Host Cell Nucleolin Is Required To Maintain the Architecture of Human Cytomegalovirus Replication Compartments

Drastic reorganization of the nucleus is a hallmark of herpesvirus replication. This reorganization includes the formation of viral replication compartments, the subnuclear structures in which the viral DNA genome is replicated. The architecture of replication compartments is poorly understood. However, recent work with human cytomegalovirus (HCMV) showed that the viral DNA polymerase subunit UL44 concentrates and viral DNA synthesis occurs at the periphery of these compartments. Any cellular factors involved in replication compartment architecture are largely unknown. Previously, we found that nucleolin, a major protein component of nucleoli, associates with HCMV UL44 in infected cells and is required for efficient viral DNA synthesis. Here, we show that nucleolin binds to purified UL44. Confocal immunofluorescence analysis demonstrated colocalization of nucleolin with UL44 at the periphery of replication compartments. Pharmacological inhibition of viral DNA synthesis prevented the formation of replication compartments but did not abrogate association of UL44 and nucleolin. Thus, association of UL44 and nucleolin is unlikely to be a nonspecific effect related to development of replication compartments. No detectable colocalization of 5-ethynyl-2′-deoxyuridine (EdU)-labeled viral DNA with nucleolin was observed, suggesting that nucleolin is not directly involved in viral DNA synthesis. Small interfering RNA (siRNA)-mediated knockdown of nucleolin caused improper localization of UL44 and a defect in EdU incorporation into viral DNA. We propose a model in which nucleolin anchors UL44 at the periphery of replication compartments to maintain their architecture and promote viral DNA synthesis

Hsc70 Focus Formation at the Periphery of HSV-1 Transcription Sites Requires ICP27

The cellular chaperone protein Hsc70, along with components of the 26S proteasome and ubiquitin-conjugated proteins have been shown to be sequestered in discrete foci in the nuclei of herpes simplex virus 1 (HSV-1) infected cells. We recently reported that cellular RNA polymerase II (RNAP II) undergoes proteasomal degradation during robust HSV-1 transcription, and that the immediate early protein ICP27 interacts with the C-terminal domain and is involved in the recruitment of RNAP II to viral transcription/replication compartments.Here we show that ICP27 also interacts with Hsc70, and is required for the formation of Hsc70 nuclear foci. During infection with ICP27 mutants that are unable to recruit RNAP II to viral replication sites, viral transcript levels were greatly reduced, viral replication compartments were poorly formed and Hsc70 focus formation was curtailed. Further, a dominant negative Hsc70 mutant that cannot hydrolyze ATP, interfered with RNAP II degradation during HSV-1 infection, and an increase in ubiquitinated forms of RNAP II was observed. There was also a decrease in virus yields, indicating that proteasomal degradation of stalled RNAP II complexes during robust HSV-1 transcription and replication benefits viral gene expression.We propose that one function of the Hsc70 nuclear foci may be to serve to facilitate the process of clearing stalled RNAP II complexes from viral genomes during times of highly active transcription

Mammalian microRNA: an important modulator of host-pathogen interactions in human viral infections

MicroRNAs (miRNAs), which are small non-coding RNAs expressed by almost all metazoans, have key roles in the regulation of cell differentiation, organism development and gene expression. Thousands of miRNAs regulating approximately 60æ% of the total human genome have been identified. They regulate genetic expression either by direct cleavage or by translational repression of the target mRNAs recognized through partial complementary base pairing. The active and functional unit of miRNA is its complex with Argonaute proteins known as the microRNA-induced silencing complex (miRISC). De-regulated miRNA expression in the human cell may contribute to a diverse group of disorders including cancer, cardiovascular dysfunctions, liver damage, immunological dysfunction, metabolic syndromes and pathogenic infections. Current day studies have revealed that miRNAs are indeed a pivotal component of host-pathogen interactions and host immune responses toward microorganisms. miRNA is emerging as a tool for genetic study, therapeutic development and diagnosis for human pathogenic infections caused by viruses, bacteria, parasites and fungi. Many pathogens can exploit the host miRNA system for their own benefit such as surviving inside the host cell, replication, pathogenesis and bypassing some host immune barriers, while some express pathogen-encoded miRNA inside the host contributing to their replication, survival and/or latency. In this review, we discuss the role and significance of miRNA in relation to some pathogenic viruses

Analysis of the intranuclear localization of essential herpes simplex virus type 1 replication proteins

