Use of real-time PCR as an alternative to conventional genotyping methods for the laboratory detection of lymphogranuloma venereum (LGV).

Lymphogranuloma venereum (LGV) can be differentiated from non-LGV chlamydial infection using Sanger sequencing or molecular assays, including those that are commercially-available internationally. Here, we describe the performance of a rapid real-time PCR (RT-PCR)-based strategy in differentiating Chlamydia trachomatis infections associated with LGV or non-LGV serovars. One hundred three rectal swabs, previously genotyped using Sanger sequencing of the ompA gene as a reference method, were tested in the RT-PCR assays. All non-LGV specimens were correctly identified, but the RT-PCR failed to detect 1 LGV specimen, resulting in a sensitivity of 87.5% for the non-LGV/LGV RT-PCR assay. Additional performance characteristics (e.g., specificity, accuracy, and reproducibility) were all between 93% and 100% with a limit of detection ≤100 copies/reaction. Thus, this rapid RT-PCR method for LGV detection in clinical specimens is comparable to the reference method.

Progressive accumulation of activated ERK2 within highly stable ORF45-containing nuclear complexes promotes lytic gammaherpesvirus infection.

De novo infection with the gammaherpesvirus Rhesus monkey rhadinovirus (RRV), a close homolog of the human oncogenic pathogen, Kaposi's sarcoma-associated herpesvirus (KSHV), led to persistent activation of the MEK/ERK pathway and increasing nuclear accumulation of pERK2 complexed with the RRV protein, ORF45 (R45) and cellular RSK. We have previously shown that both lytic gene expression and virion production are dependent on the activation of ERK [1]. Using confocal microscopy, sequential pull-down assays and FRET analyses, we have demonstrated that pERK2-R45-RSK2 complexes were restricted to the nucleus but that the activated ERK retained its ability to phosphorylate nuclear substrates throughout infection. Furthermore, even with pharmacologic inhibition of MEK beginning at 48 h p.i., pERK2 but not pERK1, remained elevated for at least 10 h, showing first order decay and a half-life of nearly 3 hours. Transfection of rhesus fibroblasts with R45 alone also led to the accumulation of nuclear pERK2 and addition of exogenous RSK augmented this effect. However, knock down of RSK during bona fide RRV infection had little to no effect on pERK2 accumulation or virion production. The cytoplasmic pools of pERK showed no co-localization with either RSK or R45 but activation of pERK downstream targets in this compartment was evident throughout infection. Together, these observations suggest a model in which R45 interacts with pERK2 to promote its nuclear accumulation, thereby promoting lytic viral gene expression while also preserving persistent and robust activation of both nuclear and cytoplasmic ERK targets.

Distinct roles for extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the structure and production of a primate gammaherpesvirus.

During their progression from intranuclear capsids to mature trilaminar virions, herpesviruses incorporate an extensive array of viral as well as a smaller subset of cellular proteins. Our laboratory previously reported that rhesus monkey rhadinovirus (RRV), a close homolog of the human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), is comprised of at least 33 different virally encoded proteins. In the current study, we found that RRV infection activated the extracellular signal-regulated kinase (ERK) pathway and nascent virions preferentially incorporated the activated form of ERK2 (pERK2) into the tegument. This was evident even in the face of greatly diminished stores of intracellular ERK2, suggesting a clear bias toward the incorporation of pERK2 into the RRV particle. Similar to earlier findings with KSHV, activation of ERK was essential for the production of lytic viral proteins and virions. Knockdown of intracellular ERK, however, failed to inhibit virus production, likely due to maintenance of residual pools of intracellular pERK2. Paradoxically, selective knockdown of ERK1 enhanced virion production nearly 5-fold and viral titers more than 10-fold. These data are the first to implicate ERK1 as a negative regulator of lytic replication in a herpesvirus and the first to demonstrate the incorporation of an activated signaling molecule within a herpesvirus. Together, the results further our understanding of how herpesviruses interact with host cells during infection and demonstrate how this family of viruses can exploit cellular signal transduction pathways to modulate their own replication.