Tripartite Motif 22 (TRIM22) protein restricts herpes simplex virus 1 by epigenetic silencing of viral immediate-early genes.

Intrinsic resistance is a crucial line of defense against virus infections, and members of the Tripartite Ring Interaction Motif (TRIM) family of proteins are major players in this system, such as cytoplasmic TRIM5α or nuclear promyelocytic leukemia (PML/TRIM19) protein. Previous reports on the antiviral function of another TRIM protein, TRIM22, emphasized its innate immune role as a Type I and Type II interferon-stimulated gene against RNA viruses. This study shows that TRIM22 has an additional intrinsic role against DNA viruses. Here, we report that TRIM22 is a novel restriction factor of HSV-1 and limits ICP0-null virus replication by increasing histone occupancy and heterochromatin, thereby reducing immediate-early viral gene expression. The corresponding wild-type equivalent of the virus evades the TRIM22-specific restriction by a mechanism independent of ICP0-mediated degradation. We also demonstrate that TRIM22 inhibits other DNA viruses, including representative members of the ß- and γ- herpesviruses. Allelic variants in TRIM22 showed different degrees of anti-herpesviral activity; thus, TRIM22 genetic variability may contribute to the varying susceptibility to HSV-1 infection in humans. Collectively, these results argue that TRIM22 is a novel restriction factor and expand the list of restriction factors functioning in the infected cell nucleus to counter DNA virus infection.

RNA polymerase I (Pol I) passage through nucleosomes depends on Pol I subunits binding its lobe structure.

RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I-associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here, we used in vitro transcription assays to study purified WT and mutant Pol I variants from the yeast Saccharomyces cerevisiae and compare their abilities to pass a nucleosomal barrier with those of yeast Pol II and Pol III. Under identical conditions, purified Pol I and Pol III, but not Pol II, could transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. Our findings suggest that the lobe-binding subunits Rpa34.5/Rpa49 and Rpa12.2 facilitate passage through nucleosomes, suggesting possible cooperation among these subunits. We discuss the contribution of Pol I-specific subunit domains to efficient Pol I passage through nucleosomes in the context of transcription rate and processivity.

Mechanisms of Host IFI16, PML, and Daxx Protein Restriction of Herpes Simplex Virus 1 Replication.

The initial events after DNA virus infection involve a race between epigenetic silencing of the incoming viral DNA by host cell factors and expression of viral genes. Several host gene products, including the nuclear domain 10 (ND10) components PML (promyelocytic leukemia) and Daxx (death domain-associated protein 6), as well as IFI16 (interferon-inducible protein 16), have been shown to restrict herpes simplex virus 1 (HSV-1) replication. Whether IFI16 and ND10 components work together or separately to restrict HSV-1 replication is not known. To determine the combinatorial effects of IFI16 and ND10 proteins on viral infection, we depleted Daxx or PML in primary human foreskin fibroblasts (HFFs) in the presence or absence of IFI16. Daxx or IFI16 depletion resulted in higher ICP0 mutant viral yields, and the effects were additive. Surprisingly, small interfering RNA (siRNA) depletion of PML in the HFF cells led to decreased ICP0-null virus replication, while short hairpin RNA (shRNA) depletion led to increased ICP0-null virus replication, arguing that different PML isoforms or PML-related proteins may have restrictive or proviral functions. In normal human cells, viral DNA replication increases expression of all classes of HSV-1 genes. We observed that IFI16 repressed transcription from both parental and progeny DNA genomes. Taken together, our results show that the mechanisms of action of IFI16 and ND10 proteins are independent, at least in part, and that IFI16 exerts restrictive effects on both input and replicated viral genomes. These results raise the potential for distinct mechanisms of action of IFI16 on parental and progeny viral DNA molecules.IMPORTANCE Many human DNA viruses transcribe their genomes and replicate in the nucleus of a host cell, where they exploit the host cell nuclear machinery for their own replication. Host factors attempt to restrict viral replication by blocking such events, and viruses have evolved mechanisms to neutralize the host restriction factors. In this study, we provide information about the mechanisms of action of three host cell factors that restrict replication of herpes simplex virus (HSV). We found that these factors function independently and that one acts to restrict viral transcription from parental and progeny viral DNA genomes. These results provide new information about how cells counter DNA virus replication in the nucleus and provide possible approaches to enhance the ability of human cells to resist HSV infection.

Thiolutin is a zinc chelator that inhibits the Rpn11 and other JAMM metalloproteases.

Thiolutin is a disulfide-containing antibiotic and anti-angiogenic compound produced by Streptomyces. Its biological targets are not known. We show that reduced thiolutin is a zinc chelator that inhibits the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease Rpn11, a deubiquitinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome; AMSH, which regulates ubiquitin-dependent sorting of cell-surface receptors; and BRCC36, a K63-specific deubiquitinase of the BRCC36-containing isopeptidase complex and the BRCA1-BRCA2-containing complex. We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.

Analysis of Yeast RNAP I Transcription of Nucleosomal Templates In Vitro.

Nuclear eukaryotic RNA polymerases (RNAPs) transcribe a chromatin template in vivo. Since the basic unit of chromatin, the nucleosome, renders the DNA largely inaccessible, RNAPs have to overcome the nucleosomal barrier for efficient RNA synthesis. Gaining mechanistical insights in the transcription of chromatin templates will be essential to understand the complex process of eukaryotic gene expression. In this article we describe the use of defined in vitro transcription systems for comparative analysis of highly purified RNAPs I-III from S. cerevisiae (hereafter called yeast) transcribing in vitro reconstituted nucleosomal templates. We also provide a protocol to study promoter-dependent RNAP I transcription of purified native 35S ribosomal RNA (rRNA) gene chromatin.

