Absence Of Other Viral Proteins Research Articles

The P7-1 protein of southern rice black-streaked dwarf virus, a fijivirus, induces the formation of tubular structures in insect cells

Southern rice black-streaked dwarf virus (SRBSDV), an insect- and plant-infecting reovirus of the genus Fijivirus, induced the formation of virus-containing tubules in infected plant and insect vector cells. Expression of the nonstructural protein P7-1 of SRBSDV in insect cells by a recombinant baculovirus resulted in the formation of tubules with dimensions and appearance similar to those found in SRBSDV-infected cells. These tubules protruded from the cell surface, supporting the hypothesis that the P7-1 protein contains two putative transmembrane domains that are necessary for the formation of tubules. Furthermore, the self-interaction of SRBSDV P7-1 protein indicates that this protein has the capacity to form homodimers or oligomers to assemble the proposed helical symmetry structure of tubules. Taken together, our results indicate that SRBSDV P7-1 has the intrinsic ability of self-interaction to form tubules growing from the cell surface in the absence of other viral proteins.

Mutagenesis of tyrosine and di-leucine motifs in the HIV-1 envelope cytoplasmic domain results in a loss of Env-mediated fusion and infectivity

BackgroundThe gp41 component of the Human Immunodeficiency Virus (HIV) envelope glycoprotein (Env) contains a long cytoplasmic domain (CD) with multiple highly conserved tyrosine (Y) and dileucine (LL) motifs. Studies suggest that the motifs distal to major endocytosis motif (Y712HRL), located at residues 712-715 of Env, may contribute to Env functionality in the viral life cycle. In order to examine the biological contribution of these motifs in the biosynthesis, transport, and function of Env, we constructed two panels of mutants in which the conserved Y- and LL-motifs were sequentially substituted by alternative residues, either in the presence or absence of Y712. Additional mutants targeting individual motifs were then constructed.ResultsAll mutant Envs, when expressed in the absence of other viral proteins, maintained at least WT levels of Env surface staining by multiple antibodies. The Y712 mutation (Y712C) contributed to at least a 4-fold increase in surface expression for all mutants containing this change. Sequential mutagenesis of the Y- and LL-motifs resulted in a generally progressive decrease in Env fusogenicity. However, additive mutation of dileucine and tyrosine motifs beyond the tyrosine at residue 768 resulted in the most dramatic effects on Env incorporation into virions, viral infectivity, and virus fusion with target cells.ConclusionsFrom the studies reported here, we show that mutations of the Y- and LL-motifs, which effectively eliminate the amphipathic nature of the lytic peptide 2 (LLP2) domain or disrupt YW and LL motifs in a region spanning residues 795-803 (YWWNLLQYW), just C-terminal of LLP2, can dramatically interfere with biological functions of HIV-1 Env and abrogate virus replication. Because these mutant proteins are expressed at the cell surface, we conclude that tyrosine and di-leucine residues within the cytoplasmic domain of gp41 play critical roles in HIV-1 replication that are distinct from that of targeting the plasma membrane.

Role of the viral protein UL24 in nucleolar modifications induced by herpes simplex virus 1

