UL 146 variability among clinical isolates of Human Cytomegalovirus from Japan

Human Cytomegalovirus (HCMV) is a herpesvirus associated with serious diseases in immunocompromised subjects. The region between ORF UL133 and UL151 from HCMV, named ULb’ is frequently deleted in attenuated AD169 and in highly passaged laboratory strains. However, this region is conserved in low-passaged and more virulent HCMV, like the Toledo strain. The UL146 gene, which is located in the ULb’ region, encodes a CXC-chemokine analogue. The diversity of UL146 gene was evaluated among fifty-six clinical isolates of HCMV from Japan. Results show that UL146 gene was successfully amplified by the polymerase chain reaction (PCR) in only 17/56 strains (30%), while the success rate for UL145/UL147 gene was 18/56 strains (32%). After DNA sequencing, the 35 amplified strains were classified into 8 groups. When compared, variability of UL146 ranged from 25.1% to 52.9% at the DNA level and from 34.5% to 67% at the amino acid level. Seven groups had the interleukin-8 (IL-8) motif ERL (Glu-Leu-Arg) CXC and one group had only the CXC motif, suggesting the absence of the IL-8 function of UL146. In conclusion, we found that UL146 gene of HCMV is hypervariable in clinical strains from Japan suggesting the possibility of a different function in each sequence group. Key terms: a) Cytomegalovirus, b) UL146, c) Sequence, d) V. * Corresponding author: Francisco Aguayo González, Ph.D., Programa de Virología, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Fono: 56-2-9786317, Fax : 56-2-9786124, E-mail: faguayo@med.uchile.cl Received: September 8, 2010. In revised form: November 9, 2010. Accepted: November 19, 2010. INTRODUCTION Human Cytomegalovirus (HCMV) is a widespread ubiquitous herpesvirus that usually produces asymptomatic infections in immunocompetent hosts. However, serious HCMV-associated diseases might occur in immunocompromised individuals such as organ transplant recipients or in patients with acquired immunodeficiency syndrome (AIDS) and in congenitally infected newborns (Britt WJ, 1996). The severity of HCMV diseases varies widely ranging from stillborn or neonatal death to no abnormalities in congenital infections and sightand lifethreatening diseases in immunocompromised hosts. Furthermore, the prevalence of HCMV retinitis in patients with AIDS is higher than among drug users (Brantsaeter et al., 2002). Clinical and epidemiological factors associated with severe disease have been identified (PuchhammerStockl & Gorzer, 2006), however little is known about viral factors associated with the variability in susceptibility to, or severity of HCMV-associated disease. HCMV has a large and complex genome; containing approximately 235 Kb of DNA and more than 200 open reading frames (ORFs) (Dolan et al., 2004). Interestingly, in laboratory-adopted attenuated HCMV strains, such as AD169, 19 ORFs between ORF UL133 and UL151 are deleted (Cha et al., 1996), suggesting that proteins encoded by these genes are not required for HCMV replication in cell culture. Nonetheless, this region is present in the Toledo strain of HCMV (HCMVTol) and also in low-passaged laboratory isolates, suggesting that these genes or their encoded proteins play a role in the wild-type HCMV strains and may be essential for viral infection or pathogenicity in vivo. The UL146 gene, which encodes for a protein similar to α (CXC)chemokine (Penfold et al., 1999), is located between UL133 and UL151. The UL146 product, vCXCL-1, is a 117-amino acid secreted glycoprotein that functions as a selective agonist for CXCR1 and CXCR2, although with differential affinity and potency (Luttichau, 2010). Its expression correlates with the ability to attract human neutrophils. In fact, recombinant vCXCL-1 has full chemokine functions, such as the induction of calcium mobilization, chemotaxis and human neutrophils degranulation (Penfold et al., 1999). The UL146 gene has been previously reported to be hypervariable among clinical isolates of HCMV. Additionally, phylogenetic analysis reveals that different unrelated strains cluster in defined sequence groups (AravBoger et al., 2006; Hassan-Walker et al., 2004; Lurain et al., 2006). In this report, we described the variability of UL146 gene and the phylogenetic relatedness among clinical isolates of HCMV from Japan. MATERIALS AND METHODS


