Viral Nucleic Acid Synthesis Research Articles

Profiling Ribonucleotide and Deoxyribonucleotide Pools Perturbed by Remdesivir in Human Bronchial Epithelial Cells.

Remdesivir (RDV) has generated much anticipation for its moderate effect in treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the unsatisfactory survival rates of hospitalized patients limit its application to the treatment of coronavirus disease 2019 (COVID-19). Therefore, improvement of antiviral efficacy of RDV is urgently needed. As a typical nucleotide analog, the activation of RDV to bioactive triphosphate will affect the biosynthesis of endogenous ribonucleotides (RNs) and deoxyribonucleotides (dRNs), which are essential to RNA and DNA replication in host cells. The imbalance of RN pools will inhibit virus replication as well. In order to investigate the effects of RDV on cellular nucleotide pools and on RNA transcription and DNA replication, cellular RNs and dRNs concentrations were measured by the liquid chromatography-mass spectrometry method, and the synthesis of RNA and DNA was monitored using click chemistry. The results showed that the IC50 values for BEAS-2B cells at exposure durations of 48 and 72 h were 25.3 ± 2.6 and 9.6 ± 0.7 μM, respectively. Ten (10) μM RDV caused BEAS-2B arrest at S-phase and significant suppression of RNA and DNA synthesis after treatment for 24 h. In addition, a general increase in the abundance of nucleotides and an increase of specific nucleotides more than 2 folds were observed. However, the variation of pyrimidine ribonucleotides was relatively slight or even absent, resulting in an obvious imbalance between purine and pyrimidine ribonucleotides. Interestingly, the very marked disequilibrium between cytidine triphosphate (CTP) and cytidine monophosphate might result from the inhibition of CTP synthase. Due to nucleotides which are also precursors for the synthesis of viral nucleic acids, the perturbation of nucleotide pools would block viral RNA replication. Considering the metabolic vulnerability of endogenous nucleotides, exacerbating the imbalance of nucleotide pools imparts great promise to enhance the efficacy of RDV, which possibly has special implications for treatment of COVID-19.

The Role of TMPRSS2 and TMPRSS2- Inhibitors in Cell Entry Mechanism of COVID-19

The enzymes trypsin, furine and other proprotein convertases, cathepsin, transmembrane proteases (TMPRSS) and elastases play a role in the cell entry of coronaviruses (Coronaviridae). The proteases TMPRSS2 and TMPRSS11a, which are abundant in the respiratory tract and expressed on cell surfaces, promote the entry of SARS-CoV-1 viruses. For the TMPRSS protease TMPRSS11d - also known as human airway trypsin-like protease (HAT) - a proteolytic activation of the S- protein of SARS-CoV-1 was demonstrated. TMPRSS2, in turn, complexes with the ACE2 receptor, which allows efficient penetration of the virus directly at the cell surface. TMPRSS2 and TMPRSS11D activate the S protein by cleaving it into the S1 and S2 subunits, thus allowing endosome-independent cell entry at the cell membrane. Virus-based therapies include monoclonal antibodies, antiviral peptides that dock to the S protein of viruses, viral nucleic acid synthesis inhibitors and inhibitors for docking to other viral structures and accessory proteins.

Targeting viral genome synthesis as broad-spectrum approach against RNA virus infections.

Zoonotic spillover, i.e. pathogen transmission from animal to human, has repeatedly introduced RNA viruses into the human population. In some cases, where these viruses were then efficiently transmitted between humans, they caused large disease outbreaks such as the 1918 flu pandemic or, more recently, outbreaks of Ebola and Coronavirus disease. These examples demonstrate that RNA viruses pose an immense burden on individual and public health with outbreaks threatening the economy and social cohesion within and across borders. And while emerging RNA viruses are introduced more frequently as human activities increasingly disrupt wild-life eco-systems, therapeutic or preventative medicines satisfying the “one drug-multiple bugs”-aim are unavailable. As one central aspect of preparedness efforts, this review digs into the development of broadly acting antivirals via targeting viral genome synthesis with host- or virus-directed drugs centering around nucleotides, the genomes’ universal building blocks. Following the first strategy, selected examples of host de novo nucleotide synthesis inhibitors are presented that ultimately interfere with viral nucleic acid synthesis, with ribavirin being the most prominent and widely used example. For directly targeting the viral polymerase, nucleoside and nucleotide analogues (NNAs) have long been at the core of antiviral drug development and this review illustrates different molecular strategies by which NNAs inhibit viral infection. Highlighting well-known as well as recent, clinically promising compounds, structural features and mechanistic details that may confer broad-spectrum activity are discussed. The final part addresses limitations of NNAs for clinical development such as low efficacy or mitochondrial toxicity and illustrates strategies to overcome these.

