Viral DNA Synthesis Research Articles

The BOLF1 gene is necessary for effective Epstein–Barr viral infectivity

The Epstein–Barr virus (EBV) is a causative agent of infectious mononucleosis and several malignancies. Here, we focused on an EBV lytic protein, BOLF1, which is conserved throughout the herpesvirus family and is reported to be a virion tegument protein. We first constructed BOLF1-deficient viruses using the bacterial artificial chromosome (BAC) and CRISPR/Cas9 systems. Although the loss of BOLF1 had almost no effect on viral protein expression, DNA synthesis, or extracellular progeny release, EBV infectivity was significantly reduced. Further analysis showed that nuclear transportation of the incoming virus was decreased by the disruption of BOLF1. Our results indicate that BOLF1enhances the infectious potential of progeny virions, at least partly by increasing nuclear transportation of incoming nucleocapsids. We also found that BOLF1 interacted with BKRF4, and the BOLF1 and BKRF4 proteins were localized in the nucleus and perinuclear area, during the viral lytic cycle.

A high rate of polymerization during synthesis of mouse mammary tumor virus DNA alleviates hypermutation by APOBEC3 proteins.

Retroviruses have evolved multiple means to counteract host restriction factors such as single-stranded DNA-specific deoxycytidine deaminases (APOBEC3s, A3s). These include exclusion of A3s from virions by an A3-unreactive nucleocapsid or expression of an A3-neutralizing protein (Vif, Bet). However, a number of retroviruses package A3s and do not encode apparent vif- or bet-like genes, yet they replicate in the presence of A3s. The mode by which they overcome deleterious restriction remains largely unknown. Here we show that the prototypic betaretrovirus, mouse mammary tumor virus (MMTV), packages similar amounts of A3s as HIV-1ΔVif, yet its proviruses carry a significantly lower level of A3-mediated deamination events than the lentivirus. The G-to-A mutation rate increases when the kinetics of reverse transcription is reduced by introducing a mutation (F120L) to the DNA polymerase domain of the MMTV reverse transcriptase (RT). A similar A3-sensitizing effect was observed when the exposure time of single-stranded DNA intermediates to A3s during reverse transcription was lengthened by reducing the dNTP concentration or by adding suboptimal concentrations of an RT inhibitor to infected cells. Thus, the MMTV RT has evolved to impede access of A3s to transiently exposed minus DNA strands during reverse transcription, thereby alleviating inhibition by A3 family members. A similar mechanism may be used by other retroviruses and retrotransposons to reduce deleterious effects of A3 proteins.

Lentiviral Vpx induces alteration of mammalian cell nuclear envelope integrity

Vpx, a virion-associated protein of Human Immunodeficiency Virus 2 (HIV-2) and Simian Immunodeficiency Virus (SIV) counteracts host restriction factor SAMDH1 for efficient viral DNA synthesis in the cytoplasm and mediates subsequent nuclear translocation of the viral genome. Vpx was found to be indispensable in the viral infection of terminally differentiated target cells and macaques infected with virions carrying truncated Vpx showed delayed pathogenesis, suggesting multiple roles of Vpx at different steps in the virus life cycle. The current study demonstrates a novel function of SIVsmPBj1.9 Vpx on the integrity of the nuclear envelope in HeLa cells. Results from the Super-Resolution Structured Illumination Microscopy (SR-SIM) analysis showed that Vpx puncta alter HeLa cell nuclear envelope assembly. Furthermore, three-dimensional (3D) SIM analysis of such regions suggests that Vpx is primed in a specific way to disrupt the nuclear envelope integrity. The nuclear incursion of cytoplasmic proteins through Vpx mediated ruptured nuclear envelope regions suggest that these events might play a critical role in the nuclear entry of otherwise cytoplasmically sequestered molecules and theirby may be assisting Vpx functions including the transport of viral genome into the nucleus, which is critical for the establishment of virus infection and pathogenesis.

Herpes Simplex Virus 1 Lytic Infection Blocks MicroRNA (miRNA) Biogenesis at the Stage of Nuclear Export of Pre-miRNAs.

