Viral DNA Synthesis Research Articles

NS1-mediated DNMT1 degradation regulates human bocavirus 1 replication and RNA processing.

Methylation of the DNA genome plays an important role in viral gene inactivation. However, the role of DNA methylation in human bocavirus (HBoV) remains unclear. In this study, the HBoV1 genomic DNA was found extensively methylated at the CHG and CHH sites. Inhibiting DNA methylation with 5-aza-2'-deoxycytidine (DAC) altered the methylation status and reduced viral DNA production, while enhanced the RNA splicing at D1 and D3 sites and the polyadenylation at the proximal polyadenylation site, (pA)p. Knockdown of DNA methyltransferase 1 (DNMT1) had the same effect on viral DNA synthesis and RNA processing as the DAC treatment, indicating that DNMT1 is the major host methyltransferase involved in viral DNA methylation. In addition, the nonstructural protein NS1 promoted DNMT1 degradation through the ubiquitin-proteasome pathway to regulate viral replication and RNA processing. Collectively, the results suggest that DNA methylation and DNMT1 facilitate HBoV replication and are essential for appropriate NS1 localization in the nucleus. DNMT1 degradation through NS1 promotes the virus RNA processing, leading to viral protein expression.

Identifying the amino acid domains of ORF59 responsible for interactions with ORF57 and PAN RNA during KSHV lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA polymerase processivity factor, ORF59, is a lytic protein essential for viral DNA synthesis as part of the core replication complex. The multifunctional nature of ORF59 has prompted the investigation into its various functional domains. Prior studies of ORF59 have identified dimerization, DNA interaction, and polymerase interaction domains. The regions of ORF59 responsible for the interaction with the viral mRNA transport accumulation protein (MTA/ORF57) and the viral long non-coding polyadenylated nuclear (PAN) RNA have not been explored. Using a series of previously characterized ORF59 deletion KSHV BACmid mutants, we identified the domains of ORF59 that interact with ORF57 and PAN RNA. Interestingly, amino acids 51-100 were essential for interacting with both ORF57 and PAN RNA. Using this information, we generated a plasmid that expresses a DsRed-tagged polypeptide spanning amino acids 30-100 of ORF59. When the 30-100 aa DsRed-tagged polypeptide expression plasmid was transfected into KSHV wild-type iSLK cells prior to lytic reactivation, a dominant-negative inhibition of virus replication was observed, resulting in a decrease of infectious virus production. Our data suggest that interactions between ORF59 with ORF57 and PAN RNA are important to successful lytic replication.IMPORTANCETo better understand the Kaposi's sarcoma-associated herpesvirus (KSHV) DNA polymerase processivity factor ORF59, we investigated the interaction of ORF59 with ORF57 and polyadenylated nuclear (PAN) RNA. We used a previously characterized KSHV BACmid containing internal deletions of ORF59 to identify the domains of ORF59 that interact with ORF57 and PAN RNA. Our study revealed multiple domains of ORF59 that are essential for its association with PAN RNA. These domains span amino acids 51-100, 251-300, and 351-396. Additional experiments confirmed amino acids 51-100 are critical for the interaction between ORF59 and ORF57. Using this information, we generated an expression plasmid encompassing the ORF57 and PAN RNA interaction domains of ORF59. The ORF59 polypeptide expression plasmid of amino acids 30-100 functioned as a dominant negative inhibitor during viral reactivation and caused a decrease in virus production. These findings provide valuable insights into the key domains of ORF59, essential for its functionality, and ultimately the production of infectious viruses.

Pistacia vera L. as natural source against antimicrobial and antiviral resistance.

Increased global research is focused on the development of novel therapeutics to combat antimicrobial and antiviral resistance. Pistachio nuts represent a good source of protein, fiber, monounsaturated fatty acids, minerals, vitamins, and phytochemicals (carotenoids, phenolic acids, flavonoids and anthocyanins). The phytochemicals found in pistachios are structurally diverse compounds with antimicrobial and antiviral potential, demonstrated as individual compounds, extracts and complexed into nanoparticles. Synergistic effects have also been reported in combination with existing drugs. Here we report an overview of the antimicrobial and antiviral potential of pistachio nuts: studies show that Gram-positive bacterial strains, such as Staphylococcus aureus, are the most susceptible amongst bacteria, whereas antiviral effect has been reported against herpes simplex virus 1 (HSV-1). Amongst the known pistachio compounds, zeaxanthin has been shown to affect both HSV-1 attachment penetration of human cells and viral DNA synthesis. These data suggest that pistachio extracts and derivatives could be used for the topical treatment of S. aureus skin infections and ocular herpes infections.