Herpes simplex virus type 1 (HSV-1) is a large, double-stranded DNA virus which replicates in the nuclei of infected cells. Seven virus-encoded proteins are essential for replication; a heterotrimeric helicase-primase complex (UL5, UL8, UL52), a heterodimeric polymerase complex (UL30, UL42), an origin-binding protein (UL9) and a single-stranded DNA binding protein (UL29). During infection, viral replication occurs in nuclear domains called replication compartments (RCs). RCs had previously been shown to contain UL29, UL30, UL42 and UL9. I have shown that they also contain UL5, UL8, UL52. Early during infection and in cells treated with polymerase inhibitors, UL29 localizes to numerous punctate sites, prereplicative sites (PRs), which have been proposed to be RC precursors. My thesis deals with determining which factors are necessary for PR and RC formation. To determine which viral factors are necessary for PR formation, I examined the localization of UL29 in cells infected with viral mutants which failed to express UL5, UL8, UL52 or UL9. Cells infected with these mutants contained UL29 in a diffuse staining pattern that was converted to a PR pattern by adding polymerase inhibitors. To determine if there was redundancy amongst the proteins for PR formation, viruses lacking both UL5 and UL9 or both UL5 and UL30 were generated. Analysis of cells infected with these double mutants suggested that neither UL5 nor UL9 are necessary for PR formation and that certain polymerase inhibitors stimulate the localization of UL29 to PRs by affecting cellular polymerases. I determined the minimal requirements for RC formation by transfecting cells with constructs expressing the essential viral replication proteins and a plasmid containing a viral origin of replication. RCs formed which were morphologically identical to those found in infected cells and their formation did not require UL9 or an origin of replication. These structures localized adjacent to preexisting nuclear matrix sites known as ND10s, as is the case during viral infection. I also found that UL29 localizes independently to the vicinity of ND10s and so may play a role in organizing the viral replication apparatus at the nuclear matrix.

The Herpes Simplex Virus Type 1 Transactivator ICP0 Mediates Aberrant Intracellular Localization of the Viral Helicase/Primase Complex Subunits

AbstractThe infected cell polypeptide 0 (ICP0) protein of herpes simplex virus type 1 (HSV-1) is a promiscuous transactivator. When expressed by transfection, ICP0 forms spherical structures in the nucleus. Using a double-label immunofluorescence assay, we have found that the HSV-1 helicase/primase complex subunits accumulate within ICP0 structures in cotransfected cells. This phenomenon was also observed in cells coexpressing ICP0 and UL6, a protein thought to be involved in the cleavage and/or packaging of viral genomes. ICP0 structures were found to be proteinaceous by immunoelectron microscopy. These results suggest that ICP0 may interact nonspecifically with a variety of viral proteins

Formation of herpes simplex virus type 1 replication compartments by transfection: requirements and localization to nuclear domain 10

During infection, the seven essential herpes simplex virus type 1 (HSV-1) replication proteins are found in globular nuclear structures called replication compartments. Replication compartments form adjacent to ND10, nuclear matrix-bound domains which are present in most cell types but whose function is unknown (G. G. Maul, I. M. Ishov, and R. D. Everett, Virology 217:67-75, 1996). We now demonstrate that replication compartments can be formed by cotransfecting Vero cells with constructs expressing the seven essential viral replication proteins and a plasmid containing an HSV-1 origin of DNA replication. Like replication compartments in infected cells, replication compartments formed by cotransfection contain all of the essential viral replication proteins, are sites of DNA synthesis, and are found adjacent to ND10. However, neither the viral origin-binding protein nor a plasmid containing an HSV-1 origin of DNA replication is individually required for the formation of transfection replication compartments, although the presence of each increases the efficiency of replication compartment formation. Further, we provide evidence that UL29 independently localizes adjacent to ND10 and so may play a role in directing replication compartments to these preexisting nuclear structures.</jats:p

Characterization of nuclear structures in cells infected with herpes simplex virus type 1 in the absence of viral DNA replication

Herpes simplex virus type 1 DNA replication occurs in nuclear domains termed replication compartments, which are areas of viral single-stranded DNA-binding protein (UL29) localization (M.P. Quinlan, L. B. Chen, and D. M. Knipe, Cell 36:857-868). In the presence of herpesvirus-specific polymerase inhibitors, UL29 localizes to punctate nuclear foci called prereplicative sites. Using versions of the helicase-primase complex proteins containing short peptide epitopes which can be detected in an immunofluorescence assay, we have found that the helicase-primase complex localizes to prereplicative sites and replication compartments. To determine if prereplicative site formation is dependent upon these and other essential viral replication proteins, we have studied UL29 localization in cells infected with replication-defective viruses. Cells infected with viruses that fail to express one of the three helicase-primase subunits or the origin-binding protein show a diffuse nuclear staining for UL29. However, in the presence of polymerase inhibitors, mutant-infected cells contain UL29 in prereplicative sites. Replication-defective viruses containing subtle mutations in the helicase or origin-binding proteins behaved identically to their null mutant counterparts. In contrast, cells infected with viral mutants which fail to express the polymerase protein contain prereplicative sites in the absence and presence of polymerase inhibitors. We propose that active viral polymerase prevents the formation of prereplicative sites. Models of the requirement of essential viral replication proteins in the assembly of prereplicative sites are presented.</jats:p