Herpes simplex virus 1 (HSV-1) switches between two infection programs, productive ("lytic") and latent infection. Some HSV-1 microRNAs (miRNAs) have been hypothesized to help control this switch, and yet little is known about regulation of their expression. Using Northern blot analyses, we found that, despite inherent differences in biogenesis efficiency among six HSV-1 miRNAs, all six exhibited high pre-miRNA/miRNA ratios during lytic infection of different cell lines and, when detectable, in acutely infected mouse trigeminal ganglia. In contrast, considerably lower ratios were observed in latently infected ganglia and in cells transduced with lentiviral vectors expressing the miRNAs, suggesting that HSV-1 lytic infection blocks miRNA biogenesis. This phenomenon is not specific to viral miRNAs, as a host miRNA expressed from recombinant HSV-1 also exhibited high pre-miRNA/miRNA ratios late during lytic infection. The levels of most of the mature miRNAs remained stable during infection in the presence of actinomycin D, indicating that the high ratios are due to inefficient pre-miRNA conversion to miRNA. Cellular fractionation experiments showed that late (but not early) during infection, pre-miRNAs were enriched in the nucleus and depleted in the cytoplasm, indicating that nuclear export was blocked. A mutation eliminating ICP27 expression or addition of acyclovir reduced pre-miRNA/miRNA ratios, but mutations drastically reducing Us11 expression did not. Thus, HSV-1 lytic infection inhibits miRNA biogenesis at the step of nuclear export and does so in an ICP27- and viral DNA synthesis-dependent manner. This mechanism may benefit the virus by reducing expression of repressive miRNAs during lytic infection while permitting elevated expression during latency.IMPORTANCE Various mechanisms have been identified by which viruses target host small RNA biogenesis pathways to achieve optimal infection outcomes. Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen whose successful persistence in the host entails both productive ("lytic") and latent infection. Although many HSV-1 miRNAs have been discovered and some are thought to help control the lytic/latent switch, little is known about regulation of their biogenesis. By characterizing expression of both pre-miRNAs and mature miRNAs under various conditions, this study revealed striking differences in miRNA biogenesis between lytic and latent infection and uncovered a regulatory mechanism that blocks pre-miRNA nuclear export and is dependent on viral protein ICP27 and viral DNA synthesis. This mechanism represents a new virus-host interaction that could limit the repressive effects of HSV-1 miRNAs hypothesized to promote latency and may shed light on the regulation of miRNA nuclear export, which has been relatively unexplored.

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells.

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Quantitative monitoring of the cytoplasmic release of NCp7 proteins from individual HIV-1 viral cores during the early steps of infection

Fluorescence microscopy imaging of individual HIV-1 viruses necessitates a specific labeling of viral structures that minimally perturbs the infection process. Herein, we used HIV-1 pseudoviruses containing NCp7 fused to a tetracystein (TC) tag, labeled by a biarsenical fluorescein derivative (FlAsH) to quantitatively monitor the NCp7 protein concentration in the viral cores during the early stages of infection. Single particle imaging of individual pseudoviruses with defined ratios of TC-tagged to non tagged NCp7 proteins, together with theoretical modeling of energy transfer between FlAsH dyes, showed that the high packaging of TC-tagged proteins in the viral cores causes a strong fluorescence quenching of FlAsH and that the fluorescence intensity of individual viral complexes is an appropriate parameter to monitor changes in the amount of NCp7 molecules within the viral particles during infection. Interestingly, we observed a dramatic fluorescence increase of individual FlAsH-labeled pseudoviruses containing 100% TC-tagged NCp7 proteins in infected cells at 8 and 16 h post-infection. This effect was significantly lower for pseudoviruses expressing TC-tagged integrase. Therefore, this fluorescence increase is likely related to the cytoplasmic viral transformation and the release of NCp7 molecules from the viral complexes. This loss of quenching effect is largely reduced when reverse transcriptase is inhibited, showing that NCp7 release is connected to viral DNA synthesis. A spatial analysis further revealed that NCp7-TC release is more pronounced in the perinuclear space, where capsid disassembly is thought to be completed. Quantification of NCp7-TC content based on fluorescence quenching presented in this study evidences for the first time the cytoplasmic release of NCp7 during the remodeling of HIV-1 viral particles on their journey toward the nucleus. The developed approach can be applied to quantify dye concentrations in a wide range of nano-objects by fluorescence microscopy techniques.

