Viral DNA Molecules Research Articles

Plant-Derived Epi-Nutraceuticals as Potential Broad-Spectrum Anti-Viral Agents.

Although the COVID-19 pandemic appears to be diminishing, the emergence of SARS-CoV-2 variants represents a threat to humans due to their inherent transmissibility, immunological evasion, virulence, and invulnerability to existing therapies. The COVID-19 pandemic affected more than 500 million people and caused over 6 million deaths. Vaccines are essential, but in circumstances in which vaccination is not accessible or in individuals with compromised immune systems, drugs can provide additional protection. Targeting host signaling pathways is recommended due to their genomic stability and resistance barriers. Moreover, targeting host factors allows us to develop compounds that are effective against different viral variants as well as against newly emerging virus strains. In recent years, the globe has experienced climate change, which may contribute to the emergence and spread of infectious diseases through a variety of factors. Warmer temperatures and changing precipitation patterns can increase the geographic range of disease-carrying vectors, increasing the risk of diseases spreading to new areas. Climate change may also affect vector behavior, leading to a longer breeding season and more breeding sites for disease vectors. Climate change may also disrupt ecosystems, bringing humans closer to wildlife that transmits zoonotic diseases. All the above factors may accelerate the emergence of new viral epidemics. Plant-derived products, which have been used in traditional medicine for treating pathological conditions, offer structurally novel therapeutic compounds, including those with anti-viral activity. In addition, plant-derived bioactive substances might serve as the ideal basis for developing sustainable/efficient/cost-effective anti-viral alternatives. Interest in herbal antiviral products has increased. More than 50% of approved drugs originate from herbal sources. Plant-derived compounds offer diverse structures and bioactive molecules that are candidates for new drug development. Combining these therapies with conventional drugs could improve patient outcomes. Epigenetics modifications in the genome can affect gene expression without altering DNA sequences. Host cells can use epigenetic gene regulation as a mechanism to silence incoming viral DNA molecules, while viruses recruit cellular epitranscriptomic (covalent modifications of RNAs) modifiers to increase the translational efficiency and transcript stability of viral transcripts to enhance viral gene expression and replication. Moreover, viruses manipulate host cells' epigenetic machinery to ensure productive viral infections. Environmental factors, such as natural products, may influence epigenetic modifications. In this review, we explore the potential of plant-derived substances as epigenetic modifiers for broad-spectrum anti-viral activity, reviewing their modulation processes and anti-viral effects on DNA and RNA viruses, as well as addressing future research objectives in this rapidly emerging field.

Constrained evolution of overlapping genes in viral host adaptation: Acquisition of glycosylation motifs in hepadnaviral precore/core genes.

Hepadnaviruses use extensively overlapping genes to expand their coding capacity, especially the precore/core genes encode the precore and core proteins with mostly identical sequences but distinct functions. The precore protein of the woodchuck hepatitis virus (WHV) is N-glycosylated, in contrast to the precore of the human hepatitis B virus (HBV) that lacks N-glycosylation. To explore the roles of the N-linked glycosylation sites in precore and core functions, we substituted T77 and T92 in the WHV precore/core N-glycosylation motifs (75NIT77 and 90NDT92) with the corresponding HBV residues (E77 and N92) to eliminate the sequons. Conversely, these N-glycosylation sequons were introduced into the HBV precore/core gene by E77T and N92T substitutions. We found that N-glycosylation increased the levels of secreted precore gene products from both HBV and WHV. However, the HBV core (HBc) protein carrying the E77T substitution was defective in supporting virion secretion, and during infection, the HBc E77T and N92T substitutions impaired the formation of the covalently closed circular DNA (cccDNA), the critical viral DNA molecule responsible for establishing and maintaining infection. In cross-species complementation assays, both HBc and WHV core (WHc) proteins supported all steps of intracellular replication of the heterologous virus while WHc, with or without the N-glycosylation sequons, failed to interact with HBV envelope proteins for virion secretion. Interestingly, WHc supported more efficiently intracellular cccDNA amplification than HBc in the context of either HBV or WHV. These findings reveal novel determinants of precore secretion and core functions and illustrate strong constraints during viral host adaptation resulting from their compact genome and extensive use of overlapping genes.

