Phosphorylation of the RNA polymerase II carboxyl-terminal domain in human cytomegalovirus-infected cells and in vitro by the viral UL97 protein kinase

Phosphorylation of the RNA polymerase II carboxyl-terminal domain in human cytomegalovirus-infected cells and in vitro by the viral UL97 protein kinase

The gH glycoprotein of herpesviruses is located on the cell surface in viral-infected cells but is retained in the endoplasmic reticulum (ER) when expressed separately from a recombinant expression vector. Among these, the human cytomegalovirus, human chorionic gonadotropin α, and somatostatin promoters were found to be very active, approximately 11-, 9-, and 0.9-fold as active as the SV40 early promoter, respectively. Although the internal volume of the HCMV capsid is approximately 17 % larger than that of HSV-1, this slight increase in volume does not provide adequate space to encapsidate the full length HCMV genome at the same packing density as HSV-1. They are degraded with a half-time of less than 1 min. Interestingly, the 3,5-dimethoxy regioisomer inhibited cell replication (mean CC50 33 μM) and was inactive as a selective anti-herpes agent. MT4 lymphocytes and cord blood mononuclear cells (CBMC) were used to evaluate the lectin effect on HHV-6 following the same technique. Our findings suggest that expression of immunoregulatory viral gene products could be a potential strategy to prolong transgene expression in vivo.

This makes it more common than Down’s Syndrome. Current drugs target nucleotide metabolism for treatment of both viruses. As a result, most people infected with CMV are unaware that they harbor the virus – a condition that is of most concern among pregnant women, who are at risk for transmitting the virus to the fetus. During the initiation and elongation phases of transcription, the CTD cycles through various phosphorylation states of which the major two forms (RNAP IIA and IIO) can be distinguished by electrophoretic mobility (reviewed in Hirose and Manley, 2000, Kobor and Greenblatt, 2002, Maniatis and Reed, 2002, Oelgeschläger, 2002 and Palancade and Bensaude, 2003). In particular, RNAP II whose CTD is hypophosphorylated (RNAP IIA) is preferentially recruited to preinitiation complexes at a promoter. Human cytomegalovirus (HCMV) is one of eight human herpesviruses and is a serious, life-threatening, opportunistic pathogen in immunocompromised patients (organ recipients or AIDS patients) (8, 19). However, their use is associated with toxicities to the bone marrow (ganciclovir [GCV]) and kidneys (foscarnet and cidofovir) (27, 29).

Strategies for development of anti-HCMV agents include the identification of both viral and cellular targets that can abrogate virus replication (“anticellular antiviral” approach) (8, 10, 51). These specific phosphorylation events mediate different transcriptional activities of the CTD and couple transcription to posttranscriptional processes such as capping, splicing, and polyadenylation. HCMV proteins alone will also drive immune reactions to nonself-peptides. Many viruses induce alterations in host cell gene expression. The human cytomegalovirus (CMV) is a member of the herpes family of viruses that contain double-stranded DNA as their genome. One well-studied example is the recruitment of CDK9/cyclin T1 by the human immunodeficiency virus (HIV) Tat protein, which results in hyperphosphorylation of the CTD and stimulation of elongation of HIV transcripts (reviewed in Taube et al., 1999). A second example occurs during herpes simplex virus (HSV) infection.

Early during the course of viral infection, the phosphorylation state of the largest subunit of RNAP II is altered, such that the IIO and IIA forms are depleted and replaced with alternately phosphorylated forms of intermediate electrophoretic mobility (Rice et al., 1994). Virus-associated UL76 presumably plays a role in the modulation of gene expression once delivered into the cell at a very early stage of viral infection. The HSV protein kinase UL13 and viral immediate early protein ICP22 are each required for the efficient formation of RNAP IIi, for the efficient depletion of the IIA form, and for the efficient expression of certain viral genes Long et al., 1999 and Purves et al., 1993. The microsatellite regions consist of iterated motifs of one to six bases that occur in the genomes of eukaryotes and some prokaryotes and represent potential sites of mutation (14, 21). Among CMV-seropositive subjects, the association of these immunological responses with markers of Alzheimer disease pathology were evaluated. The HCMV homolog of HSV UL13 is UL97 (Chee et al., 1989). Patients and study design.
Phosphorylation of the RNA polymerase II carboxyl-terminal domain in human cytomegalovirus-infected cells and in vitro by the viral UL97 protein kinase