INTRODUCTION
Human Cytomegalovirus (HCMV) is a widespread ubiquitous herpesvirus that usually produces asymptomatic infections in immunocompetent hosts.However, serious HCMV-associated diseases might occur in immunocompromised individuals such as organ transplant recipients or in patients with acquired immunodeficiency syndrome (AIDS) and in congenitally infected newborns (Britt WJ, 1996).The severity of HCMV diseases varies widely ranging from stillborn or neonatal death to no abnormalities in congenital infections and sight-and lifethreatening diseases in immunocompromised hosts.Furthermore, the prevalence of HCMV retinitis in patients with AIDS is higher than among drug users (Brantsaeter et al., 2002).Clinical and epidemiological factors associated with severe disease have been identified (Puchhammer-Stockl & Gorzer, 2006), however little is known about viral factors associated with the variability in susceptibility to, or severity of HCMV-associated disease.
HCMV has a large and complex genome; containing approximately 235 Kb of DNA and more than 200 open reading frames (ORFs) (Dolan et al., 2004).Interestingly, in laboratory-adopted attenuated HCMV strains, such as AD169, 19 ORFs between ORF UL133 and UL151 are deleted (Cha et al., 1996), suggesting that proteins encoded by these genes are not required for HCMV replication in cell culture.Nonetheless, this region is present in the Toledo strain of HCMV (HCMV Tol ) and also in low-passaged laboratory isolates, suggesting that these genes or their encoded proteins play a role in the wild-type HCMV strains and may be essential for viral infection or pathogenicity in vivo.The UL146 gene, which encodes for a protein similar to α (CXC)chemokine (Penfold et al., 1999), is located between UL133 and UL151.The UL146 product, vCXCL-1, is a 117-amino acid secreted glycoprotein that functions as a selective agonist for CXCR1 and CXCR2, although with differential affinity and potency (Luttichau, 2010).Its expression correlates with the ability to attract human neutrophils.In fact, recombinant vCXCL-1 has full chemokine functions, such as the induction of calcium mobilization, chemotaxis and human neutrophils degranulation (Penfold et al., 1999).The UL146 gene has been previously reported to be hypervariable among clinical isolates of HCMV.Additionally, phylogenetic analysis reveals that different unrelated strains cluster in defined sequence groups (Arav-Boger et al., 2006;Hassan-Walker et al., 2004;Lurain et al., 2006).In this report, we described the variability of UL146 gene and the phylogenetic relatedness among clinical isolates of HCMV from Japan.

Cells and viruses
Human embryonic lung fibroblasts (HEL) were grown in Eagle's minimum essential medium (Nissui Pharmaceutical Co., LTD., Tokyo) supplemented with 10% fetal bovine serum (Flow Laboratories Inc., Irvine, VA), 60 μg/mL of kanamycin and 0.12 % NaHCO3.HEL cells were used for HCMV isolation and propagation.Thirty-three clinical isolates were obtained from urine, 2 from amniotic fluid, 2 from saliva, 4 from lymphocytes, 1 from mother's milk, 1 from throat swab, 1 from tears and 9 from other human sources.Almost all the specimens used in this study were obtained from subjects with congenital HCMV infection, except for one liver and four renal post-transplanted patients.All the clinical strains were passaged as a cellassociated virus 5 to 8 times before preparation of viral DNA.

Preparation of viral DNA
Viral DNA was prepared from the infected cells following the Hirt's procedure (Hirt, 1967).The Hirt's supernatant was treated with Proteinase K (50 μg/mL) at 56ºC for 2 hours, followed by phenol/chloroform extraction and ethanol precipitation.Finally, the DNA was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA).

Polymerase chain reaction for the UL146 sequence
The Hirt's supernatant DNA was diluted in sterilized water to optimal concentrations for use as templates for PCR amplification of specific regions of the HCMV genome.All the samples were shown to be HCMV positive through IE1 region amplification.

