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Key Words:
Stealth, Cytomegalovirus, Chemokine, US28, Virus

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e-mail wj_martin@hotmail.com
Running Title: HCMV US28 Sequences in a Stealth Virus

ABSTRACT

The US28 gene of human cytomegalovirus (HCMV) codes a cell surface receptor for both beta-chemokine and fractalkine molecules. This receptor facilitates HCMV induced cell fusion, virus dissemination and influences susceptibility to infection with other viruses, including the human immunodeficiency virus (HIV). Five adjacent but divergent open reading frames that potentially code for molecules related to the US28 protein of HCMV are present in an African green monkey simian cytomegalovirus (SCMV) derived stealth virus. This finding implies a role for chemokines in the pathogenicity of at least some stealth-adapted viruses. It may also help explain the apparent therapeutic benefit achieved in certain stealth virus infected patients treated with agents that down regulate chemokine production.

INTRODUCTION

Chemokines are a subset of cell signaling protein molecules that, in addition to other biological activities, have the capacity to promote cellular migration (Zlotnik et al.1999). Four families of chemokines can be distinguished by the arrangement of cysteine amino acids involved in disulphide bond formation. Beta chemokines, which primarily attract monocytes, contain two adjacent cysteines (abbreviated CC) that form two cystine-to-cystine disulphide bonds. Alpha chemokines, which primarily attract polymorphonuclear cells, have a single amino acid separating the two cysteines and are designated CXC. Fractalkines are cell bound proteins in which the initial two cysteines are separated by three amino acids and are referred to as CX3C. They are extensively expressed within the brain and may mediate neuron-glial cell interactions (Harrison et al.1998; Nishiyori et al.1998). A remaining type of chemokine contains only a single disulphide bond and is abbreviated C.

Chemokine receptors also constitute an array of molecules with varying specificity for individual members of each of the chemokine families (Zlotnik et al.1999) Certain receptors also allow for cross binding of chemokines belonging to different family groups. Typically the chemokine receptors loop in and out of the cell membrane with seven transmembrane segments and an intracellular tail that is coupled to a G protein signal transducing molecular complex.

Several viruses encode for molecules with chemokine and chemokine receptor like structures (McFadden et al.1998; Penfold et al.1999; Bais et al.1999; Bugert et al.1998). Moreover, some viruses use chemokine receptors to facilitate intracellular entry (Lalani et al.1999; Pleskoff et al.1998; Rucker et al.1997). Most notable is the use of certain CC, CXC and CX3C receptors by HIV (Berger 1999; Combadiere et al.1998). Among the viral coded chemokine receptors, the unique short segment coded US28 molecule of HCMV has been extensively studied (Gao and Murphy 1994; Kuhn et al.1995; Billstrom et al.1998). It binds to CC chemokines, and even more avidly to CX3C fractalkine (Kledal et al.1998).

Stealth adaptation is a proposed mechanism that allows viruses to evade immune elimination through the deletion of antigens targeted by the cellular immune system (Martin 1994; 1999c). A prototype stealth virus, derived from SCMV, has been cloned and partially sequenced (Martin et al. 1994;1995; Martin 1996). The virus has retained numerous sequences that match to various genes of HCMV, and where the comparison can be made, match even more closely to SCMV (Martin et al. 1995; Martin 1999c). In addition, the virus has acquired numerous cellular sequences from infected cells (Martin 1998; 1999b) and also sequences of bacterial origin (Martin 1999a). The presence of bacterial sequences within certain stealth viruses has led to the designation of such microorganisms as "viteria" (Martin 1999a).

This paper describes the presence of five adjacent, yet divergent, nucleotide sequences within a cloned region of the prototype stealth virus. The sequences potentially code for proteins that show a statistically significant homology to the US28 chemokine receptor of HCMV. The remaining portions of the stealth virus clone code for two proteins that are respectively highly homologous to the US24 and US26 proteins of HCMV (Chee et al.1990).

MATERIALS AND METHODS

The prototype stealth virus was isolated from a patient with the chronic fatigue syndrome. Cultured virus was subsequently obtained from filtered lysate of frozen thawed infected MRC-5 cells (Martin et al. 1995) DNA was extracted from material pelleted by ultracentifugation. It was fractionated on an agarose gel, where it migrated as a single band of approximately 20 kilobase pairs (Martin et al. 1994). The DNA was cut with SacI restriction enzyme and cloned in pBluescript to yield the C16 series of clones. DNA sequencing was performed by Lark Technology, Houston TX. Clone C16246 comprised 8,259 nucleotides. The 3’ end of the clone showed a 736 nucleotide overlap (99% identity) with a previously sequenced 897 nucleotide EcoRI restricted clone. The sequence of this clone allowed for a 161 nucleotide extension of the sequence provided by clone C16246. The extended C16246 sequence is available from the National Center for Biotechnology Information (NCBI) using GenBank identifier 903463.