In vitro growth kinetics and gene expression analysis of the turkey adenovirus 3, a siadenovirus

Turkey adenovirus 3 (TAdV-3) belongs to the genus Siadenovirus, family Adenoviridae. Previously, nucleotide sequencing and annotation of the Virginia avirulent strain (VAS) of TAdV-3 genome, isolated in our laboratory, indicated the presence of a total of 23 genes and open reading frames (ORFs). The goals of this study were 1) to delineate the growth kinetics of the virus using a qPCR-based infectivity assay, and 2) to determine the virus gene expression profile during the early and late phases of infection in target B lymphocytes. The one-step growth curve experiment demonstrated 3 phases of virus replication cycle: a lag phase lasted for 12–18 h post-infection (h.p.i.), in which the virus titer declined; a log phase from 18 to 120 h.p.i., in which the number of infectious virus particles increased over 20,000 folds, and a brief decline phase thereafter. Southern blot analysis indicated that the synthesis of new viral DNA started by 8 h.p.i. Gene-specific RT-PCR analysis revealed the expression of mRNAs from the 23 TAdV-3 genes/ORFs. According to the temporal transcriptional profiling of TAdV-3 genome, genes could be divided into 3 groups based on the time of transcription initiation: group 1 showed detectable levels of transcription at 2 h.p.i and included 7 genes, i.e., hyd, III, pX, pVI, II, 100 K, and 33 K; group 2 included 12 genes whose mRNAs were detected for the first time at 4 h.p.i., i.e., ORF1, IVa2, pol, pTP, pIIIa, EP, DBP, E3, U exon, IV, ORF7, and ORF8; group 3 of transcripts were detectable starting 8 h.p.i. and included only 4 genes, i.e., 52 K, 22 K, pVII, and pVIII. Our data suggest that the transcriptional kinetics of genus Siadenovirus differ from that observed in other adenoviral genera; however, a few TAdV-3 genes showed similar expression patterns to their adenoviral homologs.

Everolimus delayed and suppressed cytomegalovirus DNA synthesis, spread of the infection, and alleviated cytomegalovirus infection

Everolimus is an inhibitor of mammalian target of rapamycin (mTOR) and reduces the risk of cytomegalovirus (CMV) infection in transplant recipients. Everolimus inhibits mTOR complex 1, which regulates factors involved in several crucial cellular functions and is required for CMV replication. However, it is not clear how everolimus regulates CMV replication and prevents and alleviates CMV infection. Effects of everolimus on CMV infection, spread, and DNA synthesis and release from infected cells were assessed by plaque formation, infectious centre assay, real-time PCR of infected cells, and culture supernatant in CMV-infected cultures with and without everolimus. Everolimus enhanced plaque formation by 3.6 times, but the size of the plaques was reduced to 36.4% of untreated cultures in the absence of a pretreatment period. Everolimus reduced viral adsorption but enhanced the replication efficiency of inoculated virus, resulting in an increase in plaque number in the early phase of infection. Preinfection treatment of cells with everolimus efficiently exhibited its antiviral efficacy, and everolimus delayed and suppressed viral DNA synthesis and release from infected cells. Everolimus had suppressed the spread of infection and reduced the number of total infected cells to 40% of untreated cells on day 9, indicating reduction of the size of CMV lesions to one-sixth in 2–3 replication cycles. Preinfection treatment of the cells with everolimus augmented its suppressive effect on CMV infection and replication. Everolimus reduced the total number of infected cells and limited the CMV lesions, and this reduction in the spread of CMV infection would alleviate CMV infection in transplant recipients.