Herpes simplex virus 1 (HSV-1) infection induces multiple modifications to nucleoli. We and others have observed the redistribution of nucleolar proteins such as nucleolin, fibrillarin, B23 (nucleophosmin), upstream binding factor (UBF) and RNA polymerase I (RNAPI) during HSV-1 infection. UL24 is one of a limited number of HSV-1 proteins that localizes to nucleoli. In a murine model of ocular HSV-1 infection, viruses that do not express the UL24 protein exhibit reduced viral titers in the eye and in trigeminal ganglia, and a reduction in clinical signs of disease. UL24 is conserved among Herpesviridae. Specifically, the N-terminal region of the protein contains several stretches of highly conserved residues. Bioinformatics studies have identified an endonuclease motif in the N-terminal portion of UL24, although as yet, no nuclease activity has been demonstrated for the protein. Previously, we discovered that UL24 transiently localizes to nucleoli during infection, and is both necessary and sufficient to induce the dispersal of nucleolin from nucleoli throughout the nucleus. In contrast, the redistribution of fibrillarin is a UL24-independent event. Moreover, the largest subunit of RNAPI and the transcription factor UBF are redistributed to viral replication compartments and these effects are also UL24-independent. We recently investigated whether UL24 was implicated in other HSV-1-induced nucleolar modifications. We discovered that UL24 was involved in the redistribution of B23 during infection. Furthermore, expression of UL24 in the absence of other viral proteins was sufficient to induce the relocalisation of B23. Similar to what we found for nucleolin, the conserved N-terminal portion of UL24 was important for its effect on B23 redistribution. The impact of the mutant vUL24-E99A/K101A, in which the endonuclease motif has been altered, and of vUL24-G121A, which harbours an amino acid substitution outside of this motif, were tested. While the G121A mutation had only a modest effect on the ability the virus to induce the relocalisation of B23, the E99A/K101A mutation appeared to abolish this function. Interestingly, the E99A/K101A mutation was previously shown to have a large impact on viral pathogenesis in mice. We found that the effect of HSV-1 on the redistribution of B23 was similar in non-immortalized cells such as human foreskin fibroblasts, as in immortalized cell lines such as vero cells. The similarities with regard to the impact of UL24 on the spatial distribution of nucleolin and B23 suggest that these effects may be related to the same function of UL24 during HSV-1 infection.

Mechanistic Characterization of the nuclear import and export signals of VZV ORF9

The ORF9 protein, a VZV-encoded late protein consisting of 302 amino acid (aa) residues, is a member of the highly conserved alphaherpesvirus UL49 gene family but shares limited identity with the UL49 prototype, HSV-1 VP22. As the orthologue of HSV-1 VP22, ORF9 is believed to be a major constituent of the VZV virion tegument. HSV-1 VP22 has been extensively studied; however, the functional properties of ORF9 are less well understood, despite the fact that its transcript is the most abundant viral message expressed during VZV lytic infection. As an important step toward understanding the detailed functions of ORF9 in vivo is to determine its precise subcellular localization, in the work presented here, to avoid the flaw of fixation protocol, living cells fluorescence microscopy technique, which is widely applied and developed in our group, was applied to deeply analyze the intracellular distribution of ORF9 protein and, to characterize its functional nuclear localization signal (NLS) and nuclear export signal (NES), as well as its transport mechanism in living cells. Transient expression of ORF9 fused to enhanced yellow fluorescent protein (EYFP) in COS-7 cells showed the predominantly cytoplasmic localization in the absence of other viral proteins. Time-lapse examination of ORF9-EYFP demonstrated that the ORF9 was found to possess a pronounced cytoplasmic stage early post transfection, followed by translocation to the nucleus at a later time, which was consistent with the fact that VP22 is predominantly localized in the cytoplasm early in infection and accumulates in the nucleus late in infection, and the nuclear targeting of VP22 is independent of other viral factors. By sequence analysis and constructing a series of deletion derivatives of ORF9 fused to EYFP and fluorescence microscopy analysis in live cells, a bona fide bipartite NLS of ORF9 was for the first time determined and mapped to amino acids (aa) 16 to 32 (RRKTTPSYSGQYRTARR), which containing two arginine-rich motifs RRK and RTARR. Additionally, the NES was identified to locate between the leucine-rich region at amino acids 103 to 117 (LRHELVEDAVYENPL). Besides, ORF9 protein was demonstrated to target to the cytoplasm through the functional NES by Ran and chromosomal region maintenance 1 (CRM1)-dependent pathway, and to the nucleus through importin β-dependent mechanism that does not require importin α5.