Sequence analysis was performed using the BLASTN, BLASTX and BLASTP programs of NCBI (Altschul et al. 1997). The probability of random matching of GenBank sequences is given as an "Expect" value, rather than as a "p" value using the descriptor "e" as log₁₀. The identification of open reading frames (ORF) was provided using the ORF Finder program of NCBI and the Map program of the Genetic Computer Group (GCG), Madison WI. Sequence comparisons were made using the BLAST 2 Sequence program of NCBI and the PileUp and PrettyPlot programs of GCG.

RESULTS

BLASTN analysis of the 8,420 nucleotide sequence showed that it contained a small region of 195 nucleotides with 157 nucleotide identity (Expect 2e-13) with the region of the AD169 isolate of HCMV extending from nucleotides 214,110 to 214,304. The region is part of the coding sequence for the US24 protein of HCMV. In contrast to the relatively limited homology revealed by nucleotide matching, BLASTX analysis showed two relatively long regions with striking homologies to the US24 and US26 of HCMV, respectively, followed by five regions that matched to the US28 gene of HCMV.

The ORF Finder program of NCBI was used to further identify the seven ORF that corresponded to the BLASTX-determined homologous regions. The sequence of each ORF was analyzed using the BLASTP program of NCBI. The best matching GenBank sequences are shown in Table 1. An initial ORF coded on frame –1 (complementary strand) matched to HCMV US24 protein (GenBank identifier 137156 ) with 65% amino acid identity and 81% similarity in amino acids (recorded as positives in Table 1) over a stretch of 465 amino acids. The overlap covered the region from amino acid number 30 to the end of the 500 amino acid HCMV US24 protein. The high degree of homology is beyond statistical calculation and is recorded as 0.0. The second ORF matched to HCMV US26 (GenBank identifier 137158) with 47% amino acid identity and an Expect value of e-152. Interestingly, the size of ORF #2 (452 amino acids) was less than that of HCMV US26 (603 amino acids) and the matching did not cover the initial 140 amino acids of the HCMV US26 molecule. ORF # 3-7 all matched best to an amino acid sequence derived from the US28 gene present in several independently derived HCMV isolates (GenBank identifier 306304). The Expect values ranged from 4e-13 to 7e-30 with identity ranging from 23 to 34%. The lower value applied to ORF # 7 for which the complete sequence at the 3’ end was not available.

ORF # 3-7 also matched to a large number of chemokine receptor molecules, most notably to the CX3C fractalkine receptor and various CC receptors. The most significant non-HCMV matching proteins for each of the 7 ORF are shown in the last column of Table 1. The highest matching sequences (ORF #4) corresponded to a CX3C fractalkine receptor molecule of the mouse (GenBank identifier 3851709). This receptor also provided the second best matches for ORF #3 and #6, both of which showed a slightly better match to a "probable G-protein coupled receptor, GPRD, expressed in rat spinal cord and brain related to chemokine receptors" (Harrison et al.1994; GenBank identifier 548703). ORF # 6 also matched well (Expect value 5e-12) to an broadly responsive alpha chemokine receptor (Gobl et al.1997; high affinity interleukin 8 receptor B, GenBank identifier 1352455). ORF #5 best matched to a CC receptor identified in the baboon Papio cynocephalus papio (GenBank identifier 5713056) whereas ORF #7 best matched to a CC receptor of the rat (Jiang et al.1998; GenBank identifier 2897073).

ORF # 3-7 were similarly identified using the Map Program of GCG. The amino acid sequences of each of the ORF were aligned along with the sequences of HCMV US28 molecule, rat GPRD chemokine receptor and mouse CX3C fractalkine receptor, using the PileUp program of GCG. Figure 1 shows a plot of the alignment. Identical or equivalent amino acids shared by four or more of the sequences at the same location are boxed. This analysis showed marked diversity, yet retention of several stretches of amino acid identity (or close similarity) between one or more of the OFR and the cellular and/or HCMV proteins. The observed diversity is consistent with the essential lack of significant nucleotide matching using the BLAST 2 Sequences comparison of the nucleotides coding ORF # 3-7 (data not shown).