Identification of a novel bipartite nuclear localization signal in the DNA polymerase of the betabaculovirus Pieris rapae granulovirus.

DNA polymerase (DNApol) is highly conserved in all baculoviruses and plays an essential role in viral DNA replication. Previous results showed that the DNApol of the betabaculovirus Pieris rapae granulovirus (PiraGV) can localize in the nucleus. However, it is not clear how the DNApol is transported into the nucleus. Bioinformatic and GFP localization analysis showed that PiraGV DNApol contains a nuclear localization signal (NLS) at aa 4-25 (LFKRKLDEPPTDHTLVKAIKLS) of the N-terminus that does not match either the classical monopartite or the bipartite NLS consensus sequence. Multiple-point-substitution analysis confirmed that the NLS is required for transport of PiraGV DNApol into the nucleus. We also substituted the NLS of the PiraGV DNApol for that of the alphabaculovirus Spodoptera litura nuclear polyhedrosis virus (SpltNPV) DNApol. A viral growth curve and quantitative real-time PCR revealed that the substitution impaired viral DNA replication and resulted in a reduction in virus production. Together, our results show that PiraGV contains a novel NLS and that the NLS cannot efficiently replace that of SpltNPV DNApol for viral DNA synthesis and virus production.

Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients.

Extracellular HBV RNA has been detected in both HBV-replicating cell culture media and sera from chronic hepatitis B (CHB) patients, but its exact origin and composition remain controversial. Here, we demonstrated that extracellular HBV RNA species were of heterogeneous lengths, ranging from the length of pregenomic RNA to a few hundred nucleotides. In cell models, these RNAs were predominantly associated with naked capsids, although virions also harbored a minority of them. Moreover, HBV RNAs in hepatitis B patients' blood circulation were localized in unenveloped capsids in the form of capsid-antibody complexes (CACs) and in virions. Furthermore, we showed that extracellular HBV RNAs could serve as the template for viral DNA synthesis. In conclusion, extracellular HBV RNAs mainly consist of pgRNA or the pgRNA species degraded by the RNase H domain of the polymerase in the process of viral DNA synthesis and circulate as CACs and virions. Their presence in blood circulation of CHB patients may be exploited to develop novel biomarkers for HBV persistence.IMPORTANCE Although increasing evidence suggests the presence of extracellular HBV RNA species, their origin and molecular forms are still under debate. In addition to the infectious virions, HBV is known to secrete several species of incomplete viral particles, including hepatitis B surface antigen (HBsAg) particles, naked capsids, and empty virions, during its replication cycle. Here, we demonstrated that extracellular HBV RNAs were associated with naked capsids and virions in HepAD38 cells. Interestingly, we found that unenveloped capsids circulate in the blood of hepatitis B patients in the form of CACs and, together with virions, serve as vehicles carrying these RNA molecules. Moreover, extracellular HBV RNAs are heterogeneous in length and represent either pregenomic RNA (pgRNA) or products of incomplete reverse transcription during viral replication. These findings provide a conceptual basis for further application of extracellular RNA species as novel biomarkers for HBV persistence.

Marek's Disease Virus Activates the PI3K/Akt Pathway Through Interaction of Its Protein Meq With the P85 Subunit of PI3K to Promote Viral Replication.

It is known that viruses can active the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in host cells to support cell survival and viral replication; however, the role of PI3K/Akt signaling in the pathogenic mechanisms induced by Marek’s disease virus (MDV) which causes a neoplastic Marek’s disease in poultry, remains unknown. In this study, we showed that MDV activated the PI3K/Akt pathway in chicken embryo fibroblasts (CEFs) at the early phase of infection, whereas treatment with a PI3K inhibitor LY294002 prior to MDV infection decreased viral replication and DNA synthesis. Flow cytometry analysis showed that inhibition of the PI3K/Akt pathway could significantly increase apoptosis in MDV-infected host cells, indicating that activation of PI3K/Akt signaling could facilitate viral replication through support of cell survival during infection. Evaluation of the underlying molecular mechanism by co-immunoprecipitation and laser confocal microscopy revealed that a viral protein Meq interacted with both p85α and p85β regulatory subunits of PI3K and could induce PI3K/Akt signaling in Meq-overexpressing chicken fibroblasts. Our results showed, for the first time, that MDV activated PI3K/Akt signaling in host cells through interaction of its Meq protein with the regulatory p85 subunit of PI3K to delay cell apoptosis and promote viral replication. This study provides clues for further studies of the molecular mechanisms underlying MDV infection and pathogenicity for the host.