Parvovirus B19 Replication and Expression in Differentiating Erythroid Progenitor Cells.

The pathogenic Parvovirus B19 (B19V) is characterized by a strict adaptation to erythroid progenitor cells (EPCs), a heterogeneous population of differentiating cells with diverse phenotypic and functional properties. In our work, we studied the dynamics of B19V infection in EPCs in dependence on the cell differentiation stage, in terms of distribution of infected cells, synthesis of viral nucleic acids and production of infectious virus. EPCs at early differentiation stage led to an abortive infection, without viral genome replication and a very low transcriptional activity. EPCs at later stages were permissive, with highest levels of viral replicative activity at day 9 (+3.0 Log from 2 to 48 hpi) and lower levels at day 18 (+1.5 Log from 2 to 48 hpi). B19V DNA increment was in accordance with the percentage of cells positive to flow-FISH assay (41.4% at day 9, 1.1% at day 18). Quantitation of total RNA indicated a close association of genome replication and transcription with viral RNA accumulation within infected cells related to viral DNA increase during the course of infection. Analysis of the different classes of mRNAs revealed two distinct pattern of genome expression profile with a fine regulation in the frequency utilization of RNA processing signals: an early phase, when cleavage at the proximal site leading to a higher relative production of mRNA for NS protein, and a late phase, when cleavage at the distal site was more frequent leading to higher relative abundance of mRNA for VP and 11 kDA proteins. Infectious virus was released from cells at day 6–15, but not at day 18. Our results, providing a detailed description of B19V replication and expression profile in differentiating EPCs, highlight the very tight adaptation of B19V to a specific cellular target defined both by its erythroid lineage and its differentiation stage.

Functional analysis and quantitative determination of the expression profile of human parvovirus B19

Comprehension of the pathogenetic potential of human parvovirus B19 requires the definition of the complete spectrum of cellular tropism and a functional analysis of the viral genome in infected cells. In this study, we carried out a systematic functional analysis of B19 virus genome in the course of infection of susceptible bone marrow mononuclear cells and myeloblastoid UT7/EpoS1 cells, in terms of dynamics of nucleic acid synthesis. A PCR array was designed and a comprehensive analysis was performed by quantitative PCR and RT-PCR, yielding extended information on the presence and abundance of the diverse classes of viral nucleic acids, on the temporal regulation of genome expression and on its relationship with the cell cycle. The analysis performed indicate that the synthesis of viral nucleic acids is correlated to the progression through the S phase of the cell cycle, that an extended pattern of transcriptional activity occurs throughout the course of infection, with a maximal rate of transcription preceding the onset of S-phase dependent replication of the viral genome, and that utilization of transcript processing signals is relatively constant throughout the course of infection. The information obtained led to the definition of a unified model of functional and expression profiling of parvovirus B19 genome.

Characterization and cell cycle regulation of the major Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) latent genes and their promoter.

Retinoblastoma tumor suppressor protein (pRB) inhibition by tumor virus oncoproteins has been attributed to the need for these viruses to promote lytic viral nucleic acid synthesis by unscheduled entry into the S phase of the cell cycle. Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8) encodes a functional cyclin (vCYC) which is expressed during latency and can direct phosphorylation of pRB. We mapped the two major latent transcripts encoding vCYC, latent transcript 1 (LT1) and LT2, by cDNA sequencing, 5' rapid amplification of cDNA ends, and primer extension analyses. Both LT1 and LT2 transcripts are spliced, originate from the same start site, and encode ORF K13 (vFLIP) as well as ORF72 (vCYC). The latency-associated nuclear antigen (LANA, ORF73) is encoded by LT1 but spliced from LT2. While differential expression of the two transcripts was not found, the promoter controlling LT1/LT2 transcription is regulated in a cell cycle-dependent manner. Activities of both KSHV LT1/LT2 and huCYC D1 luciferase promoter reporters transfected into NIH 3T3 cells increase 11- and 4-fold, respectively, after release from cell cycle arrest by serum starvation. Further, vCYC and huCYC D2 mRNA levels are low in naturally infected BCBL-1 cells arrested in late G1 with L-mimosine but increase in parallel during a 24-h period after release from cell cycle arrest. Cell cycle regulation of KSHV vCYC expression mimics cellular D cyclin regulation and may maintain infected cell cycling. This is consistent with an alternative hypothesis that tumor viruses have developed specific responses to innate cellular defenses against latent virus infection that include pRB-induced cell cycle arrest.