DISCUSSION

Viral encoded chemokine receptors are likely to play important roles in viral host interactions (Murayama et al.1998; Gershengorn et al.1998; Monini et al.1999; Yurochko and Huang 1999; Chang et al. 2000). . They can render a viral infected cell subject to growth regulation by host and/or virus derived chemokines, and may possibly promote malignant transformation (Burger et al.1999). They can also conceivably act as a drain on the availability of chemokines required for various physiological networks (Bodaghi et al.1998; Billstrom et al.1999). Chemokine receptors can also provide important attachment molecules to facilitate infection by other viruses, including HIV (Rucker et al.1997). The presumed sequence homology between viral and cellular encoded chemokine genes may also allow for genetic recombination with an altered function of the resulting gene product. Finally, chemokine networks involved in viral activation can provide a potential target for therapeutic intervention in virus induced diseases.

The process of stealth-adaptation involves the loss of genes required for effective anti-viral cellular immunity. The mechanism whereby such viruses can retain and/or regain their cytopathic activity is largely unknown, but appears to involve the acquisition of new genetic sequences (Martin 1998, 1999a,b). It is likely that, through a selection process, genes that favor viral survival and/or replication would be preserved and even amplified. This may be the case with the genes that potentially encode chemokine receptor proteins described in this paper. It has not been shown, however, that these genes are, in fact, transcribed and that the proteins actually function in chemokine binding and/or G protein activation. In favor of the functionality of the presumptive stealth virus proteins, is the presence of various amino acids common to both the cellular chemokine receptors and the US28 protein (Figure I).

As previously described, there is considerable microheterogeneity between stealth viral clones that match to similar regions of the HCMV genome (Martin 1996). It is likely, therefore, that through an ongoing mutational process, both functional and non-functional (or even inhibitory) stealth virus coded chemokine receptor-related proteins may exist. As such, the extended C16246 clone may not reflect the entire repertoire of the stealth virus genes encoding US28 related sequences.

The homologies between the deduced amino acid sequences in extended clone C16246 that correspond to the US24 and US26 genes of HCMV exceed the homologies of the five regions of the clone that matched to the US28 protein of HCMV. This difference may reflect a greater divergence of US28, compared to both US24 and US26, between SCMV and HCMV. This can not be the entire answer, however, since the five US28 matching regions were quite distinct from each other. Whether the five genes were originally viral or cellular in origin, and whether derived from a single incorporated gene that was subsequently amplified, or from different genes, cannot be answered at present. It is also not known whether SCMV contains genes encoding US25 and US27 genes. If so then these genes have been deleted from at least parts of the stealth virus genome. Similarly, if the US26 gene of SCMV is comparable in length to that of HCMV, the corresponding stealth virus protein is significantly truncated at its amino terminal end.

As previously reported (Martin 1999b), the prototype stealth virus has acquired three copies of an alpha (CXC) chemokine, related to melanoma growth stimulatory activity/Gro-alpha (MGSA/Gro-alpha). With the possible exception of ORF # 6, it seems unlikely that alpha chemokines would engage the US28-related receptors described in the present paper. Still the detection of amplified copies of genes coding chemokines and chemokine receptors clearly highlight the potential role of chemokine mediated pathways in stealth virus replication and pathogenesis. In this regard, it is worth noting that certain therapeutic modalities commonly prescribed for patients with chronic fatigue syndromes, and with other stealth virus related conditions, may be achieving their beneficial effects by regulating chemokine levels. These compounds include various antibiotics (Matsuoka et al.1996), a broad range of agents referred to as DMARD (disease modifying anti-rheumatic drugs; Conaghan et al.1997; Danning and Boumpas 1998), certain neuropsychiatric medications (Taupin et al.1991) and a range of dietary supplements (Jobin et al.1999; Ishikawa et al.1999). Culturing stealth viruses provides a useful in vitro system to help assess the capacity of these and other agents to suppress any chemokine-mediated virus activation. This approach may prove particularly useful to help identify adjunct therapy for patients in whom stealth virus infection may be linked to malignancy (Gollard et al.1996; Martin and Anderson 1997).

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Table 1. BLASTP Analysis of Predicted Amino Acid Sequences of Open Reading Frames in Clone of SCMV-Derived Stealth Virus

Legend to Figure 1. GCG PileUp computer generated alignment of the amino acid sequences of a chemokine receptor identified in rat brain (GPRD); CX3C receptor of murine origin, US28 protein of HCMV and ORF # 3-7 identified in extended clone C16246 obtained from a SCMV-derived stealth virus. The boxed areas show regions in which 4 or more of the sequences share the same or an closely related amino acid. Default parameters were used without manual adjustments which could yield somewhat better alignments. For example the cysteine at position 175 of GPRD could be aligned with five rather than two additional cysteines.