Negative detection of HCMV may be attributed to several factors. The use of microarrays revealed many biological pathways that are significantly altered during infection and established an important progress in our understanding of how HCMV exploits cellular pathways during infection [6–11]. It is a serine/threonine protein kinase with several unusual properties including a high pH optimum, resistance to high salt concentrations, and an uncommon preference for peptide substrates with basic residues at the P + 5 position Baek et al., 2002b and He et al., 1997. Hence, the development of vaccines against HCMV is considered a top priority [3]. UL97 is critical for viral replication Krosky et al., 2003b, Prichard et al., 1999 and Wolf et al., 2001. gB is essential for entry into all cell types and is conserved among herpesviruses [8]. Interestingly, the modest replication defect of an HSV UL13 mutant can be repaired by HCMV UL97 (Ng et al., 1996).

Homologs of these genes exist in HCMV and are UL104, UL89, UL77, UL56, UL52, and UL51, respectively (18). To investigate this possibility, we studied the effects of infection by wild-type HCMV on the electrophoretic mobility of the largest subunit of RNAP II. In the absence of other viral proteins, pp28 was not associated with the ER, Golgi apparatus, TGN, or lysosomal compartments but was restricted to the ER-Golgi-intermediate compartment (ERGIC). First, the 5′ portion of the UL69 open reading frame (the ATG codon to the MluI site) was amplified from HCMV strain AD169 DNA by PCR with the PFU enzyme (Stratagene) and 5′-CGGGATCCCCATGGAGCTGC-3′ and 5′CGGAATTCCACGCGTCGCTTGAAAG-3′ primers, which introducedBamHI and EcoRI recognition sites into the 5′ and 3′ ends, respectively, of the amplified DNA. During HSV infection, the abundance of hypo- and hyperphosphorylated forms of the largest subunits of RNA polymerase II (RNAP IIA and RNAP IIO, respectively) declines and the abundance of intermediately migrating forms (RNAP IIi) increases (Rice et al., 1994). gB is encoded by the UL55 gene of the AD169 strain of human CMV and is synthesized as a 906-amino-acid precursor molecule in infected cells (3). The monoclonal antibody, ARNA 3a, recognizes both IIA and IIO, while the monoclonal antibody 8WG16, whose binding is blocked by phosphorylation of Ser-2, recognizes the hypophosphorylated form, IIA, but not the hyperphosphorylated form, IIO Cho et al., 2001 and Patturajan et al., 1998.

To demonstrate released phenotypically complemented virus, supernatants of exemplary cotransfections were harvested 2 weeks after transfection, clarified by centrifugation at 5,000 rpm for 10 min, and distributed onto two parallel cultures of fresh fibroblasts grown in 24-well dishes containing cover slips. The relevance of these alterations to viral infection is not yet understood. The construct containing the UL77-specific sequence, a 500-bp SalI-SacI fragment (nucleotides 112129 to 112629), was designated pRB-C. BADsubUL99 was used to make BADpmUL99 and BADrevUL99 by using pGS284-ATG-TTG/UL99 and pGS284WT/UL99, respectively, by screening for kanamycin sensitivity. HCMV strain AD169 was used as the wild-type virus. Chemicals.BDCRB (30) and UMJD 1311 (22) were synthesized in the laboratory of L. During the same time period, there was little change in the amount of RNAP II in mock-infected cells (data not shown).

FAM/TAMRA-labeled probes were manufactured by Biosearch Technologies (Novato, Calif.). The gels were quantified by phosphorimager analysis (Storm 840; Amersham Biosciences). [11]) or human embryonic lung (HEL) fibroblasts were used for all experiments (96). After a 1-h incubation, the wells were washed and the plate was incubated for 16 h. These antibodies appear to be bound to the hexons, have a rough Y shape when examined closely (Fig. The desired transgene of interest was cloned into the pShuttle EF1α construct as a BamHI fragment into a BglII site. Sinzger (Tübingen, Germany), respectively.

The UL112-113 locus is capable of activating the KSHV RTA gene promoter, which is the KSHV gene responsible for initiating KSHV lytic gene expression.

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