SAMHD1 Suppression of Antiviral Immune Responses.

SAMHD1 is a host triphosphohydrolase that degrades intracellular deoxynucleoside triphosphates (dNTPs) to a lower level that restricts viral DNA synthesis, and thus prevents replication of diverse viruses in nondividing cells. Recent progress indicates that SAMHD1 negatively regulates antiviral innate immune responses and inflammation through interacting with various key proteins in immune signaling and DNA damage-repair pathways. SAMHD1 can also modulate antibody production in adaptive immune responses. In this review, we summarize how SAMHD1 regulates antiviral immune responses through distinct mechanisms, and discuss the implications of these new functions of SAMHD1. Furthermore, we propose important new questions and future directions that can advance functional and mechanistic studies of SAMHD1-mediated immune regulation during viral infections.

Ivermectin inhibits DNA polymerase UL42 of pseudorabies virus entrance into the nucleus and proliferation of the virus in vitro and vivo

Pseudorabies virus (PRV) is an important viral pathogen of pigs that causes huge losses in pig herds worldwide. Ivermectin is a specific inhibitor of importin-α/β-dependent nuclear transport and shows antiviral potential against several RNA viruses by blocking the nuclear localization of viral proteins. Since the replication of DNA viruses is in the nucleus, ivermectin may be functional against DNA virus infections if the DNA polymerase or other important viral proteins enter the nucleus via the importin-α/β-mediated pathway. Here, we determined whether ivermectin suppresses PRV replication in hamster kidney BHK-21 cells and investigated the effect of ivermectin on the subcellular localization of the PRV UL42 protein, the accessory subunit of PRV DNA polymerase. Also, an in vivo anti-PRV assay was conducted in mice. Our data demonstrate that ivermectin treatment inhibits PRV infection in cells in a dose-dependent manner. Treatment of PRV-infected cells with ivermectin significantly suppressed viral DNA synthesis and progeny virus production. Ivermectin disrupted the nuclear localization of UL42 by targeting the nuclear localization signal of the protein in transfected cells. Ivermectin treatment increased the survival rates of mice infected with PRV and relieved infection as indicated by lower clinical scores and fewer gross lesions in the brain. Together, our results suggest that ivermectin may be a therapeutic or preventative agent against PRV infection.

Hepatitis B Virus Serum DNA andRNA Levels in Nucleos(t)ide Analog-Treated or Untreated Patients During Chronic and Acute Infection.

Treatment of chronic hepatitis B (CHB) patients with nucleos(t)ide analogs (NAs) suppresses hepatitis B virus (HBV) DNA synthesis but does not affect synthesis of HBV pregenomic RNA (pgRNA). Hepatitis B virus pgRNA is detectable in the serum during NA treatment and has been proposed as a marker of HBV covalently closed circular DNA activity within the infected hepatocyte. We developed an automated assay for the quantification of serum HBV pgRNA using a dual-target real-time quantitative PCR approach on the Abbott m2000sp/rt system. We demonstrate accurate detection and quantification of serum HBV RNA. Hepatitis B virus DNA was quantified using the Abbott RealTime HBV viral load assay. We further compared serum nucleic acid levels and kinetics in HBV-positive populations. Samples included on-therapy CHB samples (n = 16), samples (n = 89) from 10 treatment naïve CHB subjects receiving 12 weeks of NA treatment with 8-week follow-up, hepatitis B surface antigen-positive blood donor samples (n = 102), and three seroconversion series from plasmapheresis donors (n = 79 samples). Conclusion: During NA treatment of CHB subjects, we observed low correlation of HBV DNA to pgRNA levels; pgRNA concentration was generally higher than HBV DNA concentrations. In contrast, when NA treatment was absent we observed serum pgRNA at concentrations that correlated to HBV DNA and were approximately 2 log lower than HBV DNA. Importantly, we observe this trend in untreated subject samples from both chronic infections and throughout seroconversion during acute infection. Results demonstrate that the presence of pgRNA in serum is part of the HBV lifecycle; constant relative detection of pgRNA and HBV DNA in the serum is suggestive of a linked mechanism for egress for HBV DNA or pgRNA containing virions.