Roles of two distinct alphasatellites modulating geminivirus pathogenesis

BackgroundAlphasatellites are small coding DNA satellites frequently associated with a begomovirus/betasatellite complex, where they are known to modulate virulence and symptom development. Two distinct alphasatellites, namely, Cotton leaf curl Multan alphasatellite (CLCuMuA), and Gossypium darwinii symptomless alphasatellite (GDarSLA) associated with Cotton leaf curl Multan virus-India (CLCuMuV-IN) and Ludwigia leaf distortion betasatellite (LuLDB) were found to be associated with yellow mosaic disease of hollyhock (Alcea rosea) plants. In this study, we show that alphasatellites CLCuMuA and GDarSLA attenuate and delay symptom development in Nicotiana benthamiana. The presence of either alphasatellites reduce the accumulation of the helper virus CLCuMuV-IN. However, the levels of the associated betasatellite, LuLDB, remains unchanged. These results suggest that the alphasatellites could contribute to the host defence and understanding their role in disease development is important for developing resistance strategies.MethodsTandem repeat constructs of two distinct alphasatellites, namely, CLCuMuA and GDarSLA associated with CLCuMuV-IN and LuLDB were generated. N. benthamiana plants were co-agroinoculated with CLCuMuV and its associated alphasatellites and betasatellite molecules and samples were collected at 7, 14 and 21 days post inoculation (dpi). The viral DNA molecules were quantified in N. benthamiana plants by qPCR. The sequences were analysed using the MEGA-X tool, and a phylogenetic tree was generated. Genetic diversity among the CLCuMuA and GDarSLA was analysed using the DnaSP tool.ResultsWe observed a reduction in symptom severity and accumulation of helper virus in the presence of two alphasatellites isolated from naturally infected hollyhock plants. However, no reduction in the accumulation of betasatellite was observed. The phylogenetic and genetic variability study revealed the evolutionary dynamics of these distinct alphasatellites , which could explain the role of hollyhock-associated alphasatellites in plants.ConclusionsThis study provides evidence that alphasatellites have a role in symptom modulation and suppress helper virus replication without any discernible effect on the replication of the associated betasatellite.

Deficient uracil base excision repair leads to persistent dUMP in HIV proviruses during infection of monocytes and macrophages.

Non-dividing cells of the myeloid lineage such as monocytes and macrophages are target cells of HIV that have low dNTP pool concentrations and elevated levels of dUTP, which leads to frequent incorporation of dUMP opposite to A during reverse transcription (“uracilation”). One factor determining the fate of dUMP in proviral DNA is the host cell uracil base excision repair (UBER) system. Here we explore the relative UBER capacity of monocytes (MC) and monocyte-derived macrophages (MDM) and the fate of integrated uracilated viruses in both cell types to understand the implications of viral dUMP on HIV diversification and infectivity. We find that the kinetics for MC infection is compatible with their lifetime in vivo and their near absence of hUNG2 activity is consistent with the retention of viral dUMP at high levels at least until differentiation into macrophages, where UBER becomes possible. Overexpression of human uracil DNA glycosylase in MDM prior to infection resulted in rapid removal of dUMP from HIV cDNA and near complete depletion of dUMP-containing viral copies. This finding establishes that the low hUNG2 expression level in these cells limits UBER but that hUNG2 is restrictive against uracilated viruses. In contrast, overexpression of hUNG2 after viral integration did not accelerate the excision of uracils, suggesting that they may poorly accessible in the context of chromatin. We found that viral DNA molecules with incorporated dUMP contained unique (+) strand transversion mutations that were not observed when dUMP was absent (G→T, T→A, T→G, A→C). These observations and other considerations suggest that dUMP introduces errors predominantly during (-) strand synthesis when the template is RNA. Overall, the likelihood of producing a functional virus from in vitro infection of MC is about 50-fold and 300-fold reduced as compared to MDM and activated T cells. The results implicate viral dUMP incorporation in MC and MDM as a potential viral diversification and restriction pathway during human HIV infection.

