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Defects in the HCMV assembly complex: Vacuolization occurs by blocking either viral or host kinases -Virology Laboratory-Medical research center

Mohammad Qawasmi, Maysa Salah, Mohammad Kurdi, Lina Qurei, Fatima Hamadeh, Zaidoun Salah and Maysa Azzeh

The cytoplasmic assembly complex (AC) in HCMV-infected human foreskin fibroblasts (HFF) is a unique "bulb"-like juxtanuclear structure.  Morphology of the AC is dependent on the viral-encoded kinase, pUL97.  We reported earlier that this morphology is also altered when wt-HCMV infected cells are treated with kinase inhibitors: NGIC-I, a kinase C inhibitor, or Staurosporine, a serine/threonine kinase inhibitor.  Infection with a UL97 deletion mutant simulated the inhibition with NGIC-I or Staurosporine of wt-HCMV infection, resulting in a less compact and a vacuole-rich AC.  In all three cases the viral titer was reduced 2-4 logs.  The rims of the vacuoles stained for WGA, a Golgi marker, as well as for pp28, pp65 (HCMV tegument proteins), gB and UL16 (HCMV glycoproteins).  Here, we investigated the building of the vacuoles in the AC.  Our kinetic experiments using Staurosporine revealed that the vacuoles were clearly detectable at 60hpi and that the viral titer was strikingly reduced at 72hpi.  The modified AC structure and the rims of the vacuoles stained additionally for trans- and cis-Golgi markers, TGN46 and GOLPH4, respectively.  However, employing non-kinase inhibitors or inducing ER stress did not result in such AC changes or in the creation of vacuoles.  In reference to the nature of these vacuoles, we observed damage of the AC structure and vacuoles with Brefeldin A treatment.  On the other hand, pre-treatment of HCMV-infected HFF with siRNA against secretory carrier membrane proteins also disrupted the AC, the vacuoles and reduced the viral titer.  Finally, our immunoprecipitation experiments indicated that pp28 co-immunoprecipitated with TGN.  All these data provide evidence for Golgi fabrication of the AC and vacuoles observed with kinase inhibition.  Furthermore, our results provide possible therapeutic roles for kinase inhibitors as anti HCMV drugs.