Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily manifesting as neurological disorders. HCMV infection alters expression of cellular microRNAs (miRs) and induces cell cycle arrest, which in turn modifies the cellular environment to favor virus replication. Previous observations found that HCMV infection reduces miR-21 expression in neural progenitor/stem cells (NPCs). Here, we show that infection of NPCs and U-251MG cells represses miR-21 while increasing the levels of Cdc25a, a cell cycle regulator and known target of miR-21. These opposing responses to infection prompted an investigation of the relationship between miR-21, Cdc25a, and viral replication. Overexpression of miR-21 in NPCs and U-251MG cells inhibited viral gene expression, genome replication, and production of infectious progeny, while shRNA-knockdown of miR-21 in U-251MG cells increased viral gene expression. In contrast, overexpression of Cdc25a in U-251MG cells increased viral gene expression and production of infectious progeny and overcame the inhibitory effects of miR-21 overexpression. Three viral gene products—IE1, pp71, and UL26—were shown to inhibit miR-21 expression at the transcriptional level. These results suggest that Cdc25a promotes HCMV replication and elevation of Cdc25a levels after HCMV infection are due in part to HCMV-mediated repression of miR-21. Thus, miR-21 is an intrinsic antiviral factor that is modulated by HCMV infection. This suggests a role for miR-21 downregulation in the neuropathogenesis of HCMV infection of the developing CNS.IMPORTANCEHuman cytomegalovirus (HCMV) is a ubiquitous pathogen and has very high prevalence among population, especially in China, and congenital HCMV infection is a major cause for birth defects. Elucidating virus-host interactions that govern HCMV replication in neuronal cells is critical to understanding the neuropathogenesis of birth defects resulting from congenital infection. In this study, we confirm that HCMV infection downregulates miR-21 but upregulates Cdc25a. Further determined the negative effects of cellular miRNA miR-21 on HCMV replication in neural progenitor/stem cells and U-251MG glioblastoma/astrocytoma cells. More importantly, our results provide the first evidence that miR-21 negatively regulates HCMV replication by targeting Cdc25a, a vital cell cycle regulator. We further found that viral gene products of IE1, pp71, and UL26 play roles in inhibiting miR-21 expression, which in turn causes increases in Cdc25a and benefits HCMV replication. Thus, miR-21 appears to be an intrinsic antiviral factor that represents a potential target for therapeutic intervention.

In this report we describe the genomic sequence of guinea pig cytomegalovirus (GPCMV) assembled from a tissue culture-derived bacterial artificial chromosome clone, plasmid clones of viral restriction fragments, and direct PCR sequencing of viral DNA. The GPCMV genome is 232,678 bp, excluding the terminal repeats, and has a GC content of 55%. A total of 105 open reading frames (ORFs) of > 100 amino acids with sequence and/or positional homology to other CMV ORFs were annotated. Positional and sequence homologs of human cytomegalovirus open reading frames UL23 through UL122 were identified. Homology with other cytomegaloviruses was most prominent in the central ~60% of the genome, with divergence of sequence and lack of conserved homologs at the respective genomic termini. Of interest, the GPCMV genome was found in many cases to bear stronger phylogenetic similarity to primate CMVs than to rodent CMVs. The sequence of GPCMV should facilitate vaccine and pathogenesis studies in this model of congenital CMV infection.

An effective cytomegalovirus (CMV) vaccine could prevent the majority of birth defects caused by congenital CMV infections. Candidate vaccines in clinical evaluation include live attenuated, protein subunit, DNA, and viral-vectored approaches. Subunit approaches have focused on the CMV proteins pp65 and IE1 as important inducers of cytotoxic T cells and glycoprotein B (gB) as an important inducer of neutralizing antibodies. A vaccine comprised of recombinant gB protein with MF59 adjuvant reduced the incidence of primary infection by 50%. Recent revelations regarding CMV entry pathways into different cell types suggest a possible course for improvement. A 5-subunit pentameric complex is uniquely required for endothelial and epithelial cell entry. Sera from naturally infected subjects contain high-potency neutralizing activities specific for this complex, whereas the gB/MF59 vaccine fails to induce comparable neutralizing activities. A vaccine's ability to induce salivary antibodies that neutralize epithelial cell entry may be especially important for preventing oral transmission as the first cells infected are presumably epithelial cells of the oral mucosa. In addition, recent evidence suggests that antibodies can inhibit postentry CMV spread between endothelial and epithelial cells. Such activities may serve to limit viral replication in tissues or impair dissemination to the placenta and fetus. Thus, inclusion of epitopes derived from the pentameric complex may provide enhanced efficacy by inducing potent neutralizing/spread-inhibiting antibodies that target virus replication in a broad spectrum of cell types. Next-generation vaccine candidates in preclinical development incorporate peptides, subunits, or multisubunit complexes representing parts or all of the pentameric complex. Approaches include peptides, recombinant proteins, DNA, replication-defective viral vectors, genetically disabled CMV, and inactivated CMV virions. The diversity of novel strategies under development engenders optimism that a successful candidate will emerge.