Further characterization of HIV RNA synthesis early after cell-to-cell transmission infection.

Using a one-step model for cell-to-cell transmission of HIV infection we have identified two distinct phases of HIV RNA synthesis. The first phase (4 h-12 h p.i.) was marked by an increase in only the full-length 9 kb RNA, while the second phase (24 h p.i. onwards) comprised a significant increase in the levels of all three species of viral RNA. We now report that while the continual presence of actinomycin D at 50 micrograms/ml abolished all detectable viral nucleic acid synthesis when virus donor H3B cells were pre-treated with 50 micrograms/ml of actinomycin D (AmD), washed free of unbound drug (a procedure which inhibited > 99% of total cellular RNA transcription) then mixed with untreated recipient Hut 78 cells, normal amounts of full length linear unintegrated viral DNA were produced and the first phase of viral RNA transcription was unaffected. Similarly, when both the virus donor cells and recipient cells were arrested in the late G1 phase of the cell cycle by aphidicolin and then mixed, linear unintegrated viral DNA was the major viral DNA species produced. The appearance of circular viral DNA and progeny virus was inhibited, but the first phase of induced viral RNA synthesis was unaffected. When AZT was added at 2 h or 4 h after cell-cell mixing, the level of 9 kb RNA detected was significantly lower, corresponding to reduction in the level of viral DNA. These and previous results indicate that the template for the first phase of viral RNA synthesis was likely to be newly synthesized, linear unintegrated viral DNA and not pre-existing proviral DNA present in the donor cells. Taken together, our results suggest that there exists a yet to be fully characterized pathway of concurrent viral DNA and RNA synthesis early after cell to cell transmission of HIV infection.

Human immunodeficiency virus neurotropism: an analysis of viral replication and cytopathicity for divergent strains in monocytes and microglia.

Productive replication of human immunodeficiency virus type 1 (HIV-1) in brain macrophages and microglia is a critical component of viral neuropathogenesis. However, how virus-macrophage interactions lead to neurological disease remains incompletely understood. Possibly, a differential ability of virus to replicate in brain tissue macrophages versus macrophages in other tissues underlies HIV-1 neurovirulence. To these ends, we established systems for the isolation and propagation of pure populations of human microglia and then analyzed the viral life cycles of divergent HIV-1 strains in these cells and in cultured monocytes by using identical viral inocula and indicator systems. The HIV-1 isolates included those isolated from blood, lung tissue, cerebrospinal fluids (CSF), and brain tissues of infected subjects: HIV-1(ADA) and HIV-1(89.6) (from peripheral blood mononuclear cells), HIV-1(DJV) and HIV-1(JR-FL) (from brain tissue), HIV-1(SF162) (from CSF), and HIV-1(BAL) (from lung tissue). The synthesis of viral nucleic acids and viral mRNA, cytopathicity, and release of progeny virions were assessed. A significant heterogeneity among macrophage-tropic isolates for infection of monocytes and microglia was demonstrated. Importantly, a complete analysis of the viral life cycle revealed no preferential differences in the abilities of the HIV-1 strains tested to replicate in microglia and/or monocytes. Macrophage tropism likely dictates the abilities of HIV-1 to invade, replicate, and incite disease within its microglial target cells.

Efficient synthesis of viral nucleic acids following monocyte infection by HIV-1

The replication of human immunodeficiency virus type 1 in mononuclear phagocytes (blood monocytes, tissue macrophages,and dendritic cells) is an important feature of HIV-1 pathogenesis. Although most primary HIV-1 isolates are able to productively infect monocytes, some reports suggest that rates of viral DNA synthesis and virus replication are reduced in HIV-1-infected monocytes as compared to infected T cells. In this study we compare kinetics of viral DNA synthesis in CD4 + T cells and monocytes following HIV-1 infection. Our results indicate that reverse transcription of viral nucleic acids following infection of monocytes occurs at rates equal to or greater than that observed following infection of T cells. These studies reveal no postentry restrictions to HIV-1 replication following infection in monocytes. Moreover, the results support the notion that both monocytes and CD4 + T cells are equally permissive for virus replication in infected individuals.

