Nuclear DNA Viruses Research Articles

Sp140L Is a Novel Herpesvirus Restriction Factor.

Herpesviruses, including the oncogenic Epstein-Barr Virus (EBV), must bypass host DNA sensing mechanisms to establish infection. The first viral latency protein expressed, EBNA-LP, is essential for transformation of naïve B cells, yet its role in evading host defenses remains unclear. Using single-cell RNA sequencing of EBNA-LP-Knockout (LPKO)-infected B cells, we reveal an antiviral response landscape implicating the 'speckled proteins' as key restriction factors countered by EBNA-LP. Specifically, loss of SP100 or the primate-specific SP140L reverses the restriction of LPKO, suppresses a subset of canonically interferon-stimulated genes, and restores viral gene transcription and cellular proliferation. Notably, we also identify Sp140L as a restriction target of the herpesvirus saimiri ORF3 protein, implying a role in immunity to other DNA viruses. This study reveals Sp140L as a restriction factor that we propose links sensing and transcriptional suppression of viral DNA to an IFN-independent innate immune response, likely relevant to all nuclear DNA viruses.

Nuclear interferon-stimulated gene product maintains heterochromatin on the herpes simplex viral genome to limit lytic infection.

Interferons (IFN) are expressed in and secreted from cells in response to virus infection, and they induce the expression of a variety of genes called interferon-stimulated genes (ISGs) in infected and surrounding cells to block viral infection and limit spread. The mechanisms of action of a number of cytoplasmic ISGs have been well defined, but little is known about the mechanism of action of nuclear ISGs. Constitutive levels of nuclear interferon-inducible protein 16 (IFI16) serve to induce innate signaling and epigenetic silencing of herpes simplex virus (HSV), but only when the HSV infected cell protein 0 (ICP0) E3 ligase, which promotes IFI16 degradation, is inactivated. In this study, we found that following IFN induction, the pool of IFI16 within the infected cell remains high and can restrict wild-type viral gene expression and replication due to both the induced levels of IFI16 and the IFI16-mediated repression of ICP0 levels. Restriction of viral gene expression is achieved by IFI16 promoting the maintenance of heterochromatin on the viral genome, which silences it epigenetically. These results indicate that a nuclear ISG can restrict gene expression and replication of a nuclear DNA virus by maintaining or preventing the removal of repressive heterochromatin associated with the viral genome.

Genome-Wide Estimation of the Spontaneous Mutation Rate of Human Adenovirus 5 by High-Fidelity Deep Sequencing.

Rates of spontaneous mutation determine the ability of viruses to evolve, infect new hosts, evade immunity and undergo drug resistance. Contrarily to RNA viruses, few mutation rate estimates have been obtained for DNA viruses, because their high replication fidelity implies that new mutations typically fall below the detection limits of Sanger and standard next-generation sequencing. Here, we have used a recently developed high-fidelity deep sequencing technique (Duplex Sequencing) to score spontaneous mutations in human adenovirus 5 under conditions of minimal selection. Based on >200 single-base spontaneous mutations detected throughout the entire viral genome, we infer an average mutation rate of 1.3 × 10−7 per base per cell infection cycle. This value is similar to those of other, large double-stranded DNA viruses, but an order of magnitude lower than those of single-stranded DNA viruses, consistent with the possible action of post-replicative repair. Although the mutation rate did not vary strongly along the adenovirus genome, we found several sources of mutation rate heterogeneity. First, two regions mapping to transcription units L3 and E1B-IVa2 were significantly depleted for mutations. Second, several point insertions/deletions located within low-complexity sequence contexts appeared recurrently, suggesting mutational hotspots. Third, mutation probability increased at GpC dinucleotides. Our findings suggest that host factors may influence the distribution of spontaneous mutations in human adenoviruses and potentially other nuclear DNA viruses.

A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication.

The Herpes Simplex Virus Virion Host Shutoff Protein Enhances Translation of Viral True Late mRNAs Independently of Suppressing Protein Kinase R and Stress Granule Formation.

