A quantitative real-time PCR assay was developed to measure human cytomegalovirus (HCMV) DNA load in peripheral blood leukocytes (PBLs). The HCMV DNA load in PBLs was normalized by means of the quantification of a cellular gene (albumin). The results of the real-time PCR assay correlated with those of the HCMV pp65-antigenemia assay (P < 0.0001).Human cytomegalovirus (HCMV) infection is characterized by a primary infection leading to a lifelong persistence of the viral genome. Periodically, the virus reactivates from latency and recovers its ability to multiply. HCMV is a major cause of morbidity and mortality in bone marrow or solid-organ transplant recipients and in AIDS patients. Early diagnosis of HCMV infection in high-risk patients is essential in order to start preemptive treatments (3,13,15). The detection of the pp65 antigen in leukocytes is a sensitive method widely used for the early diagnosis of HCMV infection, but it is laborintensive, requires immediate processing, and relies on a subjective interpretation of the slides (1, 7). Qualitative PCR detection of HCMV DNA in leukocytes or plasma is considered the most sensitive method, but it lacks specificity for the diagnosis of HCMV disease (2, 6, 16). Quantification of HCMV DNA has been proposed to be more specifically associated with the disease (1, 18). Real-time PCR based on the TaqMan technology (4, 5) provides an accurate means to quantify viral DNA with the major advantage of avoiding post-PCR handling that can be the source of DNA carryover. Recent studies report the utility of real-time PCR for the quantification of HCMV DNA (9,11,12,14,17). In these studies, the primers used for PCR were located in the major immediate-early gene (12,14,17), the US17 gene (9), or the glycoprotein B gene (17). As suggested by Yun et al., the sensitivity of quantitative PCR may be dependent on the viral target gene (17); however, the most appropriate region for amplification has not been established, as the sequence diversity of clinical isolates remains to be characterized. In the present study, a real-time PCR assay was developed to quantify HCMV DNA in peripheral blood leukocytes (PBLs) using a target sequence located in the UL83 gene which codes for the lower matrix protein detected in the pp65 antigenemia test. The HCMV DNA load in PBLs was normalized by means of the quantification of a cellular gene (albumin) and the results were compared to those of the pp65 antigenemia assay.DNA extractions were performed in all experiments using the QIAamp blood kit (QIAGEN S. A., Courtaboeuf, France) according to the manufacturer's recommendations, except that DNA was eluted in 200 l of distilled water. To amplify HCMV DNA, primers and probe were defined in the UL83 region as follows: pp549s (direct primer), 5Ј-GTCAGCGTTC GTGTTTCCCA-3Ј; pp812as (reverse primer), 5Ј-GGGACAC AACACCGTAAAGC-3Ј; and pp770s (fluorogenic probe), 5ЈFAM-CCCGCAACCCGCAACCCTTCATG-3ЈTAMRA. No cross-reactivity was observed when the specificity of the primers and probe was tested for other human herp...

Efficient translation of most eukaryotic mRNAs results from synergistic cooperation between the 5 m 7 GpppN cap and the 3 poly(A) tail. In contrast to such mRNAs, the polyadenylated genomic RNAs of picornaviruses are not capped, and translation is initiated internally, driven by an extensive sequence termed IRES (for internal ribosome entry segment). Here we have used our recently described poly(A)-dependent rabbit reticulocyte lysate cell-free translation system to study the role of mRNA polyadenylation in IRESdriven translation. Polyadenylation significantly stimulated translation driven by representatives of each of the three types of picornaviral IRES (poliovirus, encephalomyocarditis virus, and hepatitis A virus, respectively). This did not result from a poly(A)-dependent alteration of mRNA stability in our in vitro translation system but was very sensitive to salt concentration. Disruption of the eukaryotic initiation factor 4G-poly(A) binding protein (eIF4G-PABP) interaction or cleavage of eIF4G abolished or severely reduced poly(A) tail-mediated stimulation of picornavirus IRES-driven translation. In contrast, translation driven by the flaviviral hepatitis C virus (HCV) IRES was not stimulated by polyadenylation but rather by the authentic viral RNA 3 end: the highly structured X region. X region-mediated stimulation of HCV IRES activity was not affected by disruption of the eIF4G-PABP interaction. These data demonstrate that the protein-protein interactions required for synergistic cooperativity on capped and polyadenylated cellular mRNAs mediate 3-end stimulation of picornaviral IRES activity but not HCV IRES activity. Their implications for the picornavirus infectious cycle and for the increasing number of identified cellular IRES-carrying mRNAs are discussed.The initiation of protein synthesis on most mRNAs in eukaryotes follows binding of the 40S ribosomal subunit near the capped 5Ј end of the mRNA and subsequent migration of this subunit along the mRNA in a 5Ј-to-3Ј direction until a suitable initiation codon is selected (for a review, see reference 29). Recognition of the mRNA 5Ј end and 40S subunit recruitment requires the eukaryotic initiation factor (eIF) 4F complex (for reviews, see references 35 and 43). The eIF4F complex comprises the cap binding protein (eIF4E) and an ATP-dependent RNA helicase (eIF4A) bound, respectively, toward the N and C termini of a scaffold protein, eIF4G (for a review, see references 14 and 35). The C-terminal half of eIF4G is also thought to associate with the multisubunit eIF3 complex, which binds the 40S ribosomal subunit directly thus bridging the gap between the mRNA 5Ј end and the 40S subunit (reviewed in reference 17).The vast majority of eukaryotic mRNAs are not only capped at their 5Ј end but are also polyadenylated at their 3Ј end. Aside from a role in mRNA metabolism (see reference 45 for a review), the poly(A) tail functions as a translational enhancer and interacts synergistically with the 5Ј cap to stimulate translation initiation (12,23,42,43). This co...

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to synergistically stimulate translation initiation in vivo. We recently described mammalian cytoplasmic extracts which, following ultracentrifugation to partially deplete them of ribosomes and associated initiation factors, reproduce cap-poly(A) synergy in vitro. Using these systems, we demonstrate that synergy requires interaction between the poly(A)-binding protein (PABP) and the eukaryotic initiation factor (eIF) 4F holoenzyme complex, which recognises the 5' cap. Here we further characterise the requirements and constraints of cap-poly(A) synergy in reticulocyte lysates by evaluating the effects of different parameters on synergy. The extent of extract depletion and the amounts of different initiation factors in depleted extracts were examined, as well as the effects of varying the concentrations of KCl, MgCl(2) and programming mRNA and of adding a cap analogue. The results presented demonstrate that maximal cap-poly(A) synergy requires: (i) limiting concentrations of ribosome-associated initiation factors; (ii) precise ratios of mRNA to translation machinery (low concentrations of ribosome-associated initiation factors and low, non-saturating mRNA concentrations); (iii) physiological concentrations of added KCl and MgCl(2). Additionally, we show that the eIF4G-PABP interaction on mRNAs which are capped and polyadenylated significantly increases the affinity of eIF4E for the 5' cap.