Potency of a small molecule that targets the molluscum contagiosum virus processivity factor increases when conjugated to a tripeptide

We recently developed compound FC-7269 for targeting the Molluscum contagiosum virus processivity factor (mD4) and demonstrated its ability to inhibit viral processive DNA synthesis in vitro and cellular infection of an mD4-dependent virus (Antiviral Res 211, 2023,105520). However, despite a thorough medicinal chemistry campaign we were unable to generate a potent second analog as a requisite for drug development. We overcame this impasse, by conjugating a short hydrophobic trivaline peptide to FC-7269 to produce FC-TriVal-7269 which significantly increased antiviral potency and reduced cellular toxicity.

Significance of hepatitis B virus capsid dephosphorylation via polymerase

BackgroundIt is generally believed that hepatitis B virus (HBV) core protein (HBc) dephosphorylation (de-P) is important for viral DNA synthesis and virion secretion. HBV polymerase contains four domains for terminal protein, spacer, reverse transcriptase, and RNase H activities.MethodsHBV Polymerase mutants were transfected into HuH-7 cells and assayed for replication and HBc de-P by the Phos-tag gel analysis. Infection assay was performed by using a HepG2-NTCP-AS2 cell line.ResultsHere, we show that a novel phosphatase activity responsible for HBc de-P can be mapped to the C-terminal domain of the polymerase overlapping with the RNase H domain. Surprisingly, while HBc de-P is crucial for viral infectivity, it is essential for neither viral DNA synthesis nor virion secretion. The potential origin, significance, and mechanism of this polymerase-associated phosphatase activity are discussed in the context of an electrostatic homeostasis model. The Phos-tag gel analysis revealed an intriguing pattern of “bipolar distribution” of phosphorylated HBc and a de-P HBc doublet.ConclusionsIt remains unknown if such a polymerase-associated phosphatase activity can be found in other related biosystems. This polymerase-associated phosphatase activity could be a druggable target in clinical therapy for hepatitis B.

A case report on tenofovir induced osteoporosis

The antiretroviral drug tenofovir, is a nucleotide analogue which is used in the treatment of HIV and hepatitis B infections. This drug works by inhibiting the HIV reverse transcriptase enzyme and prevent the synthesis of viral DNA. It is administered as a single 300 mg tablet once daily. This drug is mostly used in the combination with lamivudine, dolutegravir (TLD regimen). Although there has been success in the use of tenofovir in treating HIV infection, there have been some adverse effects associated with it. Multiple studies have implicated tenofovir associated bone loss and renal failure. In order to treat the adverse effects which are caused due to tenofovir, an alternate drug should be administered.

The conserved RNP motif of the herpes simplex virus 1 family B DNA polymerase is crucial for viral DNA synthesis but not polymerase activity

The herpes simplex virus 1 DNA polymerase contains a highly conserved structural motif found in most family B polymerases and certain RNA-binding proteins. To investigate its importance within cells, we constructed a mutant virus with substitutions in two residues of the motif and a rescued derivative. The substitutions resulted in severe impairment of plaque formation, yields of infectious virus, and viral DNA synthesis while not meaningfully affecting expression of the mutant enzyme, its co-localization with the viral single-stranded DNA binding protein at intranuclear punctate sites in non-complementing cells or in replication compartments in complementing cells, or viral DNA polymerase activity. Taken together, our results indicate that the RNA binding motif plays a crucial role in herpes simplex virus 1 DNA synthesis through a mechanism separate from effects on polymerase activity, thus identifying a distinct essential function of this motif with implications for hypotheses regarding its biochemical functions.