Poliovirus 2A<sup>pro</sup> induces the nucleic translocation of poliovirus 3CD and 3C′ proteins

Poliovirus genomic RNA replication, protein translation, and virion assembly are performed in the cytoplasm of host cells. However, this does not mean that there is no relationship between poliovirus infection and the cellular nucleus. In this study, recombinant fluorescence-tagged poliovirus 3CD and 3C' proteins were shown to be expressed mainly in the cytoplasm of Vero cells in the absence of other viral proteins. However, upon poliovirus infection, many of these proteins redistributed to the nucleus, as well as to the cytoplasm. A series of transfection experiments revealed that the poliovirus 2A(pro) was responsible for the same redistribution of 3CD and 3C' proteins to the nucleus. Furthermore, a mutant 2A(pro) protein lacking protease activity abrogated this effect. The poliovirus 2A(pro) protein was also found to co-localize with the Nup153 protein, a component of the nuclear pore complexes on the nuclear envelope. These data provide further evidence that there are intrinsic interactions between poliovirus proteins and the cell nucleus, despite that many processes in the poliovirus replication cycle occur in the cytoplasm.

Characterization of the nuclear import and export signals, and subcellular transport mechanism of varicella-zoster virus ORF9

Varicella-zoster virus (VZV) open reading frame 9 (ORF9) mRNA is one of the most abundantly expressed messages during VZV infection. However, little is known concerning the function of ORF9 protein. Here, we found that transient expression of ORF9 fused to enhanced yellow fluorescent protein (EYFP) in COS-7 cells showed a predominantly cytoplasmic localization in the absence of other viral proteins. By constructing a series of ORF9 variants fused to EYFP, a bona fide bipartite nuclear localization signal of ORF9 was, for the first time, determined and mapped to aa 16-32 (RRKTTPSYSGQYRTARR). Additionally, the nuclear export signal (NES) was identified and found to be in a leucine-rich region at aa 103-117 (LRHELVEDAVYENPL). Finally, ORF9 was demonstrated to be targeted to the cytoplasm through the functional NES by Ran and the chromosomal region maintenance 1-dependent pathway, and to the nucleus via an importin β-dependent pathway that does not require importin α5.

Adenovirus E1B 55-Kilodalton Protein Is a p53-SUMO1 E3 Ligase That Represses p53 and Stimulates Its Nuclear Export through Interactions with Promyelocytic Leukemia Nuclear Bodies

Oncogenic transformation by adenovirus E1A and E1B-55K requires E1B-55K inhibition of p53 activity to prevent E1A-induced apoptosis. During viral infection, E1B-55K and E4orf6 substitute for the substrate-binding subunits of the host cell cullin 5 class of ubiquitin ligases, resulting in p53 polyubiquitinylation and proteasomal degradation. Here we show that E1B-55K alone also functions as an E3 SUMO1-p53 ligase. Fluorescence microscopy studies showed that E1B-55K alone, in the absence of other viral proteins, causes p53 to colocalize with E1B-55K in promyelocytic leukemia (PML) nuclear bodies, nuclear domains with a high concentration of sumoylated proteins. Photobleaching experiments with live cells revealed that E1B-55K tethering of p53 in PML nuclear bodies decreases the in vivo nuclear mobility of p53 nearly 2 orders of magnitude. E1B-55K-induced p53 sumoylation contributes to maximal inhibition of p53 function since mutation of the major p53 sumoylation site decreases E1B-55K-induced p53 sumoylation, tethering in PML nuclear bodies, and E1B-55K inhibition of p53 activity. Mutation of the E1B-55K sumoylation site greatly inhibits E1B-55K association with PML nuclear bodies and the p53 nuclear export to cytoplasmic aggresomes observed in E1A-E1B-transformed cells. Purified E1B-55K and p53 form high-molecular-weight complexes potentially through the formation of a network of E1B-55K dimers bound to the N termini of p53 tetramers. In support of this model, a p53 mutation that prevents tetramer formation greatly reduces E1B-55K-induced tethering in PML nuclear bodies and p53 nuclear export. These data indicate that E1B-55K's association with PML nuclear bodies inactivates p53 by first sequestering it in PML nuclear bodies and then greatly facilitating its nuclear export.

Dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis

Dengue virus (DENV) modifies cellular membranes to establish its sites of replication. Although the 3D architecture of these structures has recently been described, little is known about the cellular pathways required for their formation and expansion. In this report, we examine the host requirements for DENV replication using a focused RNAi analysis combined with validation studies using pharmacological inhibitors. This approach identified three cellular pathways required for DENV replication: autophagy, actin polymerization, and fatty acid biosynthesis. Further characterization of the viral modulation of fatty acid biosynthesis revealed that a key enzyme in this pathway, fatty acid synthase (FASN), is relocalized to sites of DENV replication. DENV nonstructural protein 3 (NS3) is responsible for FASN recruitment, inasmuch as (i) NS3 expressed in the absence of other viral proteins colocalizes with FASN and (ii) NS3 interacts with FASN in a two-hybrid assay. There is an associated increase in the rate of fatty acid biosynthesis in DENV-infected cells, and de novo synthesized lipids preferentially cofractionate with DENV RNA. Finally, purified recombinant NS3 stimulates the activity of FASN in vitro. Taken together, these experiments suggest that DENV co-opts the fatty acid biosynthetic pathway to establish its replication complexes. This study provides mechanistic insight into DENV membrane remodeling and highlights the potential for the development of therapeutics that inhibit DENV replication by targeting the fatty acid biosynthetic pathway.

Poxvirus Complement Control Proteins Are Expressed on the Cell Surface through an Intermolecular Disulfide Bridge with the Viral A56 Protein

The vaccinia virus (VACV) complement control protein (VCP) is an immunomodulatory protein that is both secreted from and expressed on the surface of infected cells. Surface expression of VCP occurs though an interaction with the viral transmembrane protein A56 and is dependent on a free N-terminal cysteine of VCP. Although A56 and VCP have been shown to interact in infected cells, the mechanism remains unclear. To investigate if A56 is sufficient for surface expression, we transiently expressed VCP and A56 in eukaryotic cell lines and found that they interact on the cell surface in the absence of other viral proteins. Since A56 contains three extracellular cysteines, we hypothesized that one of the cysteines may be unpaired and could therefore form a disulfide bridge with VCP. To test this, we generated a series of A56 mutants in which each cysteine was mutated to a serine, and we found that mutation of cysteine 162 abrogated VCP cell surface expression. We also tested the ability of other poxvirus complement control proteins to bind to VACV A56. While the smallpox homolog of VCP is able to bind VACV A56, the ectromelia virus (ECTV) VCP homolog is only able to bind the ECTV homolog of A56, indicating that these proteins may have coevolved. Surface expression of poxvirus complement control proteins may have important implications in viral pathogenesis, as a virus that does not express cell surface VCP is attenuated in vivo. This suggests that surface expression of VCP may contribute to poxvirus pathogenesis.

The Lack of an Inherent Membrane Targeting Signal Is Responsible for the Failure of the Matrix (M1) Protein of Influenza A Virus To Bud into Virus-Like Particles

The matrix protein (M1) of influenza A virus is generally viewed as a key orchestrator in the release of influenza virions from the plasma membrane during infection. In contrast to this model, recent studies have indicated that influenza virus requires expression of the envelope proteins for budding of intracellular M1 into virus particles. Here we explored the mechanisms that control M1 budding. Similarly to previous studies, we found that M1 by itself fails to form virus-like-particles (VLPs). We further demonstrated that M1, in the absence of other viral proteins, was preferentially targeted to the nucleus/perinuclear region rather than to the plasma membrane, where influenza virions bud. Remarkably, we showed that a 10-residue membrane targeting peptide from either the Fyn or Lck oncoprotein appended to M1 at the N terminus redirected M1 to the plasma membrane and allowed M1 particle budding without additional viral envelope proteins. To further identify a functional link between plasma membrane targeting and VLP formation, we took advantage of the fact that M1 can interact with M2, unless the cytoplasmic tail is absent. Notably, native M2 but not mutant M2 effectively targeted M1 to the plasma membrane and produced extracellular M1 VLPs. Our results suggest that influenza virus M1 may not possess an inherent membrane targeting signal. Thus, the lack of efficient plasma membrane targeting is responsible for the failure of M1 in budding. This study highlights the fact that interactions of M1 with viral envelope proteins are essential to direct M1 to the plasma membrane for influenza virus particle release.