HSV-1 DNA polymerase 3'-5' exonuclease-deficient mutant D368A exhibits severely reduced viral DNA synthesis and polymerase expression.

Herpesviruses, including herpes simplex virus-1, encode and express a DNA polymerase that is required for replication of their dsDNA genomes. The catalytic subunit of this enzyme contains a 3'-5' exonuclease that is involved in proofreading during replication. Although certain mutations that severely impair exonuclease activity are not lethal to the virus, it was reported that virus containing the substitution of alanine for aspartate 368 (D368A), which ablates exonuclease activity, could not be recovered, raising the possibility that this activity is essential for viral replication. To investigate this issue, we produced virus containing this mutation (D368A Pol) using a complementing cell line. D368A Pol virus was unable to form plaques on non-complementing cells. Viral DNA synthesis and polymerase activity were severely inhibited in D368A-infected cells, as was expression of the enzyme, suggesting that effects on polymerase expression rather than on exonuclease activity per se largely explain the lethal phenotype of this mutation.

The aryl hydrocarbon receptor and interferon gamma generate antiviral states via transcriptional repression.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor whose activation induces the expression of numerous genes, with many effects on cells. However, AhR activation is not known to affect the replication of viruses. We show that AhR activation in macrophages causes a block to HIV-1 and HSV-1 replication. We find that AhR activation transcriptionally represses cyclin-dependent kinase (CDK)1/2 and their associated cyclins, thereby reducing SAMHD1 phosphorylation, cellular dNTP levels and both HIV-1 and HSV-1 replication. Remarkably, a different antiviral stimulus, interferon gamma (IFN-γ), that induces a largely non-overlapping set of genes, also transcriptionally represses CDK1, CDK2 and their associated cyclins, resulting in similar dNTP depletion and antiviral effects. Concordantly, the SIV Vpx protein provides complete and partial resistance to the antiviral effects of AhR and IFN-γ, respectively. Thus, distinct antiviral signaling pathways converge on CDK/cyclin repression, causing inhibition of viral DNA synthesis and replication.

MODERN ETHIOTROPIC CHEMOTHERAPY OF HERPESVIRUS INFECTIONS: ADVANCES, NEW TRENDS AND PERSPECTIVES. ALPHAHERPESVIRUSES (PART II).

A key role in the treatment of herpesviral infections is played by modified nucleosides and their predecessors - acyclovir, its L-valine ester (valaciclovir) and famciclovir (prodrug of penciclovir). The biological activity of compounds of this class is determined by their similarity to natural nucleosides. After phosphorylation by viral thymidine kinase and then cell enzymes to the triphosphate forms, acyclovir and penciclovir inhibit the activity of viral DNA polymerase and synthesis of viral DNA. The increasing role of herpesvirus infections in human infectious pathology, as well as the development of drug resistance in viruses, mainly in patients with immunodeficiencies of various origins, necessitate the search for new compounds possessing anti-herpesvirus activity, using as a biological target not DNA polymerase, but other viral proteins and enzymes, unique or different from cellular proteins, performing similar functions.