Endoplasmic reticulum stress-related gene expression causes the progression of dilated cardiomyopathy by inducing apoptosis.

Background: Previous studies have shown that endoplasmic reticulum stress (ERS) -induced apoptosis is involved in the pathogenesis of dilated cardiomyopathy (DCM). However, the molecular mechanism involved has not been fully characterized. Results: In total, eight genes were obtained at the intersection of 1,068 differentially expressed genes (DEGs) from differential expression analysis between DCM and healthy control (HC) samples, 320 module genes from weighted gene co-expression network analysis (WGCNA), and 2,009 endoplasmic reticulum stress (ERGs). These eight genes were found to be associated with immunity and angiogenesis. Four of these genes were related to apoptosis. The upregulation of MX1 may represent an autocompensatory response to DCM caused by a virus that inhibits viral RNA and DNA synthesis, while acting as an autoimmune antigen and inducing apoptosis. The upregulation of TESPA1 would lead to the dysfunction of calcium release from the endoplasmic reticulum. The upregulation of THBS4 would affect macrophage differentiation and apoptosis, consistent with inflammation and fibrosis of cardiomyocytes in DCM. The downregulation of MYH6 would lead to dysfunction of the sarcomere, further explaining cardiac remodeling in DCM. Moreover, the expression of genes affecting the immune micro-environment was significantly altered, including TGF-β family member. Analysis of the co-expression and competitive endogenous RNA (ceRNA) network identified XIST, which competitively binds seven target microRNAs (miRNAs) and regulates MX1 and THBS4 expression. Finally, bisphenol A and valproic acid were found to target MX1, MYH6, and THBS4. Conclusion: We have identified four ERS-related genes (MX1, MYH6, TESPA1, and THBS4) that are dysregulated in DCM and related to apoptosis. This finding should help deepen understanding of the role of endoplasmic reticulum stress-induced apoptosis in the development of DCM.

Interaction and assembly of the DNA replication core proteins of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes six highly conserved core replication proteins essential for the viral lytic DNA synthesis, including ORF6 (single-stranded DNA-binding protein), ORF9 (DNA polymerase), ORF40/41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), and ORF59 (polymerase processivity factor). Since the protein-protein interactions among KSHV core replication proteins are largely unknown, this study aimed to decipher their interrelationships. Herein, we propose a protein-protein interaction network of these six core replication proteins according to the results obtained from confocal fluorescence microscopy, coimmunoprecipitation, and mammalian two-hybrid (GAL4/VP16) reporter assays. In this interaction network, ORF40/41 plays a central role in the connection of different replication subcomplexes. In addition to the well-conserved helicase-primase subcomplex (consisting of ORF44, ORF56, and ORF40/41) and the replisome subcomplex (consisting of ORF9 and ORF59), our data also suggest that several discrete, stable subcomplexes exist in the cell nucleus. Among these replication subcomplexes in the nucleus, the tetrameric subcomplex composed of ORF44, ORF56, ORF40/41, and ORF6 exhibited the ability to trigger a DNA damage response. By using an established GAL4/VP16-based reporter system and confocal fluorescence microscopy, we additionally found that the heat shock protein 90 (Hsp90) inhibitor, radicicol, significantly inhibited the formation of both the helicase-primase subcomplex and the replisome subcomplex in a dose-dependent manner. Collectively, these data not only provide further insights into the interaction and assembly of KSHV-encoded core replication proteins but also suggest a critical role of Hsp90 in assisting the construction of the viral core replication machinery.IMPORTANCEEukaryotic DNA replication is a highly regulated process that requires multiple replication enzymes assembled onto DNA replication origins. Due to the complexity of the cell's DNA replication machinery, most of what we know about cellular DNA replication has come from the study of viral systems. Herein, we focus our study on the assembly of the Kaposi's sarcoma-associated herpesvirus core replication complex and propose a pairwise protein-protein interaction network of six highly conserved viral core replication proteins. A detailed understanding of the interaction and assembly of the viral core replication proteins may provide opportunities to develop new strategies against viral propagation.

Mechanistic Understanding of the Antiviral Properties of Pistachios and Zeaxanthin against HSV-1.