The N-Terminal Region of Severe Acute Respiratory Syndrome Coronavirus Protein 6 Induces Membrane Rearrangement and Enhances Virus Replication

The severe acute respiratory syndrome coronavirus (SARS-CoV) accessory protein 6 (p6) is a 63-amino-acid multifunctional Golgi-endoplasmic reticulum (ER) membrane-associated protein, with roles in enhancing virus replication and in evading the innate immune response to infection by inhibiting STAT1 (signal transducer and activator of transcription factor 1) translocation to the nucleus. Here, we demonstrate that p6 has an N-terminal region-cytoplasm-C-terminal region-cytoplasm configuration with residues 2 to 37 likely membrane embedded. Expression of p6, or of its N-terminal 41-amino-acid region, in the absence of other viral proteins, induced the formation of membranous structures, some of which were similar to double membrane vesicles involved in virus replication. Consistent with a role in virus replication, p6 partially colocalized with nonstructural protein 3 (nsp3), a marker for virus replication complexes. Further, while the C-terminal region is required for preventing STAT1 translocation to the nucleus, our results also indicated that the N-terminal 18 amino acids were necessary for maximal inhibition. Collectively, these results support the notion that p6 is a two-domain protein, although the function of each is not completely independent of the other.

Interaction Domains of the UL16 and UL21 Tegument Proteins of Herpes Simplex Virus

The UL16 protein of herpes simplex virus is capsid associated and was previously identified as a binding partner of the membrane-associated UL11 tegument protein (J. S. Loomis, R. J. Courtney, and J. W. Wills, J. Virol. 77:11417-11424, 2003). In those studies, a less-prominent, approximately 65-kDa binding partner of unknown identity was also observed. Mass spectrometry studies have now revealed this species to be UL21, a tegument protein that has been implicated in the transport of capsids in the cytoplasm. The validity of the mass spectrometry results was tested in a variety of coimmunoprecipitation and glutathione S-transferase pull-down experiments. The data revealed that UL21 and UL16 can form a complex in the absence of other viral proteins, even when the assays used proteins purified from Escherichia coli. Moreover, UL11 was able to pull down UL21 only when UL16 was present, suggesting that all three proteins can form a complex. Deletion analyses revealed that the second half of UL21 (residues 268 to 535) is sufficient for the UL16 interaction and packaging into virions; however, attempts to map a subdomain of UL16 were largely unsuccessful, with only the first 40 (of 373) residues being found to be dispensable. Nevertheless, it is clear that UL16 must have two distinct binding sites, because covalent modification of its free cysteines with N-ethylmaleimide blocked binding to UL11 but not UL21. These findings should prove useful for elucidating the molecular machinery used to transmit a signal into a virion when it attaches to cells, a recently discovered mechanism in which UL16 is a central player.

HIV-1 Nef Inhibits Ruffles, Induces Filopodia, and Modulates Migration of Infected Lymphocytes

The HIV-1 Nef protein is a pathogenic factor modulating the behavior of infected cells. Nef induces actin cytoskeleton changes and impairs cell migration toward chemokines. We further characterized the morphology, cytoskeleton dynamics, and motility of HIV-1-infected lymphocytes. By using scanning electron microscopy, confocal immunofluorescence microscopy, and ImageStream technology, which combines flow cytometry and automated imaging, we report that HIV-1 induces a characteristic remodeling of the actin cytoskeleton. In infected lymphocytes, ruffle formation is inhibited, whereas long, thin filopodium-like protrusions are induced. Cells infected with HIV with nef deleted display a normal phenotype, and Nef expression alone, in the absence of other viral proteins, induces morphological changes. We also used an innovative imaging system to immobilize and visualize living individual cells in suspension. When combined with confocal "axial tomography," this technique greatly enhances three-dimensional optical resolution. With this technique, we confirmed the induction of long filopodium-like structures in unfixed Nef-expressing lymphocytes. The cytoskeleton reorganization induced by Nef is associated with an important impairment of cell movements. The adhesion and spreading of infected cells to fibronectin, their spontaneous motility, and their migration toward chemokines (CXCL12, CCL3, and CCL19) were all significantly decreased. Therefore, Nef induces complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impacts the capacity of infected cells to circulate and to encounter and communicate with bystander cells.

