A protease cleavage of MAVS on the mitochondrion-associated endoplasmic reticulum (ER) membrane (MAM). The multifunctional HCV NS3/4A serine protease is associated with intracellular membranes, including the MAM, through membrane-targeting domains within NS4A and also at the amphipathic helix α(0) of NS3. The serine protease domain of NS3 is required for both cleavage of MAVS, a tail-anchored membrane protein, and processing the HCV polyprotein. Here, we show that hydrophobic amino acids in the NS3 helix α(0) are required for selective cleavage of membrane-anchored portions of the HCV polyprotein and for cleavage of MAVS for control of RIG-I pathway signaling of innate immunity. Further, we found that the hydrophobic composition of NS3 helix α(0) is essential to establish HCV replication and infection. Alanine substitution of individual hydrophobic amino acids in the NS3 helix α(0) impaired HCV RNA replication in cells with a functional RIG-I pathway, but viral RNA replication was rescued in cells lacking RIG-I signaling. Therefore, the hydrophobic amphipathic helix α(0) of NS3 is required for NS3/4A control of RIG-I signaling and HCV replication by directing the membrane targeting of both viral and cellular substrates.

Heterogeneous Nuclear Ribonucleoprotein A2 Participates in the Replication of Japanese Encephalitis Virus through an Interaction with Viral Proteins and RNA

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is kept in a zoonotic transmission cycle between pigs and mosquitoes. JEV causes infection of the central nervous system with a high mortality rate in dead-end hosts, including humans. Many studies have suggested that the flavivirus core protein is not only a component of nucleocapsids but also an important pathogenic determinant. In this study, we identified heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) as a binding partner of the JEV core protein by pulldown purification and mass spectrometry. Reciprocal coimmunoprecipitation analyses in transfected and infected cells confirmed a specific interaction between the JEV core protein and hnRNP A2. Expression of the JEV core protein induced cytoplasmic retention of hnRNP A2 in JEV subgenomic replicon cells. Small interfering RNA (siRNA)-mediated knockdown of hnRNP A2 resulted in a 90% reduction of viral RNA replication in cells infected with JEV, and the reduction was cancelled by the expression of an siRNA-resistant hnRNP A2 mutant. In addition to the core protein, hnRNP A2 also associated with JEV nonstructural protein 5, which has both methyltransferase and RNA-dependent RNA polymerase activities, and with the 5'-untranslated region of the negative-sense JEV RNA. During one-step growth, synthesis of both positive- and negative-strand JEV RNAs was delayed by the knockdown of hnRNP A2. These results suggest that hnRNP A2 plays an important role in the replication of JEV RNA through the interaction with viral proteins and RNA.

Ethanol Enhances Hepatitis C Virus Replication through Lipid Metabolism and Elevated NADH/NAD+

Ethanol has been suggested to elevate HCV titer in patients and to increase HCV RNA in replicon cells, suggesting that HCV replication is increased in the presence and absence of the complete viral replication cycle, but the mechanisms remain unclear. In this study, we use Huh7 human hepatoma cells that naturally express comparable levels of CYP2E1 as human liver to demonstrate that ethanol, at subtoxic and physiologically relevant concentrations, enhances complete HCV replication. The viral RNA genome replication is affected for both genotypes 2a and 1b. Acetaldehyde, a major product of ethanol metabolism, likewise enhances HCV replication at physiological concentrations. The potentiation of HCV replication by ethanol is suppressed by inhibiting CYP2E1 or aldehyde dehydrogenase and requires an elevated NADH/NAD(+) ratio. In addition, acetate, isopropyl alcohol, and concentrations of acetone that occur in diabetics enhance HCV replication with corresponding increases in the NADH/NAD(+). Furthermore, inhibiting the host mevalonate pathway with lovastatin or fluvastatin and fatty acid synthesis with 5-(tetradecyloxy)-2-furoic acid or cerulenin significantly attenuates the enhancement of HCV replication by ethanol, acetaldehyde, acetone, as well as acetate, whereas inhibiting beta-oxidation with beta-mercaptopropionic acid increases HCV replication. Ethanol, acetaldehyde, acetone, and acetate increase the total intracellular cholesterol content, which is attenuated with lovastatin. In contrast, both endogenous and exogenous ROS suppress the replication of HCV genotype 2a, as previously shown with genotype 1b. Therefore, lipid metabolism and alteration of cellular NADH/NAD(+) ratio are likely to play a critical role in the potentiation of HCV replication by ethanol rather than oxidative stress.