DNA sequencing
The amplicons were purified from agarose gels using QIAEX II Gel extraction Kit (Qiagen GmmH, Hilden, Germany) according to the manufacturer's instructions.The purified PCR products were sequenced with the BigDye Terminal Cycle sequencing Kit (PE applied Biosystems, Foster City, CA), according to the manufacturer's instructions, and analyzed using the ABI310 automated sequencer (PE Applied Biosystems, Foster City, CA).

Phylogenetic and protein analysis
A phylogenetic tree inferred from the HCMV UL146 ORF was constructed by the neighbor-joining method with 354 bp nucleotides of this gene.The Clustal W and Jalview software servers were used.TreeView software was used to display the phylogenetic tree.NetNGlyc 1.0 Server was used to find glycosylation sites and NetPhos 2.0 Server was used to find phosphorylation sites in the vCXCL-1 protein.

UL146 gene amplification and sequencing in clinical strains
Only 17/56 (30.4%) strains were successfully amplified by PCR when the primer pair to HCMV UL146 Tol ORF was used.Another 18 strains were amplified using UL145F and UL147R primer pair.Thus, a total of 21 clinical strains were not successfully amplified when the herein proposed experimental strategy was used.Nonetheless, all the clinical strains were positive for IE1 gene amplification, demonstrating the presence of viral DNA (data not shown).Amplicons were sequenced, confirming that they corresponded to HCMV isolates.Phylogenetic analysis clustered the 35 isolated HCMV strains into 8 distinct groups (Fig. 1).The UL146 Tol , group 1 proved to be the most prevalent among the Japanese clinical isolates (14 strains), followed by group 8 (4 strains), group 3 (4 strains), group 5 (4 strains), group 6 (3 strains), group 4 (3 strains), group 2 (2 strains) and group 7 (1 strain).Further analysis revealed that the ratios of sequence variability among the different groups ranged from 25.1% to 52.9% (Table I).

The vCXCL-1 sequence analysis
Sequence analysis further revealed that the ratios of amino acid variability among the 8 distinct groups ranged from 34.5% to 67%.The predicted amino acid sequences were compared among 8 representative strains of each individual group (Table II).The estimated size of the vCXCL-1 protein ranged from 97 to 125 amino acids (Fig. 2).The UL146 Tol group contained the ELRCRC motif.Two strains belonging to group 2 contained the ELRCKC motif.All 16 strains belonging to groups 3, 4, 5, 6 and 7 contained the ELRCPC motif, although the variability at the amino acid level among these groups ranged from 34.5% to 66.7%.The 4 strains belonging to group 8 contained the CXC motif, but lacked the ELR motif.The variability between group 8 and other groups at the amino acid level ranged from 54.5% to 67%, suggesting a clear difference in the protein sequence between group 8 and the other groups of this study.The bioinformatics analysis of post-translational modifications showed that the sequence of group 1 and 2 exhibit three possible glycosylation sites; groups 3 to 6 do not have any predictable glycosylation sites, while groups 7 and 8 have only one possible glycosylation site.The analysis for possible serine, threonine and tyrosine phosphorylation sites were different in all 8 groups (Table III).

DISCUSSION
It was previously reported that the HCMV Toledo strain has 19 ORF between UL133 and UL151 in the UL/b' region.Strikingly, these OFR are not present in AD169, even in highly passaged attenuated laboratory viral strains (Cha et al., 1996).To us, these observations suggest that these additional genes might be candidate pathogenic markers of HCMV infection.Inside the UL/b' region, the UL146 gene that encodes a CXC-chemokine is highly variable in epidemiological studies.Thus, we were interested in analyzing the diversity of UL146 of clinical isolates of HCMV from Japan using PCR followed by UL146 sequencing.The phylogenetic analysis of the UL146 gene showed that is highly polymorphic and variants can be clustered in eight sequence groups (Fig. 1).These groups, except for four  strains corresponding to group 8, exhibited the conserved ELR/CXC domain (Fig. 2).Group 8 displayed the CTC domain, suggesting that the physiological function of these proteins may differ from that of the other seven groups.
Previously, Dolan et al. clustered UL146 sequences in 14 groups (Dolan et al., 2004), so we aligned these sequences with those reported here.Seven UL146 sequences at the amino acid level reported by Dolan can be clustered in seven groups reported in this study (group 1: Toledo; group 2: KSG, group 3: KM; group 5: Towne; group 6: FS; group 7: TB40; group 8: Davis).In addition, another six sequences cannot be clustered in the groups reported in this study (RK, AL, 6397, ML1, NT and Merlin).