Specialization of RNA Polymerase I in Comparison to Other Nuclear RNA Polymerases of Saccharomyces cerevisiae.

In archaea and bacteria the major classes of RNAs are synthesized by one DNA-dependent RNA polymerase (RNAP). In contrast, most eukaryotes have three highly specialized RNAPs to transcribe the nuclear genome. RNAP I synthesizes almost exclusively ribosomal (r)RNA, RNAP II synthesizes mRNA as well as many noncoding RNAs involved in RNA processing or RNA silencing pathways and RNAP III synthesizes mainly tRNA and 5S rRNA. This review discusses functional differences of the three nuclear core RNAPs in the yeast S. cerevisiae with a particular focus on RNAP I transcription of nucleolar ribosomal (r)DNA chromatin.

Establishment and Maintenance of Open Ribosomal RNA Gene Chromatin States in Eukaryotes.

In growing eukaryotic cells, nuclear ribosomal (r)RNA synthesis by RNA polymerase (RNAP) I accounts for the vast majority of cellular transcription. This high output is achieved by the presence of multiple copies of rRNA genes in eukaryotic genomes transcribed at a high rate. In contrast to most of the other transcribed genomic loci, actively transcribed rRNA genes are largely devoid of nucleosomes adapting a characteristic "open" chromatin state, whereas a significant fraction of rRNA genes resides in a transcriptionally inactive nucleosomal "closed" chromatin state. Here, we review our current knowledge about the nature of open rRNA gene chromatin and discuss how this state may be established.

Role for a Filamentous Nuclear Assembly of IFI16, DNA, and Host Factors in Restriction of Herpesviral Infection.

Several host cell nuclear factors are known to restrict herpes simplex virus 1 (HSV-1) replication, but their mechanisms of action remain to be defined. Interferon-inducible protein 16 (IFI16) and the nuclear domain 10-associated proteins, such as promyelocytic leukemia (PML) protein, localize to input viral genomes, but they are also capable of restricting progeny viral transcription. In this study, we used structured illumination microscopy to show that after HSV DNA replication, IFI16 forms nuclear filamentous structures on DNA within a subset of nuclear replication compartments in HSV-1 ICP0-null mutant virus-infected human cells. The ability to form filaments in different cell types correlates with the efficiency of restriction, and the kinetics of filament formation and epigenetic changes are similar. Thus, both are consistent with the filamentous structures being involved in epigenetic silencing of viral progeny DNA. IFI16 filaments recruit other restriction factors, including PML, Sp100, and ATRX, to aid in the restriction. Although the filaments are only in a subset of the replication compartments, IFI16 reduces the levels of elongation-competent RNA polymerase II (Pol II) in all replication compartments. Therefore, we propose that IFI16 filaments with associated restriction factors that form in replication compartments constitute a "restrictosome" structure that signals in cis and trans to silence the progeny viral DNA throughout the infected cell nucleus. The IFI16 filamentous structure may constitute the first known nuclear supramolecular organizing center for signaling in the cell nucleus.IMPORTANCE Mammalian cells exhibit numerous strategies to recognize and contain viral infections. The best-characterized antiviral responses are those that are induced within the cytosol by receptors that activate interferon responses or shut down translation. Antiviral responses also occur in the nucleus, yet these intranuclear innate immune responses are poorly defined at the receptor-proximal level. In this study, we explored the ability of cells to restrict infection by assembling viral DNA into transcriptionally silent heterochromatin within the nucleus. We found that the IFI16 restriction factor forms filaments on DNA within infected cells. These filaments recruit antiviral restriction factors to prevent viral replication in various cell types. Mechanistically, IFI16 filaments inhibit the recruitment of RNA polymerase II to viral genes. We propose that IFI16 filaments with associated restriction factors constitute a "restrictosome" structure that can signal to other parts of the nucleus where foreign DNA is located that it should be silenced.

Analysis of S. cerevisiae RNA Polymerase I Transcription In Vitro.

RNA polymerase I (Pol I) activity is crucial to provide cells with sufficient amounts of ribosomal RNA (rRNA). Synthesis of rRNA takes place in the nucleolus, is tightly regulated and is coordinated with synthesis and assembly of ribosomal proteins, finally resulting in the formation of mature ribosomes. Many studies on Pol I mechanisms and regulation in the model organism S. cerevisiae were performed using either complex in vitro systems reconstituted from more or less purified fractions or genetic analyses. While providing many valuable insights these strategies did not always discriminate between direct and indirect effects in transcription initiation and termination, when mutated forms of Pol I subunits or transcription factors were investigated. Therefore, a well-defined minimal system was developed which allows to reconstitute highly efficient promoter-dependent Pol I initiation and termination of transcription. Transcription can be initiated at a minimal promoter only in the presence of recombinant core factor and extensively purified initiation competent Pol I. Addition of recombinant termination factors triggers transcriptional pausing and release of the ternary transcription complex. This minimal system represents a valuable tool to investigate the direct impact of (lethal) mutations in components of the initiation and termination complexes on the mechanism and regulation of rRNA synthesis.