Comprehensive Analysis of the Effects of Ordinary Nutrients on Hepatitis C Virus RNA Replication in Cell Culture

To date, only a limited number of studies have reported finding an influence of ordinary nutrients on hepatitis C virus (HCV) RNA replication. However, the effects of other nutrients on HCV RNA replication remain largely unknown. We recently developed a reporter assay system for genome-length HCV RNA replication in hepatoma-derived HuH-7 cells (OR6). Here, using this OR6 assay system, we comprehensively examined 46 nutrients from four nutrient groups: vitamins, amino acids, fatty acids, and salts. We found that three nutrients-beta-carotene, vitamin D(2), and linoleic acid-inhibited HCV RNA replication and that their combination caused additive and/or synergistic effects on HCV RNA replication. In addition, combined treatment with each of the three nutrients and interferon alpha or beta or fluvastatin inhibited HCV RNA replication in an additive manner, while combined treatment with cyclosporine synergistically inhibited HCV RNA replication. In contrast, we found that vitamin E enhanced HCV RNA replication and negated the effects of the three anti-HCV nutrients and cyclosporine but not those of interferon or fluvastatin. These results will provide useful information for the treatment of chronic hepatitis C patients who also take anti-HCV nutrients as an adjunctive therapy in combination with interferon. In conclusion, among the ordinary nutrients tested, beta-carotene, vitamin D(2), and linoleic acid possessed anti-HCV activity in a cell culture system, and these nutrients are therefore considered to be potential candidates for enhancing the effects of interferon therapy.

Membrane Association of the RNA-Dependent RNA Polymerase Is Essential for Hepatitis C Virus RNA Replication

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), belongs to a class of integral membrane proteins termed tail-anchored proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane protein-protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored proteins.

The optimal temperature for RNA replication in cells infected by Soil-borne wheat mosaic virus is 17 degrees C.

Systemic infection of wheat plants with Soil-borne wheat mosaic virus (SBWMV) requires temperatures below 20 degrees C. Here we examine the cause of the temperature sensitivity by inoculating infectious in vitro transcripts of SBWMV RNA1 and RNA2 to barley mesophyll protoplasts. After RNA inoculation, protoplasts were incubated at temperatures between 15 and 25 degrees C for up to 48 h. Western blot analysis showed that the capsid protein accumulated most abundantly at 17 degrees C but was not detectable at 25 degrees C. Northern blot analysis showed that the wild-type RNA1 and RNA2 and their subgenomic RNAs accumulated most abundantly at 17 degrees C but were barely detectable at 25 degrees C. An RNA1 mutant in which the p152 and p211 replicase genes were placed between the 5'- and 3'-untranslated regions also replicated most efficiently at 17 degrees C but not at 25 degrees C. Thus, the requirement for temperatures lower than 20 degrees C for SBWMV infection is primarily determined by replication of RNA1, which encodes the viral RNA replicase.

Oligomeric structures of poliovirus polymerase are important for function.