Characterization of recombinant tomato leaf curl Palampur virus causing leaf curl disease of Basella alba L. in India

Malabar spinach (Basella alba L.), a leafy vegetable native to the Asian and African continents, was observed with rosetting and curling of leaves associated with begomovirus infection. PCR assay involving the universal begomovirus specific primer pair was used to detect the viral infection. Subsequently, the circular DNA molecules associated with the infected samples were enhanced through rolling circle amplification (RCA). After restriction analysis, the desired viral DNA molecules were cloned, sequenced and analyzed. Based on sequencing analysis, the bipartite tomato leaf curl Palampur virus (ToLCPalV) was found to be associated with leaf curl disease of Basella. DNA-A and DNA-B of the ToLCPalV isolate infecting basella (VNS Bas1) revealed 95.65 and 98.64% identity, respectively, with that of the ToLCPalV reported earlier from India. No evidence was found for the association of alpha- and beta-satellites through testing by PCR assay. Interestingly, DNA-A sequences of ToLCPalV infecting Basella appear to be a recombinant of sequences closely related to tomato leaf curl New Delhi virus (ToLCNDV-KY780202). To the best of our knowledge, this is the first confirmed report on the association of ToLCPalV with a leaf curl disease in a new host Basella from India.

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.

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

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

No evidence of bovine papillomavirus type 1 or 2 infection in healthy equids.

There is a large body of evidence supporting bovine papillomavirus types 1 and 2 (BPV1; BPV2) as aetiological agents of equine sarcoids. However, there is conflicting data regarding BPV1/2 infection in sarcoid-free equids. Data obtained between 2007 and 2017 by BPV1/2 screening of sarcoids and nonsarcoid tumours vs. samples from healthy equids are presented to help clarify this issue. Cross-sectional study. Tumour material obtained from horses, donkeys and mules with confirmed sarcoids (n=130), suspected sarcoids (n=120), or nonsarcoid lesions (n=70), skin biopsies from 102 tumour-free horses and dandruff/hair roots from 35 tumour-free donkeys and mules were screened for BPV1/2 infection. Sample DNA was extracted and validated by equine β-actin PCR. BPV1/2 screening was performed by BPV1/2 E5-specific PCR allowing for the detection of less than 10 viral DNA molecules. Twenty-six amplicons were bidirectionally sequenced and compared to known E5 variants using BLAST program. BPV1/2 E5 PCR scored positive for 130/130 diagnosed sarcoids, 63/120 suspected sarcoids and 13/70 nonsarcoid lesions, whereas 137/137 DNA aliquots derived from tumour-free equids tested negative. On predicted E5 protein level, six different BPV1 E5 variants were identified. The diagnosis of equine sarcoid was not confirmed in 120 lesions. Lack of BPV1/2 E5 DNA in tumour-free equids and the prevalence of sarcoid disease in young adult individuals suggest that the time span between initial infection and sarcoid development is short. This contrasts with the long phase of virus latency characterising infection of humans by carcinogenic papillomaviruses. Presence of BPV1/2 DNA in several cases of poor wound healing/hypergranulation and dermatitis points to these skin disorders being possibly co-induced by BPV1/2. PCR screening of tumour tissue/scrapings for BPV1/2 DNA represents a reliable tool for the rapid validation of a clinical diagnosis of equine sarcoid.

Endoplasmic Reticulum Transmembrane Proteins ZDHHC1 and STING Both Act as Direct Adaptors for IRF3 Activation in Teleost.

IFN regulatory factor (IRF)3 is a central regulator for IFN-β expression in different types of pathogenic infections. Mammals have various pathogenic sensors that are involved in monitoring pathogen intrusions. These sensors can trigger IRF3-mediated antiviral responses through different pathways. Endoplasmic reticulum-associated proteins stimulator of IFN gene (STING) and zinc finger DHHC-type containing 1 (ZDHHC1) are critical mediators of IRF3 activation in response to viral DNA infections. In this study, grass carp STING and ZDHHC1 were found to have some similar molecular features and subcellular localization, and both were upregulated upon stimulation with polyinosinic:polycytidylic acid, B-DNA, or Z-DNA. Based on these results, we suggest that grass carp STING and ZDHHC1 might possess some properties similar to their mammalian counterparts. Overexpression of ZDHHC1 and STING in Ctenopharyngodon idella kidney cells upregulated IFN expression, whereas knockdown of IRF3 inhibited IFN activation. In addition, coimmunoprecipitation and GST pull-down assays demonstrated that STING and ZDHHC1 can interact separately with IRF3 and promote the dimerization and nuclear translocation of IRF3. Furthermore, we also found that small interfering RNA-mediated knockdown of STING could inhibit the expression of IFN and ZDHHC1 in fish cells. Similarly, knockdown of STING resulted in inhibition of the IFN promoter. In contrast, ZDHHC1 knockdown also inhibited IFN expression but had no apparent effect on STING, which indicates that STING is necessary for IFN activation through ZDHHC1. In conclusion, STING and ZDHHC1 in fish can respond to viral DNA or RNA molecules in cytoplasm, as well as activate IRF3 and, eventually, trigger IFN expression.