The herpes simplex virus (HSV) virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs, suppresses host protein synthesis, dampens antiviral responses, and stimulates translation of viral mRNAs. vhs mutants display a host range phenotype: translation of viral true late mRNAs is severely impaired and stress granules accumulate in HeLa cells, while translation proceeds normally in Vero cells. We found that vhs-deficient virus activates the double-stranded RNA-activated protein kinase R (PKR) much more strongly than the wild-type virus does in HeLa cells, while PKR is not activated in Vero cells, raising the possibility that PKR might play roles in stress granule induction and/or inhibiting translation in restrictive cells. We tested this possibility by evaluating the effects of inactivating PKR. Eliminating PKR in HeLa cells abolished stress granule formation but had only minor effects on viral true late protein levels. These results document an essential role for PKR in stress granule formation by a nuclear DNA virus, indicate that induction of stress granules is the consequence rather than the cause of the translational defect, and are consistent with our previous suggestion that vhs promotes translation of viral true late mRNAs by preventing mRNA overload rather than by suppressing eIF2α phosphorylation. The herpes simplex virus vhs RNase plays multiple roles during infection, including suppressing PKR activation, inhibiting the formation of stress granules, and promoting translation of viral late mRNAs. A key question is the extent to which these activities are mechanistically connected. Our results demonstrate that PKR is essential for stress granule formation in the absence of vhs, but at best, it plays a secondary role in suppressing translation of viral mRNAs. Thus, the ability of vhs to promote translation of viral mRNAs can be largely uncoupled from PKR suppression, demonstrating that this viral RNase modulates at least two distinct aspects of RNA metabolism.

Viral Enhancer Mimicry of Host Innate-Immune Promoters

The inflammatory milieu is the natural habitat for a pathogenic infection, characterised by activity of pro-inflammatory signalling pathways and inflammatory cytokines. Viral entry rapidly activates a range of innate-immune signalling events such as the activation of Pattern Recognition Receptors (PRRs) [1]–[5]. A virus must therefore counteract intrinsic cellular and innate-immune responses to successfully complete the replication cycle. Frequently this is accomplished by encoding viral effector molecules that block these cellular responses by working as either structural or functional mimics of host target proteins [6]–[11]. Nuclear DNA viruses are dependent on the host transcriptional machinery to express the first viral genes; for example the immediate-early (IE) control elements of DNA viruses are by definition absolutely dependent on host transcription factors (TF) [12]. Therefore, these viruses are particularly hostage to their host transcriptional environment [13], [14]. Here we propose that mimicry of regulatory DNA sequences by viral regulatory regions may also provide an additional strategy to counteract at IE times of infection the innate-immune response. In this context, viral IE control elements might functionally mimic innate-immune enhancers, taking advantage of the activated immune signalling TFs for promoting viral IE gene expression. In other words: “If you can't beat ‘em. Join ‘em.” In exploring this possibility, we present a synopsis of the promoter-regulatory elements from seven extensively studied mammalian viruses with a DNA stage, and seven promoters representing prototypical cellular innate-immune genes. These are the SV-40 early enhancer, the E1A enhancer of HAdV5, the long terminal repeat (LTR) of HIV-1, the E6/7 long control region (LCR) of both HPV-16 and HPV-18, the major IE (MIE) enhancer of HCMV, and the enhancer-1 (Eh-1) regulatory region of HBV for viral sequences, and the enhancer regions of human IFNB1, IFNG, TNF, IRF1, IL8, IL12B, and IL1B for host sequences. First, we consider similarities between the primary sequence structures of the enhancers. Second, we present arguments for convergent evolution and structural flexibility inherent to enhancer sequences. Third, we discuss functional features and regulatory hallmarks that may be used to define viral enhancer mimicry of cellular immune enhancers.

A Novel Selective LSD1/KDM1A Inhibitor Epigenetically Blocks Herpes Simplex Virus Lytic Replication and Reactivation from Latency

ABSTRACTCellular processes requiring access to the DNA genome are regulated by an overlay of epigenetic modifications, including histone modification and chromatin remodeling. Similar to the cellular host, many nuclear DNA viruses that depend upon the host cell’s transcriptional machinery are also subject to the regulatory impact of chromatin assembly and modification. Infection of cells with alphaherpesviruses (herpes simplex virus [HSV] and varicella-zoster virus [VZV]) results in the deposition of nucleosomes bearing repressive histone H3K9 methylation on the viral genome. This repressive state is modulated by the recruitment of a cellular coactivator complex containing the histone H3K9 demethylase LSD1 to the viral immediate-early (IE) gene promoters. Inhibition of the activity of this enzyme results in increased repressive chromatin assembly and suppression of viral gene expression during lytic infection as well as reactivation from latency in a mouse ganglion explant model. However, available small-molecule LSD1 inhibitors are not originally designed to inhibit LSD1, but rather monoamine oxidases (MAO) in general. Thus, their specificity for and potency to LSD1 is low. In this study, a novel specific LSD1 inhibitor was identified that potently repressed HSV IE gene expression, genome replication, and reactivation from latency. Importantly, the inhibitor also suppressed primary infection of HSV in vivo in a mouse model. Based on common control of a number of DNA viruses by epigenetic modulation, it was also demonstrated that this LSD1 inhibitor blocks initial gene expression of the human cytomegalovirus and adenovirus type 5.IMPORTANCE Epigenetic mechanisms, including histone modification and chromatin remodeling, play important regulatory roles in all cellular processes requiring access to the genome. These mechanisms are often altered in disease conditions, including various cancers, and thus represent novel targets for drugs. Similarly, many viral pathogens are regulated by an epigenetic overlay that determines the outcome of infection. Therefore, these epigenetic targets also represent novel antiviral targets. Here, a novel inhibitor was identified with high specificity and potency for the histone demethylase LSD1, a critical component of the herpes simplex virus (HSV) gene expression paradigm. This inhibitor was demonstrated to have potent antiviral potential in both cultured cells and animal models. Thus, in addition to clearly demonstrating the critical role of LSD1 in regulation of HSV infection, as well as other DNA viruses, the data extends the therapeutic potential of chromatin modulation inhibitors from the focused field of oncology to the arena of antiviral agents.