The search for alternative clinical treatments to fight resistance and find alternative antiviral treatments for the herpes simplex virus (HSV) is of great interest. Plants are rich sources of novel antiviral, pharmacologically active agents that provide several advantages, including reduced side effects, less resistance, low toxicity, and different mechanisms of action. In the present work, the antiviral activity of Californian natural raw (NRRE) and roasted unsalted (RURE) pistachio polyphenols-rich extracts was evaluated against HSV-1 using VERO cells. Two different extraction methods, with or without n-hexane, were used. Results showed that n-hexane-extracted NRRE and RURE exerted an antiviral effect against HSV-1, blocking virus binding on the cell surface, affecting viral DNA synthesis as well as accumulation of ICP0, UL42, and Us11 viral proteins. Additionally, the identification and quantification of phenolic compounds by RP-HPLC-DAD confirmed that extraction with n-hexane exclusively accumulated tocopherols, carotenoids, and xanthophylls. Amongst these, zeaxanthin exhibited strong antiviral activity against HSV-1 (CC50: 16.1 µM, EC50 4.08 µM, SI 3.96), affecting both the viral attachment and penetration and viral DNA synthesis. Zeaxanthin is a dietary carotenoid that accumulates in the retina as a macular pigment. The use of pistachio extracts and derivates should be encouraged for the topical treatment of ocular herpetic infections.

Proliferating cell nuclear antigen inhibitors block distinct stages of herpes simplex virus infection.

Proliferating cell nuclear antigen (PCNA) forms a homotrimer that encircles replicating DNA and is bound by DNA polymerases to add processivity to cellular DNA synthesis. In addition, PCNA acts as a scaffold to recruit DNA repair and chromatin remodeling proteins to replicating DNA via its interdomain connecting loop (IDCL). Despite encoding a DNA polymerase processivity factor UL42, it was previously found that PCNA associates with herpes simplex virus type 1 (HSV-1) replication forks and is necessary for productive HSV-1 infection. To define the role that PCNA plays during viral DNA replication or a replication-coupled process, we investigated the effects that two mechanistically distinct PCNA inhibitors, PCNA-I1 and T2AA, have on the HSV-1 infectious cycle. PCNA-I1 binds at the interface between PCNA monomers, stabilizes the homotrimer, and may interfere with protein-protein interactions. T2AA inhibits select protein-protein interactions within the PCNA IDCL. Here we demonstrate that PCNA-I1 treatment results in reduced HSV-1 DNA replication, late gene expression, and virus production, while T2AA treatment results in reduced late viral gene expression and infectious virus production. To pinpoint the mechanisms by which PCNA inhibitors affect viral processes and protein recruitment to replicated viral DNA, we performed accelerated native isolation of proteins on nascent DNA (aniPOND). Results indicate that T2AA inhibits recruitment of the viral uracil glycosylase UL2 and transcription regulatory factors to viral DNA, likely leading to a defect in viral base excision repair and the observed defect in late viral gene expression and infectious virus production. In addition, PCNA-I1 treatment results in decreased association of the viral DNA polymerase UL30 and known PCNA-interacting proteins with viral DNA, consistent with the observed block in viral DNA replication and subsequent processes. Together, we conclude that inhibitors of cellular PCNA block recruitment of key viral and cellular factors to viral DNA to inhibit viral DNA synthesis and coupled processes.

Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection.

Innate immune responses are crucial for limiting virus infection. However, viruses often hijack our best defenses for viral objectives. Human Cytomegalovirus (HCMV) is a beta herpesvirus which establishes a life-long latent infection. Defining the virus-host interactions controlling latency and reactivation is vital to the control of viral disease risk posed by virus reactivation. We defined an interaction between UL138, a pro-latency HCMV gene, and the host deubiquitinating complex, UAF1-USP1. UAF1 is a scaffold protein pivotal for the activity of ubiquitin specific peptidases (USP), including USP1. UAF1-USP1 sustains an innate immune response through the phosphorylation and activation of signal transducer and activator of transcription-1 (pSTAT1), as well as regulates the DNA damage response. After the onset of viral DNA synthesis, pSTAT1 levels are elevated in infection and this depends upon UL138 and USP1. pSTAT1 localizes to viral centers of replication, binds to the viral genome, and influences UL138 expression. Inhibition of USP1 results in a failure to establish latency, marked by increased viral genome replication and production of viral progeny. Inhibition of Jak-STAT signaling also results in increased viral genome synthesis in hematopoietic cells, consistent with a role for USP1-mediated regulation of STAT1 signaling in the establishment of latency. These findings demonstrate the importance of the UL138-UAF1-USP1 virus-host interaction in regulating HCMV latency establishment through the control of innate immune signaling. It will be important going forward to distinguish roles of UAF1-USP1 in regulating pSTAT1 relative to its role in the DNA damage response in HCMV infection.