Central to the replication of poliovirus and other positive-strand RNA viruses is the virally encoded RNA-dependent RNA polymerase. Previous biochemical studies have suggested that direct polymerase- polymerase interactions might be important for polymerase function, and the structure of poliovirus polymerase has revealed two regions of extensive polymerase-polymerase interaction. To explore potential functional roles for the structurally observed polymerase-polymerase interactions, we have performed RNA binding and extension studies of mutant polymerase proteins in solution, disulfide cross-linking studies, mutational analyses in cells, in vitro activity analyses and RNA substrate modeling studies. The results of these studies indicate that both regions of polymerase-polymerase interaction observed in the crystals are indeed functionally important and, furthermore, reveal specific functional roles for each. One of the two regions of interaction provides for efficient substrate RNA binding and the second is crucial for forming catalytic sites. These studies strongly support the hypothesis that the polymerase- polymerase interactions discovered in the crystal structure provide an exquisitely detailed structural context for poliovirus polymerase function and for poliovirus RNA replication in cells.

Replication of hepatitis delta virus RNA: effect of mutations of the autocatalytic cleavage sites.

Hepatitis delta virus (HDV) contains a circular RNA genome of 1.7 kb. HDV RNA replication is thought to proceed via a rolling-circle mechanism that is dependent on autocatalytic cleavage and ligation reactions. However, it has never been established that these ribozyme activities are indeed involved in HDV RNA replication. To investigate the possible biological significance of HDV RNA self-cleavage, we constructed several HDV dimer cDNAs containing single-base substitutions of the 3' nucleotide of the genomic and the antigenomic self-cleavage sites. These mutations were known to affect self-cleavage in vitro to various extents. The effects of these mutations on HDV RNA replication were examined in hepatic and nonhepatic cell lines. The results showed that all of the mutants which had lost the in vitro self-cleavage activity could not replicate. The only mutant which retained full cleavage activity replicated as efficiently as the wild-type RNA. Thus, this study established that self-cleavage activity is required for HDV RNA replication in cells. Interestingly, the level of HDV RNA detected in cells transfected with this replication-competent mutant and that detected in cells transfected with the wild-type construct were similar in COS-7 cells but vastly different in HepG2 and Huh-7 cells, suggesting that HDV RNA self-cleavage activity may be modulated by cell-specific factors. We also compared the effects of mutations when the primary transcripts of these constructs were of either genomic or antigenomic sense. In constructs which synthesize primary transcripts of genomic sense, all of the antigenomic self-cleavage mutants produced as much hepatitis delta antigen (HDAg) as did the wild-type construct, even in the absence of detectable HDV RNA replication, whereas the genomic self-cleavage mutants produced very little HDAg. These and other data suggest that (i) the primary HDV RNA transcripts of both genomic and antigenomic polarities must first be processed to serve as a template for HDV RNA transcription, (ii) efficient cleavage at the antigenomic self-cleavage site is not required for HDAg expression, and (iii) HDV RNA replication most likely occurs by a double-rolling-circle mechanism.

INHIBITION OF HIV-1 RNA PRODUCTION BY THE DIPHTHERIA TOXIN-RELATED IL-2 FUSION PROTEINS DAB486IL-2 AND DAB389IL-2

Productive infection of cells by human immunodeficiency virus type 1 (HIV-1) is associated with the activation state of the cell and its obligatory expression of the interleukin-2 receptor (IL-2R), the latter providing a new target for antiviral therapy. A quantitative RNA-RNA hybridization assay is employed to detect production of HIV-1 RNA and to show that two IL-2 diphtheria toxin-related fusion proteins (DAB486IL-2 and its more potent, truncated form DAB389IL-2) inhibit HIV-1 RNA production in infected cells. A mutant form of DAB486IL-2 containing a single point mutation that inactivates the adenosine diphosphate ribosyltransferase activity of the toxin does not inhibit HIV RNA production, even though the molecule binds to the IL-2R. The active fusion proteins inhibit viral RNA replication in cells infected with HIV-1 clinical isolates as well as with a ZDV-resistant strain of HIV-1. These results indicate that IL-2 receptor-targeted fusion proteins can be utilized to inhibit HIV-1 replication effectively in infected human lymphocytes.