Characterization of pUL5, an HCMV protein interacting with the cellular protein IQGAP1

Human cytomegalovirus (HCMV) is a ubiquitous virus infecting the majority of adults worldwide. In healthy individuals, a strong immune response to HCMV is able to limit and contain the spread of the disease, although the virus is able to establish a lifelong lasting latency with recurrent and spontaneous reactivation. Acute disease is observed only in particular settings where the normal immune response of the patient is weak or compromised, such as in transplant or patients with Acquired Immuno-Deficiency Syndrome (AIDS) or in fetuses that acquire the infection congenitally. HCMV possesses the largest genome among herpesviruses and a lot of effort is being put on the study of the huge coding potential this virus displays in order to have a better understanding of the mechanisms behind its infection. In this work, we address the study of the protein product of UL5 open reading frame (ORF), belonging to the RL11 gene family and previously uncharacterized. UL5 is only present in human and chimpanzee cytomegaloviruses while is absent in cytomegaloviruses infecting other species, therefore is considered a ‘private’ gene. Among HCMV strains, UL5 aminoacid sequence is relatively conserved and all minor differences are concentrated in the N-terminus. In silico predictions classify pUL5 as a type Ib membrane protein with no signal peptide and no N-linked glycosylations, while 7 O-glycosylation sites are predicted. Three different mRNAs with similar lengths including UL5 and the previous ORF UL4 have already been described in literature. In order to confirm these data and to exclude the presence of other UL5-containing transcripts, we performed a Northern blot analysis on the total RNA extracted from HCMV-infected cells at different times post infection (p.i.) using an UL5-specific biotinylated DNA probe. A band around 1.5 kb was detected, excluding the presence of further transcripts. To address the study of UL5 protein product (pUL5), a two-step mutagenesis was carried out on a bacterial artificial chromosome (BAC) containing the entire genome of the TR strain of HCMV to generate a recombinant virus with the addition of an optimized combination of tags (2StrepII-2FLAG) at the C-terminus of pUL5. pUL5 expression profile was studied by immunoblot on lysates of fibroblasts infected with the recombinant pUL5-tagged virus prepared at different times p.i. using an anti-FLAG antibody. Two species of approximately 24 and 15 kDa were detected with late and early expression kinetics, respectively. Since the predicted molecular weight of the entire tagged protein is 24.5 kDa, the higher molecular weight form probably corresponds to the full-length protein. Glycosylation analysis of pUL5 both in transfection and in infection further confirmed the absence of glycosylation events. To investigate the nature of the lower molecular weight species, we performed a PCR site-directed mutagenesis to eliminate a second ATG in UL5 ORF. Western blot analysis on lysates of HEK293T cells transfected with the mutated plasmid of UL5 revealed a strong reduction in the signal of the 15 kDa species. A recombinant form of pUL5 lacking the putative transmembrane region was expressed in E. coli, purified and used to obtain an antiserum in mice. Western blot on lysates of fibroblasts infected with the wild-type virus using the mouse antiserum replicated the previous data, excluding a possible influence of the tags to the obtained results. Confocal microscopy of infected fibroblasts both with wild-type and recombinant viruses, implementing both the pUL5 antiserum and the anti-FLAG antibody, localizes pUL5 with markers of the trans-Golgi network and secretory pathways, in the compartment of viral assembly. Western blot on purified virions failed to reveal the presence of pUL5, suggesting that this protein is only expressed in the host cell during infection but it is not incorporated in the released viral progeny. To have a deeper insight in pUL5 role during infection, an UL5 deletion mutant was generated by BAC mutagenesis and its ability to replicate in human fibroblasts was assessed. The knock-out mutant virus is still able to infect and replicate in fibroblasts but with a strongly reduced replication rate, suggesting an important role of pUL5 during HCMV infection. The recombinant pUL5-tagged virus was also implemented for a pull-down experiment to isolate potential interacting partners using anti-FLAG coupled magnetic beads. Mass spectrometry analysis allowed the identification of the cellular protein IQGAP1 as a pUL5 interactor. IQGAP1 is an ubiquitously expressed cellular protein involved in many cellular mechanisms, such as cytoskeletal remodeling, cell adhesion, cell cycle regulation and Ca2+/Calmodulin signaling, and it is a known target of intracellular pathogens. This interaction was also confirmed by reciprocal immunoprecipitations in transfection, giving new hints on the possible role of pUL5 during HCMV infection.