A C-terminal “Tail” Region in the Rous Sarcoma Virus Integrase Provides High Plasticity of Functional Integrase Oligomerization during Intasome Assembly

The retrovirus integrase (IN) inserts the viral cDNA into the host DNA genome. Atomic structures of five different retrovirus INs complexed with their respective viral DNA or branched viral/target DNA substrates have indicated these intasomes are composed of IN subunits ranging from tetramers, to octamers, or to hexadecamers. IN precursors are monomers, dimers, or tetramers in solution. But how intasome assembly is controlled remains unclear. Therefore, we sought to unravel the functional mechanisms in different intasomes. We produced kinetically stabilized Rous sarcoma virus (RSV) intasomes with human immunodeficiency virus type 1 strand transfer inhibitors that interact simultaneously with IN and viral DNA within intasomes. We examined the ability of RSV IN dimers to assemble two viral DNA molecules into intasomes containing IN tetramers in contrast to one possessing IN octamers. We observed that the last 18 residues of the C terminus ("tail" region) of IN (residues 1-286) determined whether an IN tetramer or octamer assembled with viral DNA. A series of truncations of the tail region indicated that these 18 residues are critical for the assembly of an intasome containing IN octamers but not for an intasome containing IN tetramers. The C-terminally truncated IN (residues 1-269) produced an intasome that contained tetramers but failed to produce an intasome with octamers. Both intasomes have similar catalytic activities. The results suggest a high degree of plasticity for functional multimerization and reveal a critical role of the C-terminal tail region of IN in higher order oligomerization of intasomes, potentially informing future strategies to prevent retroviral integration.

The fourth central polypurine tract guides the synthesis of prototype foamy virus plus-strand DNA.

Foamy virus (FV) is a nonpathogenic retrovirus that has the potential to serve as a gene therapy vector. In retroviral replication, the central polypurine tract (cPPT) is used as a primer to synthesize plus-strand DNA. The cPPT is subsequently degraded to produce a single-stranded gap in the double-stranded viral DNA molecule. In the prototype foamy virus (PFV), four cPPT-like motifs have been previously identified, in which there is a gap with uncertain terminals. In this study, we determined the length of the PFV gap varying from 144 to 731bp. The 3' terminus of the cleavage sites is located between 6272bp and 6274bp from the first base of PFV genome, while the 5' terminus is located within a 465bp range. The start and terminal nucleotides of the gap are located on either side of the fourth cPPT element. Deletion, mutation, and replacement of the fourth cPPT with the Human immunodeficiency virus 1 (HIV-1) cPPT resulted in a significant reduction in modified PFV virions, indicating that the fourth cPPT ought to be the primer that guides the synthesis of PFV plus-strand DNA. These results improve the theoretical basis for understanding FVs replication and will help construct new FV vectors with simple genome sequences containing only the necessary cis elements.

Histones Are Rapidly Loaded onto Unintegrated Retroviral DNAs Soon after Nuclear Entry.

Chromosomal structure of nuclear DNA is usually maintained by insertion of nucleosomes into preexisting chromatin, both on newly synthesized DNA atreplication forks and at sites of DNA damage. But during retrovirus infection, a histone-free DNA copy of the viral genome is synthesized that must be loaded with nucleosomes de novo. Here, we show that core histones are rapidly loaded onto unintegrated Moloney murine leukemia virus DNAs. Loading of nucleosomes requires nuclear entry, but does not require viral DNA integration. The histones associated with unintegrated DNAs become marked by covalent modifications, with a delay relative to thetime of core histone loading. Expression from unintegrated DNA can be enhanced by modulation of the histone-modifying machinery. The data show that histone loading onto unintegrated DNAs occurs very rapidly after nuclear entry and does not require prior establishment of an integrated provirus.

Sources of Materials for Paleomicrobiology.