MicroRNA expression by an oncogenic retrovirus

Although >250 viral microRNAs (miRNAs) are expressed by a range of nuclear DNA viruses, efforts to identify miRNAs expressed by RNA viruses have so far been in vain (1, 2). In PNAS, Kincaid et al. (3) report the identification of five miRNAs encoded by the delta retrovirus bovine leukemia virus (BLV) that are expressed in BLV-transformed B cells. Interestingly, BLV uses RNA polymerase III (pol III) to directly transcribe the pre-miRNA hairpins that give rise to these viral miRNAs, thereby preventing the cleavage of viral genomic RNA and mRNA species as a result of pre-miRNA excision. Moreover, one of the BLV miRNAs, miR-B4, shares the same seed sequence as cellular miR-29 and therefore can down-regulate many of the same mRNA targets. As miR-29 overexpression can induce chronic lymphocytic leukemia (CLL) in transgenic mice (4), and BLV induces CLL-like B-cell neoplasms in infected cattle and sheep (5), it appears likely that these viral miRNAs play an important role in BLV pathogenesis.

High-resolution human cytomegalovirus transcriptome.

Deep sequencing was used to bring high resolution to the human cytomegalovirus (HCMV) transcriptome at the stage when infectious virion production is under way, and major findings were confirmed by extensive experimentation using conventional techniques. The majority (65.1%) of polyadenylated viral RNA transcription is committed to producing four noncoding transcripts (RNA2.7, RNA1.2, RNA4.9, and RNA5.0) that do not substantially overlap designated protein-coding regions. Additional noncoding RNAs that are transcribed antisense to protein-coding regions map throughout the genome and account for 8.7% of transcription from these regions. RNA splicing is more common than recognized previously, which was evidenced by the identification of 229 potential donor and 132 acceptor sites, and it affects 58 protein-coding genes. The great majority (94) of 96 splice junctions most abundantly represented in the deep-sequencing data was confirmed by RT-PCR or RACE or supported by involvement in alternative splicing. Alternative splicing is frequent and particularly evident in four genes (RL8A, UL74A, UL124, and UL150A) that are transcribed by splicing from any one of many upstream exons. The analysis also resulted in the annotation of four previously unrecognized protein-coding regions (RL8A, RL9A, UL150A, and US33A), and expression of the UL150A protein was shown in the context of HCMV infection. The overall conclusion, that HCMV transcription is complex and multifaceted, has implications for the potential sophistication of virus functionality during infection. The study also illustrates the key contribution that deep sequencing can make to the genomics of nuclear DNA viruses.

Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing

Like other eukaryotes, plants use DICER-LIKE (DCL) proteins as the central enzymes of RNA silencing, which regulates gene expression and mediates defense against viruses. But why do plants like Arabidopsis express four DCLs, a diversity unmatched by other kingdoms? Here we show that two nuclear DNA viruses (geminivirus CaLCuV and pararetrovirus CaMV) and a cytoplasmic RNA tobamovirus ORMV are differentially targeted by subsets of DCLs. DNA virus-derived small interfering RNAs (siRNAs) of specific size classes (21, 22 and 24 nt) are produced by all four DCLs, including DCL1, known to process microRNA precursors. Specifically, DCL1 generates 21 nt siRNAs from the CaMV leader region. In contrast, RNA virus infection is mainly affected by DCL4. While the four DCLs are partially redundant for CaLCuV-induced mRNA degradation, DCL4 in conjunction with RDR6 and HEN1 specifically facilitates extensive virus-induced silencing in new growth. Additionally, we show that CaMV infection impairs processing of endogenous RDR6-derived double-stranded RNA, while ORMV prevents HEN1-mediated methylation of small RNA duplexes, suggesting two novel viral strategies of silencing suppression. Our work highlights the complexity of virus interaction with host silencing pathways and suggests that DCL multiplicity helps mediate plant responses to diverse viral infections.

