Polyomavirus BK-encoded microRNA suppresses autoregulation of viral replication

Polyomavirus BK (BKV) infection is an important cause of renal allograft failure. Viral microRNAs are known to play a crucial role in viral replication. This study investigated the expression of BKV-encoded microRNAs (miR-B1) in patients with polyomavirus-associated nephropathy (PVAN) and their role in viral replication. Following BKV infection in renal proximal tubular cells, the 3p and 5p miR-B1 levels were significantly increased. Cells transfected with the vector containing the miR-B1 precursor (the miR-B1 vector) showed a significant increase in expression of 3p and 5p miR-B1 and decrease in luciferase activity of a reporter containing the 3p and 5p miR-B1 binding sites, compared to cells transfected with the miR-B1-mutated vector. Transfection of the miR-B1 expression vector or the 3p and 5p miR-B1 oligonucleotides inhibited expression of TAg. TAg-enhanced promoter activity and BKV replication were inhibited by miR-B1. In contrast, inhibition of miR-B1 expression by addition of miR-B1 antagomirs or silencing of Dicer upregulated the expression of TAg and VP1 proteins in BKV-infected cells. Importantly, patients with PVAN had significantly higher levels of 3p and 5p miR-B1 compared to renal transplant patients without PVAN. In conclusion, we demonstrated that (1) miR-B1 expression was upregulated during BKV infection and (2) miR-B1 suppressed TAg-mediated autoregulation of BKV replication. Use of miR-B1 can be evaluated as a potential treatment strategy against BKV infection.     

DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi’s sarcoma-associated herpesvirus latent replication

During latent infection, latency-associated nuclear antigen (LANA) of Kaposi’s sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/−) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.     

Substrate modulation of enzyme activity in the herpesvirus protease family

The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 A resolution structure of Kaposi's sarcoma-associated herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme. Extended substrate binding sites are induced upon peptide binding. In particular, 104 A2 of surface are buried in the newly formed S4 pocket when tyrosine binds at this site. The peptide inhibitor also induces a rearrangement of residues that stabilizes the oxyanion hole and the dimer interface. Concomitant with the structural changes, an increase in catalytic efficiency of the enzyme results upon extended substrate binding. A nearly 20-fold increase in kcat/KM results upon extending the peptide substrate from a tetrapeptide to a hexapeptide exclusively due to a KM effect. This suggests that the mechanism by which herpesvirus proteases achieve their high specificity is by using extended substrates to modulate both the structure and activity of the enzyme.     

The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence

Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.     

KSHV latent protein LANA2 inhibits sumo2 modification of p53

Tumor suppressor p53 plays a crucial antiviral role and targeting of p53 by viral proteins is a common mechanism involved in virus oncogenesis. The activity of p53 is tightly regulated at the post-translational levels through a myriad of modifications. Among them, modification of p53 by SUMO has been associated with the onset of cellular senescence. Kaposi´s sarcoma-associated herpesvirus (KSHV) expresses several proteins targeting p53, including the latent protein LANA2 that regulates polyubiquitylation and phosphorylation of p53. Here we show that LANA2 also inhibits the modification of p53 by SUMO2. Furthermore, we show that the reduction of p53-SUMO2 conjugation by LANA2, as well as the p53-LANA2 interaction, both require the SUMOylation of the viral protein and its interaction with SUMO or SUMOylated proteins in a non-covalent manner. Finally, we show that the control of p53-SUMO2 conjugation by LANA2 correlates with its ability to inhibit SUMO2- and type I interferon-induced senescence. These results highlight the importance of p53 SUMOylation in the control of virus infection and suggest that viral oncoproteins could contribute to viral infection and cell transformation by abrogating p53 SUMOylation.     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.