Herpes simplex virus (HSV) immediate-early protein ICP0 is a transcriptional activator with E3 ubiquitin ligase activity that induces the degradation of ND10 proteins, including the promyelocytic leukemia protein (PML) and Sp100. Moreover, ICP0 has a role in the derepression of viral genomes and in the modulation of the host interferon response to virus infection. Here, we report that ICP0 interacts with SIAH-1, a cellular E3 ubiquitin ligase that is involved in multiple cellular pathways and is itself capable of mediating PML degradation. This novel virus-host interaction profoundly stabilized SIAH-1 and recruited this cellular E3 ligase into ICP0-containing nuclear bodies. Moreover, SIAH-1 mediated the polyubiquitination of HSV ICP0 in vitro and in vivo. After infection of SIAH-1 knockdown cells with HSV, higher levels of ICP0 were produced, ICP0 was less ubiquitinated, and the half-life of this multifunctional viral regulatory protein was increased. These results indicate an inhibitory role of SIAH-1 during lytic infection by targeting ICP0 for proteasomal degradation.ICP0 is a member of the class of immediate-early gene products of herpes simplex virus 1 and 2 (HSV-1 and -2), with homologs in other herpesvirus subfamilies. ICP0 is required for the efficient initiation of viral lytic infection and reactivation from latently infected neurons (reviewed in references 22, 34, 35, and 65). ICP0 appears to be a multifunctional regulator of gene expression that directly and indirectly interacts with numerous viral and cellular proteins. For example, these include the viral transcriptional activator ICP4, the cellular transcription factor BMAL1, the translation initiation factor 1␣, cell cycle regulators such as cyclins, and the tumor suppressor p53 (6,(45)(46)(47)81). ICP0 is also a component of viral particles, where it can be detected in a salt-resistant fraction of the tegument, suggesting it to be an inner tegument protein closely associated with the capsid (21,53,66).Early in infection, newly synthesized ICP0 localizes to discrete subnuclear structures, which are characterized by the presence of the promyelocytic leukemia protein (PML) and are variously referred to as nuclear domain 10 (ND10), Kremer bodies, PML nuclear bodies, or PML oncogenic domains (PODs). ND10 has been implicated in a variety of cellular processes, including the regulation of growth control, senescence, apoptosis, transformation, and antiviral responses (for reviews, see references 4, 15, and 24). During the early stage of infection, HSV genomes locate preferentially in the periphery of ND10, and these nuclear areas are considered to be the sites where viral transcription initiates (23, 55). Importantly, later in infection the ND10 components PML and Sp100, which is another major and constitutive component of ND10 (7,12,27), are rearranged and disrupted by the E3 ubiquitin ligase activity of ICP0, which is mediated by a zinc-binding RING finger domain near its amino terminus (8,28,56,57). Nevertheless, ubiquitination of PML by ICP0 was not...
Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART) into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR)-specific recombinase (Tre-recombinase) has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD) from the HIV-1 Tat trans-activator or the translocation motif (TLM) of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies.
The HIV-1 Rev trans-activator is a nucleocytoplasmic shuttle protein that is essential for virus replication. Rev directly binds to unspliced and incompletely spliced viral RNA via the cis-acting Rev Response Element (RRE) sequence. Subsequently, Rev oligomerizes cooperatively and interacts with the cellular nuclear export receptor CRM1. In addition to mediating nuclear RNA export, Rev also affects the stability, translation and packaging of Rev-bound viral transcripts. Although it is established that Rev function requires the multimeric assembly of Rev molecules on the RRE, relatively little is known about how many Rev monomers are sufficient to form a trans-activation competent Rev:RRE complex, or which specific activity of Rev is affected by its oligomerization. We here analyzed by functional studies how homooligomer formation of Rev affects the trans-activation capacity of this essential HIV-1 regulatory protein. In a gain-of-function approach, we fused various heterologous dimerization domains to an otherwise oligomerization-defective Rev mutant and were able to demonstrate that oligomerization of Rev is not required per se for the nuclear export of this viral trans-activator. In contrast, however, the formation of Rev oligomers on the RRE is a precondition to trans-activation by directly affecting the nuclear export of Rev-regulated mRNA. Moreover, experimental evidence is provided showing that at least two protein activation domains are required for the formation of trans-activation competent Rev:RRE complexes. The presented data further refine the model of Rev trans-activation by directly demonstrating that Rev oligomerization on the RRE, thereby recruiting at least two protein activation domains, is required for nuclear export of unspliced and incompletely spliced viral RNA.
Diaminopropionate ammonialyase (DAPAL), a fold-type II pyridoxal 5′-phosphate-dependent enzyme, catalyzes the a,b-elimination of diaminopropionate (DAP) to pyruvate and ammonia. DAPAL was able to utilize both D-and L-DAP as substrates with almost equal efficiency. Mutational analysis of functionally important residues such as Thr385, Asp125 and Asp194 was carried out to understand the mechanism by which the isomers are hydrolyzed. Further, the putative residues involved in the formation of disulfide bond Cys271 and Cys299 were also mutated. T385S, T385D sDA-PAL were as active with DL-DAP as substrate as sDAPAL, whereas the later exhibited a threefold increase in catalytic efficiency with D-Ser as substrate. Further analysis of these mutants suggested that DAPAL might follow an anti-E 2 mechanism of catalysis that does not involve the formation of a quinonoid intermediate. Of the two mutants of Asp125, D125E showed complete loss of activity with D-DAP as substrate, whereas the reaction with L-DAP was not affected significantly, demonstrating that Asp125 was essential for abstraction of protons from the D-isomer. By contrast, mutational analysis of Asp194 showed that the residue may not be directly involved in proton abstraction from L-DAP. sDAPAL does not form a disulfide bond in solution, although the position of Cys299 and Cys271 in the modeled structure of sDAPAL favored the formation of a disulfide bond. Further, unlike eDAPAL, sDAPAL could be activated by monovalent cations. Mutation of the cysteine residues showed that Cys271 may be involved in coordinating the monovalent cation, as observed in the case of other fold-type II enzymes.
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