Table II
Alignment of the HCMV-encoded chemokine vCXCL-1 obtained from 35 HCMV strains from Japan The ELR/CXC chemokine motif plays an important role in neutrophils recruitment and promotes angiogenesis (Strieter et al., 2005).The specific absence of the ELR domain has been reported to be angiostatic (Strieter et al., 1995).In this report, we did not find clinical evidence suggesting association between ELR/CXC motif presence and outcome or progression of HCMV infection.In agreement with this observation, Arav-Boger et al. in 2006 reported that UL146 gene is polymorphic in congenitally infected newborns and no association between specific UL146 variant and the outcome of HCMV infection was found (Arav-Boger et al., 2006).Other studies have analysed the association between the variation of glycoprotein genes (gB) and the outcome of HCMV infection, however, results remain highly controversial (Rasmussen et al., 1997;Sarcinella et al., 2002).In our study, we observed differences in glycosylation and phosphorylation sites in the vCXCL-1 protein among the eight groups, suggesting that these proteins might have different functions in vivo.However, it was reported that variations in the ELR-CXC motif in UL146 ORF do not alter the functionality of the vCXCL-1 protein (Miller-Kittrell et al., 2007).
The variability of other genes in the UL133-UL151 region of HCMV has been analysed and the function of these genes is currently being investigated.It is suggested that the UL133-UL151 region is important in immune response evasion and might be involved in some of the pathogenic properties ascribed to HCMV (Wang et al., 2005).For instance, the sequence of the UL144 ORF, reported to encode a tumor necrosis factor analogue, amplified from clinical samples clustered in five defined phylogenetic groups (Lurain et al., 1999;Murayama et al., 2005;Picone et al., 2005).The UL139 gene was shown to be highly polymorphic and the UL139 sequences clustered in six groups, in which various nucleotide insertions and non-synonymous substitutions were detected (Qi et al., 2006).UL139 function is unknown; however the protein product had sequence similarity with human CD24, a signal transducer modulating B-cell activation responses (Qi et al., 2006).On the other hand, it was found that UL141 is highly conserved in clinical strains from China (Ma et al., 2006).This protein downregulates a natural killer cellactivating ligand CD155, suggesting that might be involved in NK cell evasion function (Tomasec et al., 2005).In addition, a previous study reported that the UL147 gene is highly polymorphic and the encoded protein displays a CXC motif, similar to the UL146 gene (Lurain et al., 2006).Albeit variation between HCMV strains was lower when compared to UL146 (He et al., 2006).However, some evidence established a link between these two genes, although no recombination was shown (Lurain et al., 2006).Although the HCMV genome is well conserved, the UL133-UL151 region is polymorphic and especially the UL146 gene.In the case of this protein, the variability among different groups has been reported to be very stable in vitro and in vivo and no mutations occur easily in infected individuals (Lurain et al., 2006;Stanton et al., 2005).It has been proposed that the variability in the viral chemokines helps to fine tune the neutrophil response, increasing the efficiency of HCMV dissemination (Miller-Kittrell et al., 2007).
In summary, in this report we describe that the HCMV UL146 gene amplified from Japanese clinical isolates is highly polymorphic and phylogenetic analysis clustered the gene sequences in 8 defined groups, suggesting a different function for the vCXCL-1 protein in each distinct group.Additional studies are necessary to characterize the UL146 function in infected cells and the eventual association with outcome or severity of the disease in immunocompromised patients.

Table III
Glycosylation and phosphorylation site in eight vCXCL-1 groups.
Group Glycosylation Phosphorylation Ser Thr Tyr

Table I
Nucleotide and amino-acid variability among different UL146 groups * Nucleotide variability; $ Amino acid variability