Alpha interferon-induced antiretroviral activities: restriction of viral nucleic acid synthesis and progeny virion production in human immunodeficiency virus type 1-infected monocytes

Alpha interferon (IFN-alpha) restricts multiple steps of the human immunodeficiency virus type 1 (HIV-1) life cycle. A well-described effect of IFN-alpha is in the modulation of viral nucleic acid synthesis. We demonstrate that IFN-alpha influences HIV-1 DNA synthesis principally by reducing the production of late products of reverse transcription. The magnitude of IFN-alpha-induced downregulation of HIV-1 DNA and/or progeny virion production was dependent on the IFN-alpha concentration, the duration of cytokine administration, the multiplicity of infection, the viral strain, and the cycles of viral infection. Interestingly, reductions in viral DNAs could not fully account for the observed IFN-alpha-induced abrogation of progeny virion production. These data, by our investigation of both single-cycle and spreading viral infections, support a predominant but not exclusive effect of IFN-alpha on viral DNA synthesis.

Orobol: An Inhibitor of Vesicular Stomatitis Virus that Blocks the Synthesis of Viral Nucleic Acids and the Glycosylation of G Protein

The naturally occurring isoflavonoid orobol exhibits antiviral effects against some animal viruses. Addition of the compound after virus entry inhibits the appearance of late viral protein synthesis in Vesicular Stomatitis Virus, influenza, or vaccinia virus-infected cells, but has no effect on poliovirus protein synthesis. Concentrations of the compound above 10–50 Mg ml−1are sufficient to decrease the synthesis of VSV proteins when added early during infection, but have no effect on viral translation if added later, indicating that orobol does not block VSV translation directly. The synthesis of VSV nucleic acids is one of the targets of this flavonoid. The synthesis of both minus and plus-stranded viral RNA are inhibited by orobol when added during the first 2 h of infection. In addition, this compound interferes potently with the glycosylation of VSV G protein, indicating that orobol has several targets of antiviral action. The possibility that orobol interferes with the function of the cellular vesicular system is discussed.

Short note: Possible role of macrophage metabolic products including quinolinic acid and neopterin in the pathogenesis of inflammatory brain diseases

It is hypothesized that macrophage metabolic products, including the possible neurotoxic NMDA receptor agonist quinolinic acid, and neopterin, may play a role in the pathogenesis of inflammatory brain diseases. It is suggested that the production of neopterin may have physiological roles, including to inhibit folate synthesis in intracellular pathogenic microorganisms and to inhibit viral nucleic acid synthesis. The possible role of excess neopterin production during chronic infection in the pathogenesis of inflammatory brain diseases is discussed.

Megalomycin C, a macrolide antibiotic that blocks protein glycosylation and shows antiviral activity

Megalomycin C, a natural macrolide antibiotic showed a potent antiherpetic activity. At concentrations that efficiently prevented HSV-1 multiplication, the compound had no cytotoxic or antiproliferative effects. Viral DNA and protein synthesis took place at normal levels in the presence of the antibiotic, suggesting that neither the translation of viral mRNA, nor the synthesis of viral nucleic acids was affected. The incorporation of mannose and galactosamine into viral proteins was blocked and precursor, but not mature, HSV-1 glycoproteins were detected in the presence of megalomycin C. Noninfectious HSV-1 viral particles were formed when the compound was present, but their glycoproteins were not properly glycosylated.

Chapter 12. Antiviral Agents

Publisher SummaryThis chapter discusses the agents with activity primarily against RNA viruses. The communicable diseases of the respiratory tract are probably the most common cause of symptomatic human infections. The viruses that are causative agents for human respiratory disease comprise the five taxonomically distinct families: orthomyxoviridae, paramyxoviridae, picornaviridae, coronaviridae, and adenoviridae. The influenza viruses, which consist of types A, B, and C, belong to the family orthomyxoviridae. Types A and B have been associated with significant increases in mortality during epidemics. The disease may be asymptomatic or cause symptoms ranging from the common cold to fatal pneumonia. Immunization against influenza has been recommended for high-risk groups and antiviral chemotherapy (amantadine) is available for the treatment and prophylaxis of all influenza A infections. There is both a great need for and interest in developing a chemotherapeutic agent for the treatment of these two viral, respiratory tract pathogens. The family picornaviridae contains the genus Rhinovirus that is composed of over a hundred distinct serotypes. Amantadine and rimantadine are specifically active against influenza A virus infections. The amantadine recipients reported a higher incidence of side effects largely attributed to the central nervous system (CNS) symptoms. This difference in side effects may be a pharmacokinetic phenomenon that results in higher plasma concentrations of amantadine. Significant progress continues to be made in the clinical use and development of agents active against DNA viruses. Acyclovir (9-(2-h droxyethoxymethyl)guanine) has been the subject of several reviews and of a syrnposium. Considerable progress has been made in evaluating the clinical promise of acyclovir; however, there remains much to be learned concerning the best use of this drug in clinical practice. Significant strides have been made in the development of clinically useful antiviral agents, especially against the DNA viruses of the herpes family. Most of these agents are directed against viral nucleic acid synthesis and require activation by a virus-induced thymidine kinase. Researchers have begun to focus on other strategies that may produce broader spectrum anti-viral agents with different mechanisms of action.