The Paleomicrobiology establishes the diagnosis of ancient infectious diseases by studying ancient pathogens. This recent science also analyzes the evolution of these pathogens, virulence, and their adaptation to their habitat and their vectors. The DNA persists a long time after the death of an organism despite the chemical and enzymatic degradation. The possibility of sequencing bacterial, viral, parasitic and archaeal DNA molecules persists over time.Various sources are used for these studies: frozen tissue and particularly human tissue are a exceptional source for the analysis because at very low temperatures, all biological activity is suspended. The coprolites are a source of choice for studying the human microbiome. Other sources, the ancient bones are the most abundant, however, they may contain only small amounts of DNA due to natural leaching. When the use of the tooth is possible, is a particularly interesting source because of its highly mineralized structure, which gives greater persistence than bone. The calcified tartar deposited on teeth is a source of interest for the study of oral microbiome.All these sources are subject to precautions (gloves and masks hat) at the time of sampling to avoid cross contamination and also be listed in the most precise way because they are precious and rare.

Crystal structure of the Rous sarcoma virus intasome.

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the lifecycle of retroviruses. Retrovirus integrase (IN) catalyzes insertions of both ends of the linear viral DNA into a host chromosome 1. IN from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by N- and C-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric IN-DNA complexes have been reported for IN from prototype foamy virus (PFV) featuring an additional DNA-binding domain and longer interdomain linkers 2–5, the architecture of a canonical three-domain IN bound to DNA remained elusive. Here we report a crystal structure of the three-domain IN from Rous sarcoma virus (RSV) in complex with viral and target DNAs. The structure shows an octameric assembly of IN, in which a pair of IN dimers engage viral DNA ends for catalysis while another pair of non-catalytic IN dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight IN molecules play varying roles to hold the complex together, making an extensive network of protein-DNA and protein-protein contacts that show both conserved and distinct features compared to those observed for PFV IN. Our work highlights diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.

The Tomato yellow leaf curl virus V2 protein forms aggregates depending on the cytoskeleton integrity and binds viral genomic DNA.

The spread of Tomato yellow leaf curl virus (TYLCV) was accompanied by the formation of coat protein (CP) aggregates of increasing size in the cytoplasm and nucleus of infected tomato (Solanum lycopersicum) cells. In order to better understand the TYLCV-host interaction, we investigated the properties and the subcellular accumulation pattern of the non-structural viral protein V2. CP and V2 are the only sense-oriented genes on the virus circular single-stranded DNA genome. Similar to CP, V2 localized to cytoplasmic aggregates of increasing size and as infection progressed was also found in nuclei, where it co-localized with CP. V2 was associated with viral genomic DNA molecules, suggesting that V2 functions as a DNA shuttling protein. The formation and the 26S proteasome-mediated degradation of V2 aggregates were dependent on the integrity of the actin and microtubule cytoskeleton. We propose that the cytoskeleton-dependent formation and growth of V2 aggregates play an important role during TYLCV infection, and that microtubules and actin filaments are important for the delivery of V2 to the 26S proteasome.

Lack of presence of the human cytomegalovirus in human glioblastoma

Recent reports have indicated human cytomegalovirus (HCMV) to be associated with human glioblastoma carcinogenesis. In established examples of viral carcinogenesis, viral DNA and one or more of its products have been detected in most tumor cells of biopsies in the majority of cases. To test whether HCMV is associated with human glioblastoma based on this criterion, we measured the number of viral DNA molecules per cell in both frozen and paraffin-embedded tumor biopsies from 58 patients using real-time quantitative PCR (QPCR). Immunohistochemical and fluorescence in situ hybridization (FISH) to detect HCMV proteins and genome was performed in 10 cases using formalin-fixed paraffin-embedded glioblastoma tissues. Southern blotting using DNA extracted from four glioblastoma cell lines together with immunoblotting using the four cell lines and five glioblastoma tissue samples were also performed. We further confirmed the immunoblot bands using liquid chromatography-tandem mass spectrometry assay. As a result, HCMV DNA was not detected in the tumor cells from any of the glioblastoma cases by QPCR detecting two different HCMV genes, in clear contrast to samples from patients with HCMV infection. Southern blotting and immunoblotting of cell lines and FISH using paraffin sections were all negative. However, immunoblotting and immunohistochemistry using tissue samples were partly positive, but HCMV proteins were not detected by proteomic analysis, suggesting false positivity of the analyses. As our QPCR analysis could detect 10 copies of HCMV DNA mixed with DNA extracted from 104 HCMV-negative cells, we conclude that HCMV is not persistent, at least in the tumor cells, of developed human glioblastoma.