Herpes Simplex Virus Virion Host Shutoff Protein: Immune Evasion Mediated by a Viral RNase?

Herpes simplex virus type 1 (HSV-1) is the prototypical member of the Herpesviridae, a large family of enveloped DNA viruses that infect diverse metazoans. It is also the defining example of the Alphaherpesvirinae, the neurotropic subfamily of herpesviruses. Like all herpesviruses, HSV displays both lytic and latent modes of interaction with its natural human host. Primary infection of epithelial cells produces the lytic response—virus replication followed by cell death. Progeny virus particles then infect adjacent sensory neurons, establishing a lifelong latent interaction. The latent viral genome is maintained in an extrachromosomal state in which only a restricted portion of the genome is transcribed. Latent genomes occasionally reactivate into the lytic cycle, producing a limited amount of progeny virus that gives rise to secondary infections of the epithelial sites enervated by the latently infected neurons. HSV executes a complex genetic program during lytic infection (reviewed in reference 47). Expression of most cellular genes is strongly suppressed, and three temporal classes of viral genes are sequentially activated in a regulatory cascade. Five viral immediate-early (IE) genes are expressed first, and four of these (ICP0, ICP4, ICP22, and ICP27) encode regulatory proteins that stimulate expression of the viral early (E) and late (L) genes. The E genes are activated next, giving rise to proteins required for replication of the viral genome. Viral DNA replication then ensues, augmenting IE-dependent expression of the L genes that encode the structural components of the virion. HSV differs from many other nuclear DNA viruses in that some of its key regulatory polypeptides are delivered into the host cell by the infecting virus particle. These virion regulators are located in the viral tegument—the space between the envelope and the nucleocapsid—and as such are injected into the newly infected cell immediately upon fusion of the viral envelope with the host cell plasma membrane. These proteins are therefore strategically poised to influence the very earliest events in the viral replication cycle. In the best-known case, the abundant tegument protein VP16 activates transcription of the viral IE genes, thereby contributing to the initial launch of the lytic program of gene expression (reviewed in reference 17). The tegument also contains vhs, the virion host shutoff protein encoded by HSV gene UL41. vhs is an mRNA-specific RNase that triggers rapid shutoff of host cell protein synthesis, disruption of preexisting polyribosomes, and degradation of host mRNAs in the absence of de novo viral gene expression (reviewed in reference 55). Here I summarize our present understanding of the mechanism of vhs action and discuss recent studies that point to intriguing roles in viral pathogenesis and immune evasion. Space limitations preclude an exhaustive review of the earlier literature; I therefore seek the indulgence of my colleagues and refer the interested reader to a recent review (55) and the introductory sections of two recent articles (9, 10) for more details. Unless otherwise stated below, vhs refers to the UL41 gene product of HSV-1.

Vaccinia virus transcription.

Vaccinia virus replication takes place in the cytoplasm of the host cell. The nearly 200 kbp genome owes part of its complexity to encoding most of the proteins involved in genome and mRNA synthesis. The multisubunit vaccinia virus RNA polymerase requires a separate set of virus-encoded proteins for the transcription of the early, intermediate and late classes of genes. Cell fractionation studies have provided evidence for a role for host cell proteins in the initiation and termination of vaccinia virus intermediate and late gene transcription. Vaccinia virus resembles nuclear DNA viruses in the integration of viral and host proteins for viral mRNA synthesis, yet is markedly less reliant on host proteins than its nuclear counterparts.

Assembly of adenovirus-specific nuclear inclusions in lytically infected HeLa cells: an ultrastructural and cytochemical study.