Effects of Major Capsid Proteins, Capsid Assembly, and DNA Cleavage/Packaging on the pU L 17/pU L 25 Complex of Herpes Simplex Virus 1

The U(L)17 and U(L)25 proteins (pU(L)17 and pU(L)25, respectively) of herpes simplex virus 1 are located at the external surface of capsids and are essential for DNA packaging and DNA retention in the capsid, respectively. The current studies were undertaken to determine whether DNA packaging or capsid assembly affected the pU(L)17/pU(L)25 interaction. We found that pU(L)17 and pU(L)25 coimmunoprecipitated from cells infected with wild-type virus, whereas the major capsid protein VP5 (encoded by the U(L)19 gene) did not coimmunoprecipitate with these proteins under stringent conditions. In addition, pU(L)17 (i) coimmunoprecipitated with pU(L)25 in the absence of other viral proteins, (ii) coimmunoprecipitated with pU(L)25 from lysates of infected cells in the presence or absence of VP5, (iii) did not coimmunoprecipitate efficiently with pU(L)25 in the absence of the triplex protein VP23 (encoded by the U(L)18 gene), (iv) required pU(L)25 for proper solubilization and localization within the viral replication compartment, (v) was essential for the sole nuclear localization of pU(L)25, and (vi) required capsid proteins VP5 and VP23 for nuclear localization and normal levels of immunoreactivity in an indirect immunofluorescence assay. Proper localization of pU(L)25 in infected cell nuclei required pU(L)17, pU(L)32, and the major capsid proteins VP5 and VP23, but not the DNA packaging protein pU(L)15. The data suggest that VP23 or triplexes augment the pU(L)17/pU(L)25 interaction and that VP23 and VP5 induce conformational changes in pU(L)17 and pU(L)25, exposing epitopes that are otherwise partially masked in infected cells. These conformational changes can occur in the absence of DNA packaging. The data indicate that the pU(L)17/pU(L)25 complex requires multiple viral proteins and functions for proper localization and biochemical behavior in the infected cell.

Tryptophan Residues in the Portal Protein of Herpes Simplex Virus 1 Critical to the Interaction with Scaffold Proteins and Incorporation of the Portal into Capsids

Incorporation of the herpes simplex virus 1 (HSV-1) portal vertex into the capsid requires interaction with a 12-amino-acid hydrophobic domain within capsid scaffold proteins. The goal of this work was to identify domains and residues in the UL6-encoded portal protein pUL6 critical to the interaction with scaffold proteins. We show that whereas the wild-type portal and scaffold proteins readily coimmunoprecipitated with one another in the absence of other viral proteins, truncation beyond the first 18 or last 36 amino acids of the portal protein precluded this coimmunoprecipitation. The coimmunoprecipitation was also precluded by mutation of conserved tryptophan (W) residues to alanine (A) at positions 27, 90, 127, 163, 241, 262, 532, and 596 of UL6. All of these W-to-A mutations precluded the rescue of a viral deletion mutant lacking UL6, except W163A, which supported replication poorly, and W596A, which fully rescued replication. A recombinant virus bearing the W596A mutation replicated and packaged DNA normally, and scaffold proteins readily coimmunoprecipitated with portal protein from lysates of infected cells. Thus, viral functions compensated for the W596A mutation's detrimental effects on the portal-scaffold interaction seen during transient expression of portal and scaffold proteins. In contrast, the W27A mutation precluded portal-scaffold interactions in infected cell lysates, reduced the solubility of pUL6, decreased incorporation of the portal into capsids, and abrogated viral-DNA cleavage and packaging.