Viral nucleic acid synthesis and antigen accumulation in pancreas and kidney of Pekin ducks infected with duck hepatitis B virus.

Liver, pancreas, and kidney from Pekin ducks infected with duck hepatitis B virus (DHBV) were assayed for the presence of both viral antigen and replication-specific forms of viral nucleic acid. In young congenitally infected ducks, antigen was detectable in hepatocytes and bile duct epithelia, in kidney glomeruli and tubular epithelia, and in cells localized to pancreatic acini. In older experimentally infected ducks, antigen was detectable in hepatocytes, in glomeruli and tubular epithelia, and in cells localized to presumptive pancreatic alpha-islets. All but the glomeruli-associated viral antigen appeared to be localized to the cytoplasm of antigen-positive cells. Much of the glomeruli-associated antigen appeared to be extracellular and was detected in glomeruli that were positive for the accumulation of immunoglobulin, observations suggestive of the deposition of viral antigen-antibody complexes. As analyzed with bulk tissue, replication-specific forms of viral nucleic acid were detectable in liver and pancreas from the young congenitally infected ducks and in liver and kidney from the older experimentally infected ducks.

Visna DNA synthesis and the tempo of infection in vitro

Visna virus is the prototype of the subfamily of nontransforming retroviruses that cause slow infections in vivo, and lytic infections in vitro. In this paper we present the first quantitative single cell analysis of the synthesis of viral nucleic acids, using improved methods of in situ hybridization; we identify early visna virus DNA synthesis as the rate-limiting step in transcription, virus production, and cell death in vitro; and we show that by manipulating the extent of early DNA synthesis we can slow the tempo of infection in vitro from 3 days to 3 weeks.

Inhibition of sindbis virus replication in Aedes albopictus cells by virazole (Ribavirin) and its reversal by actinomycin: A correction

The antibiotic actinomycin interferes with the antiviral action of ribavirin in Sindbis virus-infected Aedes albopictus mosquito cells. In the absence of actinomycin, ribavirin inhibits viral nucleic acid synthesis whether assayed by infectivity, incorporation of [ 3H]uridine, or ethidium bromide staining.

Viral nucleic acid synthesis in HSV infected neural cells

HSV-1 replication and synthesis of viral DNA and RNA have been examined in gliomas of human (COX) and rat origin (C6) and in mouse neuroblastomas (D2). COX cells fully support HSV-1 replication and show patterns of viral DNA and RNA synthesis similar to those seen in continuous line cells. HSV-1 also grows to high titers in D2 cells but without concomitant high levels of viral DNA and RNA synthesis in the infected cells. Finally, HSV-1 established a persistent infection in C6 cells. Viral mRNA and DNA synthesis could not be detected in these cultures. At cycles of approximately 15--20 days, the persistently infected cultures exhibited massive CPE and relatively high production of infectious HSV.

7-Deazanebularin: METABOLISM IN CULTURES OF MOUSE FIBROBLASTS AND INCORPORATION INTO CELLULAR AND VIRAL NUCLEIC ACIDS

Abstract The metabolism in mouse and chick embryo fibroblast cultures of the purine nucleoside analogue 7-deazanebularin has been studied. The analogue is cytotoxic at low concentrations, it is rapidly phosphorylated and enters the cellular nucleotide pools, and it is incorporated into cellular DNA and RNA. A substantial proportion of the incorporated material is present at the 3' termini of tRNA molecules. 7-Deazanebularin is also incorporated into the RNA of Mengovirus and the DNA of vaccinia virus, and it strongly inhibits cellular and viral nucleic acid synthesis.

The production of interferon by animal viruses.

The production of interferon by animal viruses is a response to the viral nucleic acid. At high multiplicity of infection the nucleic acid of the infecting virus is an adequate stimulus, while at lower multiplicities, viral nucleic acid synthesis is necessary for subsequent interferon formation. It is not known why viral nucleic acids are such effective interferon inducers, nor is it known how their presence within the cell leads to the formation of interferon. It is most likely that the presence of the viral nucleic acids within the cytoplasm leads to a change in the nucleus, resulting in the transcription and translation of the appropriate gene for interferon.