Adenoviruses (Ads) are nuclear DNA viruses that remodel host nuclear structure and function and induce formation of a variety of nuclear inclusions within which Ad DNA is replicated and transcribed. In this study, we have examined inclusion assembly by electron microscopy of samples stained conventionally or with bismuth to detect phosphoproteins. Small dense fibrillar bodies (DFBs) appeared very early associated with interchromatin granule (ICG) clusters. Somewhat later, similar DFBs lay near amorphous, loosely fibrillar structures that were moderately electron dense and showed little bismuth deposition. These clear fibrillar bodies (CFBs) enlarged and DFBs became embedded in their surface. At later stages, CFBs and DFBs were again dissociated. DFBs seen very early were poor in phosphoproteins, but later DFBs, whether embedded in the CFBs or lying near them, were intensely bismuth stained. DFBs and CFBs were less prominent once assembled virions were seen. At this late stage, virions were generally associated with moderately dense, slightly bismuth positive, irregularly shaped fibrillar inclusions that have previously been identified as viral genome storage sites. In addition, very dense fibrillar bodies, consisting usually of an electron-dense fibrillar shell and a less dense fibrogranular core, were observed at all but the earliest stages of infection, often at some distance from CFBs. There was also a major reorganization of host components during infection, including chromatin condensation, reduction of nucleolar volume and aggregation of the fibrillar regions at the nucleolar surface, and increased prominence of ICG clusters. A model is proposed for the assembly of Ad replication factories.

Adenovirus type 2 polypeptide synthesis in arginine-deprived cells

Among the exogenous amino acids which are essential for mammalian cell growth in vitro, only arginine (arg) is absolutely required for productive infection by nuclear DNA viruses. As previously reported, when arg-deprived KB cells are infected with adenovirus type 2, both viral DNA and viral structural proteins are synthesized at appropriate times in the virus growth cycle, but virion assembly does not occur. Quantitative study of the polypeptides made late in infection by electrophoresis under denaturing conditions show that (1) all the polypeptides synthesized in arg + cultures are also identified in arg − cultures; (2) overall synthesis is reduced 70–75%; (3) neither a disproportionate reduction of one or more polypeptides nor enhanced degradation is observed. An equivalent reduction in virus polypeptide synthesis is seen in isoleucine-starved cells as well as a similarly reduced rate of protein synthesis, but significant yields (1000 FFU/cell) of virus progeny are obtained. Since no alteration in the proportions of the various viral polypeptides could be detected and since similar amounts were adequate to support virus production in isoleucine-deficient cells, the arg-sensitive function cannot occur at the level of synthesis. This suggests the possibility that an as yet undefined arg-sensitive host function may be a critical prerequisite in virus assembly.

Virus Development in Enucleate Cells: Echovirus, Poliovirus, Pseudorabies Virus, Reovirus, Respiratory Syncytial Virus and Semliki Forest Virus

A group of RNA viruses, echovirus, poliovirus, reovirus, respiratory syncytial virus and Semliki Forest virus have been examined for ability to grow in enucleate African green monkey kidney (BSCi) cells. Semliki Forest virus produced an almost normal yield of virus but poliovirus, echovirus, reovirus and respiratory syncytial virus, although showing clear evidence of virus replication when compared with a nuclear DNA virus (pseudorabies virus) gave much lower yields than those from nucleate cells. Analysis of enucleate cells infected with echovirus and reovirus showed no evidence of a specific block in the synthesis of any virus-specified polypeptide. Infection with vesicular stomatitis virus at intervals after enucleation demonstrated a diminishing ability to support virus growth with increasing time. It is suggested that the yield of virus obtained from an enucleate cell is related to the length of the growth cycle of the virus, the reduced yield obtained with some viruses reflecting the declining ability of the enucleate cell to support virus growth.

TRANSPORT OF VIRAL RNA IN KB CELLS INFECTED WITH ADENOVIRUS TYPE 2

Messenger RNA transport was studied in KB cells infected with the nuclear DNA virus adenovirus type 2. Addition of 0.04 microg/ml of actinomycin completes the inhibition of ribosome synthesis normally observed late after infection and apparently does not alter the pattern of viral RNA synthesis: Hybridization-inhibition experiments indicate that similar viral RNA sequences are transcribed in cells treated or untreated with actinomycin. The polysomal RNA synthesized during a 2 hr labeling period in the presence of actinomycin is at least 60% viral specific. Viral messenger RNA transport can occur in the absence of ribosome synthesis. When uridine-(3)H is added to a late-infected culture pretreated with actinomycin, viral RNA appears in the cytoplasm at 10 min, but the polysomes do not receive viral RNA-(3)H until 30 min have elapsed. Only 25% of the cytoplasmic viral RNA is in polyribosomes even when infected cells have been labeled for 150 min. The nonpolysomal viral RNA in cytoplasmic extracts sediments as a broad distribution from 10S to 80S and does not include a peak cosedimenting with 45S ribosome subunits. The newly formed messenger RNA that is ribosome associated is not equally distributed among the ribosomes; by comparison to polyribosomes, 74S ribosomes are deficient at least fivefold in receipt of new messenger RNA molecules.

Metabolism of animal cells infected with nuclear DNA viruses.

Metabolism of animal cells infected with nuclear DNA viruses.