Anti-Monkeypox Infection Approaches: From Prevention to Therapeutic Lines.

Anti-Monkeypox Infection Approaches: From Prevention to Therapeutic Lines.

Monkeypox: Virology, laboratory diagnosis and therapeutic approach.

Monkeypox infection outbreaks have been observed sporadically in Africa, usually as a result of interaction with wildlife reservoirs. The genomes of the new strain range in size from 184.7 to 198.0kb and are identified with 143-214 open reading frames. Viral cores are rapidly carried on microtubules away from the cell's perimeter and deeper into the cytoplasm once the virus and cell membranes fuse. Depending on the kind of exposure, patients with monkeypox may experience a febrile prodrome 5-13 days after exposure, which frequently includes lymphadenopathy, malaise, headaches, and muscle aches. A different diagnostic approach is available for monkeypox, including histopathological analysis, electron microscopy, immunoassays, polymerase chain reaction, genome sequencing, microarrays, loop-mediated isothermal amplification technology and CRISPR (i.e., "clustered regularly interspaced short palindromic repeats"). There are currently no particular, clinically effective treatments available for the monkeypox virus. An initial treatment is cidofovir. As a monophosphate nucleotide analog, cidofovir is transformed into an inhibitor of viral DNA polymerase by cellular kinases, which is analogous to cidofovir's function in inhibiting viral DNA synthesis. The European Medicine Agency and the Food and Drug Administration have both granted permission for IMVAMUNE, a replication-deficient, attenuated third-generation modified vaccinia Ankara vaccine, to be used for the prevention of smallpox and monkeypox in adults.

Early-Phase Drive to the Precursor Pool: Chloroviruses Dive into the Deep End of Nucleotide Metabolism.

Viruses face many challenges on their road to successful replication, and they meet those challenges by reprogramming the intracellular environment. Two major issues challenging Paramecium bursaria chlorella virus 1 (PBCV-1, genus Chlorovirus, family Phycodnaviridae) at the level of DNA replication are (i) the host cell has a DNA G+C content of 66%, while the virus is 40%; and (ii) the initial quantity of DNA in the haploid host cell is approximately 50 fg, yet the virus will make approximately 350 fg of DNA within hours of infection to produce approximately 1000 virions per cell. Thus, the quality and quantity of DNA (and RNA) would seem to restrict replication efficiency, with the looming problem of viral DNA synthesis beginning in only 60-90 min. Our analysis includes (i) genomics and functional annotation to determine gene augmentation and complementation of the nucleotide biosynthesis pathway by the virus, (ii) transcriptional profiling of these genes, and (iii) metabolomics of nucleotide intermediates. The studies indicate that PBCV-1 reprograms the pyrimidine biosynthesis pathway to rebalance the intracellular nucleotide pools both qualitatively and quantitatively, prior to viral DNA amplification, and reflects the genomes of the progeny virus, providing a successful road to virus infection.