Fast High-throughput Screening of the H1N1 Virus by Parallel Detection with Multi-channel Microchip Electrophoresis

A multi-channel microchip electrophoresis (MCME) method with parallel laser-induced fluorescence (LIF) detection was developed for rapid screening of H1N1 virus. The hemagglutinin (HA) and nucleocapsid protein (NP) gene of H1N1 virus were amplified using polymerase chain reaction (PCR). The amplified PCR products of the H1N1 virus DNA (HA, 116 bp and NP, 195 bp) were simultaneously detected within 25 s in three parallel channels using an expanded laser beam and a charge-coupled device camera. The parallel separations were demonstrated using a sieving gel matrix of 0.3% poly(ethylene oxide) (<TEX>$M_r$</TEX> = 8,000,000) in <TEX>$1{\times}$</TEX> TBE buffer (pH 8.4) with a programmed step electric field strength (PSEFS). The method was ~20 times faster than conventional slab gel electrophoresis, without any loss of resolving power or reproducibility. The proposed MCME/PSEFS assay technique provides a simple and accurate method for fast high-throughput screening of infectious virus DNA molecules under 400 bp.

Increased plasma levels of extracellular mitochondrial DNA during HIV infection: A new role for mitochondrial damage-associated molecular patterns during inflammation

HIV infection is characterized by a chronic inflammatory state. Recently, it has been shown that mitochondrial DNA (mtDNA) released from damaged or dead cells can bind Toll like receptor-9 (TLR9), an intracellular receptor that responds to bacterial or viral DNA molecules. The activation of TLR9 present within monocytes or neutrophils results in a potent inflammatory reaction, with the production of proinflammatory cytokines. We measured plasma levels of mtDNA in different groups of HIV + patients, i.e., those experiencing an acute HIV infection (AHI), long term non progressors (LTNP), late presenters (LP) taking antiretroviral therapy for the first time, and healthy controls. We found that in AHI and LP mtDNA plasma levels were significantly higher than in healthy individuals or in LTNP. Plasma mtDNA levels were not correlated to peripheral blood CD4 + T cell count, nor to markers of immune activation, but had a significant correlation with plasma viral load, revealing a possible role for mtDNA in inflammation, or as a biomarker of virus-induced damage.

The mechanism of retroviral integration from X-ray structures of its key intermediates

To establish successful infection, a retrovirus must insert a DNA replica of its genome into host cell chromosomal DNA1,2. This process is carried out by the intasome, a nucleoprotein complex comprised of a tetramer of integrase (IN) assembled on the viral DNA ends3,4. The intasome engages chromosomal DNA within a target capture complex to carry out strand transfer, irreversibly joining the viral and cellular DNA molecules. Although several intasome/transpososome structures from the DDE(D) recombinase superfamily were reported4-6, the mechanics of target DNA capture and strand transfer by these enzymes have not been established. Herein, we report crystal structures of the intasome from prototype foamy virus in complex with target DNA, elucidating the pre-integration target DNA capture and post-catalytic strand transfer intermediates of the retroviral integration process. The cleft between IN dimers within the intasome accommodates chromosomal DNA in a severely bent conformation, allowing widely spaced IN active sites to access the scissile phosphodiester bonds. Our results elucidate the structural basis for retroviral DNA integration and moreover provide a framework for the design of INs with altered target sequences.

Conservation of HHV-6 DNA polymerase processivity factor sequence and predicted structure suggests it as a target for antiviral development

The replication of human herpesvirus-6 (HHV-6) DNA is catalyzed by the viral DNA polymerase pU38 and the processivity factor pU27 which stabilizes the enzyme on the DNA template. The genetic polymorphism of pU27 among 46 clinical strains of HHV-6 variant A or B and four strains resistant to antivirals was investigated. Overall, 28 amino acid changes (7.6%) and a two-amino acid deletion were identified among the 368 residues of pU27, when using the U1102 (variant A) sequence as the reference. Eleven amino acid changes (3.0%) specifically differentiated both variants. The median intravariant amino acid variability was 1.2% and 0.3% for A and B, respectively. Except for a single change, the pU27 sequence of multi-drug resistant HHV-6 strains was also conserved. Structural models of pU27 for variants A and B were derived from that of the human cytomegalovirus homologue pUL44, and showed either identical or very similar residues in the regions interacting with viral DNA polymerase and viral DNA molecule. As pU27 is both highly conserved and essential for viral replication, it might constitute an interesting target for antiviral chemotherapy.