Proline and Tyrosine Residues in Scaffold Proteins of Herpes Simplex Virus 1 Critical to the Interaction with Portal Protein and Its Incorporation into Capsids

Previous results showed that amino acids 449 to 457 of pU(L)26, a component of the scaffold of herpes simplex virus 1 capsids, were critical for interaction with the portal protein encoded by U(L)6 and for incorporation of the portal into capsids. To identify residues in this scaffold domain critical for the interaction with pU(L)6, the two proteins were coexpressed in the absence of other viral proteins and subjected to immunoprecipitation with scaffold-specific antibody. Coimmunoprecipitation of pU(L)6 was precluded by pU(L)26 mutations Y451A, P452A, and E454A but not by P449A, R456A, or Y450A. In infected cells, Y451A and P452A diminished solubilization of pU(L)6, reduced incorporation of the portal into the capsid, and precluded viral replication and DNA packaging. In contrast, E454A did not affect these parameters despite the fact that E454 is invariant in a number of different alphaherpesvirus scaffold proteins. These data suggest that the interaction between the scaffold E454A mutant and portal protein is rescued by other viral functions. Finally, we show that amino acids 448 to 459 of pU(L)26 are sufficient to interact with pU(L)6 in a glutathione S-transferase pulldown assay in the absence of other viral proteins and that this interaction is inhibited with excess peptide containing pU(L)26 amino acids 443 to 462. Together, these observations suggest that a direct interaction between this scaffold domain and portal protein mediates incorporation of the portal into the capsid.

De novo synthesis of VP16 coordinates the exit from HSV latency in vivo.

The mechanism controlling the exit from herpes simplex virus latency (HSV) is of central importance to recurrent disease and transmission of infection, yet interactions between host and viral functions that govern this process remain unclear. The cascade of HSV gene transcription is initiated by the multifunctional virion protein VP16, which is expressed late in the viral replication cycle. Currently, it is widely accepted that VP16 transactivating function is not involved in the exit from latency. Utilizing the mouse ocular model of HSV pathogenesis together with genetically engineered viral mutants and assays to quantify latency and the exit from latency at the single neuron level, we show that in vivo (i) the VP16 promoter confers distinct regulation critical for viral replication in the trigeminal ganglion (TG) during the acute phase of infection and (ii) the transactivation function of VP16 (VP16TF) is uniquely required for the exit from latency. TG neurons latently infected with the VP16TF mutant in1814 do not express detectable viral proteins following stress, whereas viruses with mutations in the other major viral transcription regulators ICP0 and ICP4 do exit the latent state. Analysis of a VP16 promoter/reporter mutant in the background of in1814 demonstrates that the VP16 promoter is activated in latently infected neurons following stress in the absence of other viral proteins. These findings support the novel hypothesis that de novo expression of VP16 regulates entry into the lytic program in neurons at all phases of the viral life cycle. HSV reactivation from latency conforms to a model in which stochastic derepression of the VP16 promoter and expression of VP16 initiates entry into the lytic cycle.

The Putative Leucine Zipper of the U L 6-Encoded Portal Protein of Herpes Simplex Virus 1 Is Necessary for Interaction with pU L 15 and pU L 28 and Their Association with Capsids

Herpes simplex virus (HSV) type 1 capsids contain a single portal vertex that is composed of 12 copies of the U(L)6 gene product (pU(L)6), which forms a pore through which DNA is inserted during packaging. This unique vertex is also believed to comprise the site with which a molecular motor, termed the terminase, associates during the DNA packaging reaction. In HSV, the terminase likely comprises the U(L)15, U(L)28, and U(L)33 proteins (pU(L)15, pU(L)28, and pU(L)33, respectively). The current study was undertaken to identify portal domains required for interaction with the terminase. Both the amino and carboxyl termini, as well as amino acids 422 to 443 of pU(L)6 forming a putative leucine zipper motif, were critical for coimmunoprecipitation with pU(L)15 in the absence of other viral proteins. Amino acids 422 to 443 were also necessary for interaction with pU(L)28 in the absence of other viral proteins. By using an engineered recombinant virus, it was further determined that although amino acids 422 to 443 were dispensable for interaction with scaffold protein and incorporation of portal protein into capsids, they were necessary for coimmunoprecipitation of pU(L)6 and pU(L)15 from infected cell lysates, association of optimal levels of pU(L)15, pU(L)28, and pU(L)33 with capsids, and DNA cleavage and packaging. These data identify a portal protein domain critical for terminase association with the capsid and suggest that both the pU(L)15- and pU(L)28-bearing terminase subunits mediate docking of the terminase with the portal vertex.