Chelerythrine inhibits HSV-1 infection by suppressing virus binding to the cells

ObjectiveHerpes simplex virus type 1 (HSV-1) is a worldwide pathogen and mainly puts immunocompromised patients at higher risk of various complications. This research aims to demonstrate the antiviral activity of chelerythrine (CHE), an alkaloid compound derived from the traditional Chinese medicine Mcleaya cordata, towards the HSV-1 F strain. MethodsBioassay-guided fractionation was carried out in order to isolate the antiviral compound CHE. The plaque reduction assay was used to evaluate the antiviral activity of CHE. The combination of quantitative PCR (qPCR) and western blot was used to elucidate whether CHE interferes with viral DNA synthesis, followed by time-of-addition assay to examine which steps of HSV-1 infection CHE affected. The immunofluorescent assay and transmission electron microscopy assay were performed to further analyze the intracellular localization and the ultrastructural features of HSV-1 virions with the treatment of CHE. The binding affinity in CHE to targeted viral glycoprotein was investigated by surface plasmon resonance (SPR) assay and docking simulation. ResultsCHE displayed a significant antiviral effect against HSV-1 at 0.312 to 2.5 µM with a viral titer reduction of 29.38% to 61.02%. CHE had an EC50 value of 1.78 µM against HSV-1, and the selectivity index (SI) for CHE was 4.07. Additionally, CHE inhibits ICP0 expression in the immediate early stage of infection, which further affected the early stage of infection, and subsequent viral replication. Results of the SPR assay showed that CHE had strong-binding affinity to gB with a KD value of 7.80 × 107 M, while gD with a value of 1.18 × 106 M. TEM revealed that CHE can clearly inhibit the virion release. In silico experiments further confirmed that CHE had low binding free energy in the activity sites of gB and gD. ConclusionThis study revealed that CHE directly targeted viral gB or gD as a potent glycoprotein inhibitor, which suggested that CHE could be a virus entry inhibitor for HSV-1 treatment.

Replicative bypass studies of l-deoxyribonucleosides in Vitro and in E. coli cell

l-nucleosides were the most important antiviral lead compounds because they can inhibit viral DNA polymerase and DNA synthesis of many viruses, whereas they may lead to mutations in DNA replication and cause genomic instability. In this study, we reported the replicative bypass of l-deoxynucleosides in recombinant DNA by restriction enzyme–mediated assays to examine their impact on DNA replication in vitro and in E. coli cells. The results showed that a template l-dC inhibited Taq DNA polymerase reaction, whereas it can be bypassed by Vent (exo-) DNA polymerase as well as in cell replication, inserting correct nucleotides opposite l-dC. l-dG can be bypassed by Taq DNA polymerase and in E. coli cells, maintaining insertion of correct incoming nucleotides, and l-dG induced mutagenic replication by Vent (exo-) DNA polymerase. In contrast, l-dA can induced mutagenic replication in vitro and in E. coli cells. MD simulations were performed to investigate how DNA polymerase affected replicative bypass and mutations when d-nucleosides replaced with l-nucleosides. This study will provide a basis for the ability to assess the cytotoxic and mutagenic properties of the l-nucleoside drugs.

156P A phase Ib/II study of nanatinostat (Nstat) plus valganciclovir (VGCV) in advanced Epstein-Barr virus positive (EBV+) solid tumors and with pembrolizumab (PEM) in recurrent/metastatic nasopharyngeal carcinoma (RM-NPC)

EBV is associated with NPC, with the virus in a latent state. Nstat is a Class-I selective oral HDAC inhibitor that induces expression of the lytic BGLF4 EBV protein kinase in EBV+ tumor cells, activating ganciclovir (GCV) via phosphorylation. This results in GCV-mediated inhibition of viral and cellular DNA synthesis and apoptosis. Nstat plus VGCV (oral prodrug of GCV) showed a favorable safety profile with anti-tumor activity in a phase 1b/2 study in R/R EBV+ lymphoma (recommended phase 2 dose [RP2D]: Nstat 20 mg daily [QD], 4 d/wk + VGCV 900 mg QD).

Anti-Herpes Simplex Virus Type-1 Activity of Kyllinga nemoralis Roots Aqueous Extract

Human Herpesvirus 1 known as herpes simplex virus type 1 (HSV-1) is belonged to the Herpesviridae family, Alphaherpesvirinae subfamily, and Simplexvirus genus. Kyllinga nemoralis, known as whitehead spike sedge, white kyllinga, white globose spike, or poverty grass, is a monocotyledonous flowering graminoid similar to grasses and rushes. Acyclovir (ACV) did not eradicate HSV-1 infection as the drug only focuses on inhibiting the production of new viral genomes by interfering with viral DNA synthesis. This study aimed to identify the cytotoxic concentration (CC50) of K. nemoralis roots aqueous extract and evaluate its antiviral activities against HSV-1. 
 