Genistein inhibits African swine fever virus replication in vitro by disrupting viral DNA synthesis

African swine fever virus (ASFV) is the causal agent of a highly-contagious and fatal disease of domestic pigs, leading to serious socio-economic consequences in affected countries. Once, neither an anti-viral drug nor an effective vaccines are available, studies on new anti-ASFV molecules are urgently need. Recently, it has been shown that ASFV type II topoisomerase (ASFV-topo II) is inhibited by several fluoroquinolones (bacterial DNA topoisomerase inhibitors), raising the idea that this viral enzyme can be a potential target for drug development against ASFV. Here, we report that genistein hampers ASFV infection at non-cytotoxic concentrations in Vero cells and porcine macrophages. Interestingly, the antiviral activity of this isoflavone, previously described as a topo II poison in eukaryotes, is maximal when it is added to cells at middle-phase of infection (8 hpi), disrupting viral DNA replication, blocking the transcription of late viral genes as well as the synthesis of late viral proteins, reducing viral progeny. Further, the single cell electrophoresis analysis revealed the presence of fragmented ASFV genomes in cells exposed to genistein, suggesting that this molecule also acts as an ASFV-topo II poison and not as a reversible inhibitor. No antiviral effects were detected when genistein was added before or at entry phase of ASFV infection. Molecular docking studies demonstrated that genistein may interact with four residues of the ATP-binding site of ASFV-topo II (Asn-144, Val-146, Gly-147 and Leu-148), showing more binding affinity (−4.62 kcal/mol) than ATP4− (−3.02 kcal/mol), emphasizing the idea that this viral enzyme has an essential role during viral genome replication and can be a good target for drug development against ASFV.

Kaposi's Sarcoma-Associated Herpesvirus mRNA Accumulation in Nuclear Foci Is Influenced by Viral DNA Replication and Viral Noncoding Polyadenylated Nuclear RNA

Kaposi's sarcoma-associated herpesvirus (KSHV), like other herpesviruses, replicates within the nuclei of its human cell host and hijacks host machinery for expression of its genes. The activities that culminate in viral DNA synthesis and assembly of viral proteins into capsids physically concentrate in nuclear areas termed viral replication compartments. We sought to better understand the spatiotemporal regulation of viral RNAs during the KSHV lytic phase by examining and quantifying the subcellular localization of select viral transcripts. We found that viral mRNAs, as expected, localized to the cytoplasm throughout the lytic phase. However, dependent on active viral DNA replication, viral transcripts also accumulated in the nucleus, often in foci in and around replication compartments, independent of the host shutoff effect. Our data point to involvement of the viral long noncoding polyadenylated nuclear (PAN) RNA in the localization of an early, intronless viral mRNA encoding ORF59-58 to nuclear foci that are associated with replication compartments.IMPORTANCE Late in the lytic phase, mRNAs from Kaposi's sarcoma-associated herpesvirus accumulate in the host cell nucleus near viral replication compartments, centers of viral DNA synthesis and virion production. This work contributes spatiotemporal data on herpesviral mRNAs within the lytic host cell and suggests a mechanism for viral RNA accumulation. Our findings indicate that the mechanism is independent of the host shutoff effect and splicing but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, PAN RNA. PAN RNA is essential for the viral life cycle, and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus.

Developing and Evaluating Inhibitors against the RNase H Active Site of HIV-1 Reverse Transcriptase.

We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 Å) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA-DNA heteroduplex. The compounds were tested using a variety of substrates: all three compounds inhibited the polymerase-independent RH activity of HIV-1 RT. Time-of-addition experiments showed that the compounds were more potent if they were bound to RT before the nucleic acid substrate was added. The compounds significantly inhibited the site-specific cleavage required to generate the polypurine tract (PPT) RNA primer that initiates the second strand of viral DNA synthesis. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RH active site. These compounds appear to be relatively specific; they do not inhibit either Escherichia coli RNase HI or human RNase H2. The compounds inhibit the replication of an HIV-1-based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to integrase strand transfer inhibitors (INSTIs), nucleoside analogs, or nonnucleoside RT inhibitors (NNRTIs), suggesting that their ability to block HIV replication is related to their ability to block RH cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds.IMPORTANCE Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and integrase), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug-resistant variants that have been selected by the current therapeutics.