The conserved N-terminal domain of herpes simplex virus 1 UL24 protein is sufficient to induce the spatial redistribution of nucleolin

UL24 is widely conserved among herpesviruses but its function during infection is poorly understood. Previously, we discovered a genetic link between UL24 and the herpes simplex virus 1-induced dispersal of the nucleolar protein nucleolin. Here, we report that in the absence of viral infection, transiently expressed UL24 accumulated in both the nucleus and the Golgi apparatus. In the majority of transfected cells, nuclear staining for UL24 was diffuse, but a minor staining pattern, whereby UL24 was present in nuclear foci corresponding to nucleoli, was also observed. Expression of UL24 correlated with the dispersal of nucleolin. This dispersal did not appear to be a consequence of a general disaggregation of nucleoli, as foci of fibrillarin staining persisted in cells expressing UL24. The conserved N-terminal region of UL24 was sufficient to cause this change in subcellular distribution of nucleolin. Interestingly, a bipartite nuclear localization signal predicted within the C terminus of UL24 was dispensable for nuclear localization. None of the five individual UL24 homology domains was required for nuclear or Golgi localization, but deletion of these domains resulted in the loss of nucleolin-dispersal activity. We determined that a nucleolar-targeting signal was contained within the first 60 aa of UL24. Our results show that the conserved N-terminal domain of UL24 is sufficient to specifically induce dispersal of nucleolin in the absence of other viral proteins or virus-induced cellular modifications. These results suggest that UL24 directly targets cellular factors that affect the composition of nucleoli.

Kaposi's Sarcoma-Associated Herpesvirus ORF57 Functions as a Viral Splicing Factor and Promotes Expression of Intron-Containing Viral Lytic Genes in Spliceosome-Mediated RNA Splicing

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 facilitates the expression of both intronless viral ORF59 genes and intron-containing viral K8 and K8.1 genes (V. Majerciak, N. Pripuzova, J. P. McCoy, S. J. Gao, and Z. M. Zheng, J. Virol. 81:1062-1071, 2007). In this study, we showed that disruption of ORF57 in a KSHV genome led to increased accumulation of ORF50 and K8 pre-mRNAs and reduced expression of ORF50 and K-bZIP proteins but had no effect on latency-associated nuclear antigen (LANA). Cotransfection of ORF57 and K8beta cDNA, which retains a suboptimal intron of K8 pre-mRNA due to alternative splicing, promoted RNA splicing of K8beta and production of K8alpha (K-bZIP). Although Epstein-Barr virus EB2, a closely related homolog of ORF57, had a similar activity in the cotransfection assays, herpes simplex virus type 1 ICP27 was inactive. This enhancement of RNA splicing by ORF57 correlates with the intact N-terminal nuclear localization signal motifs of ORF57 and takes place in the absence of other viral proteins. In activated KSHV-infected B cells, KSHV ORF57 partially colocalizes with splicing factors in nuclear speckles and assembles into spliceosomal complexes in association with low-abundance viral ORF50 and K8 pre-mRNAs and essential splicing components. The association of ORF57 with snRNAs occurs by ORF57-Sm protein interaction. We also found that ORF57 binds K8beta pre-mRNAs in vitro in the presence of nuclear extracts. Collectively our data indicate that KSHV ORF57 functions as a novel splicing factor in the spliceosome-mediated splicing of viral RNA transcripts.