 Cytotoxicity assay involved the incubation of Vero cells with 10 concentrations of extract ranging between 0.002 mg/mL and 1.000 mg/mL. MTT solution was added and absorbance was measured at 540 nm [1]. The CC50 of extract was determined from the graph of cell viability (%) against extract concentrations. Antiviral assays of post-treatment, pre-treatment and virucidal tests determined the mode of antiviral activity. The 50 % effective concentration (EC50) of extract was derived from the graph of plaque inhibition (%) against concentrations. Selectivity index (SI) was evaluated as the ratio of CC50 to EC50 [2]. 
 
 
 Post-treatment assay: Cells were inoculated with HSV-1 for 2 hours. Extract with (2% DMEM and 1% MCS) was added into cells and incubated for 48 hours. 
 Pre-treatment assay: Cells were treated with extract in 5% DMEM for 24 hours and inoculated with HSV-1 for 1 hour 30 minutes. Then, cells were overlaid with (2% DMEM and 1% MCS) and incubated for 48 hours. 
 Virucidal assay: HSV-1 was incubated with extract for 30 minutes. Then, virus-extract suspension was inoculated into cells and overlaid with 2% DMEM and 1% MCS for 48 hours. 
 
 
 After 48 hours incubation, cells were stained with crystal violet solution and incubated at room temperature for 30 minutes. The plaques were counted and percentage of plaque inhibition (%) was calculated. 
 The CC50 revealed aqueous extract concentration was able to kill at 50% of cell population. Post-treatment assay showed that 0.062 mg/mL of the extract inhibited intracellular viral replication. Pre-treatment study indicated that 0.02 mg/mL of extract produced protective effects towards cells. Finally, virucidal assay demonstrated that 0.222 mg/mL of extract initiated extracellular interaction with virus in inhibiting infection [3] as shown in Table 1. 
 The cytotoxicity study showed that CC50, 0.72 mg/mL was toxic on cells. Therefore, lower concentrations between 0.016 mg/mL to 0.5 mg/mL, that were least or non-toxic to the cells were utilized in the subsequent antiviral screenings [4]. The extract showed good inhibition of intracellular virus replication with an EC50 of 0.062 mg/mL and SI more than 10 which is 11.61. Similarly, in pre-treatment investigation, the extract produced excellent antiviral activity with an EC50 of 0.02 mg/mL and corresponding SI of 36.00. This finding highlight that the extract is most effective when administered as pre-treatment. Antiviral activity exhibited by roots extract probably results from the inhibition of host cell glycoprotein receptors or due to prevention of binding between HSV-1 virions and host cells [5]. Virucidal assay revealed a weak extracellular interaction between HSV-1 and extract. It was supported by the low SI, 3.24 as shown in Figure 1.1. The finding suggests that 30 minutes of virus-extract exposure could be insufficient for roots extract to show an effective inhibition of virus [2]. In conclusion, the present study revealed that K. nemoralis roots aqueous extract inhibits in-vitro productive HSV-1 infection through three different modes that are effective prevention of intracellular viral replication, excellent interaction with host cell surface to inhibit HSV-1 infection and moderate direct destruction the virus particles.

Replication Compartments of Eukaryotic and Bacterial DNA Viruses: Common Themes Between Different Domains of Host Cells.

Subcellular organization is essential for life. Cells organize their functions into organelles to concentrate their machinery and supplies for optimal efficiency. Likewise, viruses organize their replication machinery into compartments or factories within their host cells for optimal replicative efficiency. In this review, we discuss how DNA viruses that infect both eukaryotic cells and bacteria assemble replication compartments for synthesis of progeny viral DNA and transcription of the viral genome. Eukaryotic DNA viruses assemble replication compartments in the nucleus of the host cell while DNA bacteriophages assemble compartments called phage nuclei in the bacterial cytoplasm. Thus, DNA viruses infecting host cells from different domains of life share common replication strategies.