Experimental and Molecular Pathology
Melanoma Growth Stimulatory Activity (MGSA/GRO-alpha) Chemokine Genes Incorporated into an African Green Monkey Simian Cytomegalovirus (SCMV)-Derived Stealth Virus
W. John Martin, M.D., Ph.D.
Running Title: Chemokine coding stealth viruses
Address for Correspondence:
DNA isolated from the culture of an African green monkey simian cytomegalovirus (SCMV) derived stealth virus has been cloned. A region of the virus that contains genes coding proteins homologous to the UL141, UL144 and UL145 proteins of human cytomegalovirus, has recombined with cellular sequences encoding several distinct copies of the melanoma growth stimulatory activity (MGSA/GRO-alpha) chemokine gene. This finding illustrates the capacity of stealth viruses to capture, amplify, and mutate genes with potential oncogenic activity. The lack of introns in the assimilated cellular genes implies a role for reverse transcription in the assembly of stealth viruses.
The term "stealth" has been applied to a molecularly heterogeneous grouping of atypically structured, cytopathic viruses that can induce multi-system illnesses without evoking an anti-viral inflammatory reaction (1-2). A prototype stealth virus has a fragmented, genetically unstable genome; portions of which were derived from an African green monkey simian cytomegalovirus (SCMV). Other portions of the same stealth virus consist of genetic sequences unrelated to those of known cytomegaloviruses (3-4 ).
Ongoing sequencing studies indicate that foreign genes, including sequences of both cellular and bacterial origins, can become incorporated into replicating stealth viruses (5-6). The potential, therefore, exists for the assimilation and over expression of various cellular oncogenes. This paper provides sequence data that the SCMV-derived stealth virus has acquired several copies of a gene with a deduced amino acid sequence closely related to that of melanoma growth stimulatory activity (MGSA/GRO-alpha) chemokine (7-9 ).
Materials and Methods
The cloning of DNA isolated from cultures of stealth virus-1 has been described previously (1,4). Partial or complete sequence data have been obtained on over 400 clones, and the sequences analyzed using both BlastN and BlastX (10) of the National Center for Biotechnology Information (NCBI). The sequence data have been submitted to GenBank under the listing stealth virus-1.
The ends of five partially sequenced clones matched by BlastX analysis to the MGSA/GRO-alpha and to other closely related chemokine proteins. One of the clones, 3B516, was completely sequenced. It comprised 5,820 nucleotides. Except for matches to the other four clones from the stealth virus cultures (3B33, 3B624, 3B654 and 3B675), BlastN did not reveal significant homologies with current GenBank entries. By BlastX analysis, however, the nucleotide sequence from the left side of the clone could be translated into a string of proteins with highly significant homologies to the UL141, UL144, and UL145 proteins respectively of the Toledo strain of human cytomegalovirus (HCMV) (11). An apparent open reading frame (ORF) was situated slightly beyond the region coding for the UL145 protein. The deduced amino acid sequence of this ORF failed to show a statistically significant match to any protein entries on GenBank. It did, however, show a weak partial matching to a human cadherin tumor suppressor protein. The right side of the clone contained three discrete regions, each of which could be translated into proteins that matched to MGSA/GRO-alpha chemokine. Two of the matches were statistically highly significant. Another potential open reading frame was situated between the region of clone 3B516 that showed a weak homology to the cadherin tumor suppressor protein and the region showing an identified, but statistically insignificant, homology to the MGSA/GRO-alpha chemokine. BlastX analysis matched this region to a human alpha chemokine that is only distantly related to the MGSA/GRO-alpha chemokine. The BlastX results indicating the best matching proteins for the various regions of clone 3B516, are summarized in Table 1. The Table also shows the percent identity and calculated "p" value for the HCMV matching sequences.
The deduced amino acid sequences of the potential ORF that comprised the 4 regions of clone 3B516 that matched to a cellular chemokine gene, are shown in Figure 1. The sequences differ significantly from each other, and to varying degrees, from the amino acid sequence of the MGSA/GRO-alpha precursor protein. Also shown for comparison is the amino acid sequence of the closely related alpha chemokine, macrophage inflammatory protein-2-alpha (Gro-beta) (12). The alignments for 3 of the sequences were obtained from the BlastX program. The amino acids that match to those of the MGSA/GRO-alpha precursor protein are underlined. The cystines involved in disulphide bond formation (12) are indicated with an asterisk. Sequence 2 contained an apparent insert that separated two closely matching segments. This insert is shown beneath the site separating the matching segments. The initial codon for this insert was a stop codon indicated by the # symbol. The 4th sequence did not match to MGSA/GRO-alpha, but rather, as noted in Table 1, to a more distantly related human alpha-chemokine.
The nucleotide sequences of the 4 regions were also compared against each other and against the nucleotide sequences coding the MGSA/GRO-alpha precursor protein. In contrast to the amino acid homology, no substantial pairings of matching nucleotide sequences were obtained (data not shown).
The sequence analysis is consistent with a genetic recombination between a portion of the SCMV-derived stealth virus that encodes several cytomegalovirus related proteins and a region of the cellular genome that encodes a protein with chemokine activity.
It is unlikely that the chemokine genes were present in the original SCMV from which the stealth virus was derived. The HCMV Toledo strain in which the UL144 and UL145 coding genes have been sequenced, continues with sequential genes that code for proteins designated UL146 to UL148 (11). While the UL147 proteins of both the Toledo and Towne strains of HCMV do show weak and statistically insignificant (p value 0.15), homology to an alpha chemokine of quinea pig origin by BlastX analysis, the common sequences show very little overlap with those coded by the 3B516 clone. Moreover, this would not account for the multiple copies of the human chemokine related sequences in the stealth virus. Additional differences between the stealth virus and HCMV clinical isolates include the lack of UL142 and UL143 genes (11). Further clarification of this important issue should be forthcoming from sequencing studies on SCMV isolates.
The previously reported genetic instability of stealth virus-1 genome (4) may account for differences seen in the 4 regions of clone 3B516 that matched, by BlastX analysis, to the MGSA/GRO-alpha chemokine. One of the regions clearly has a major insert, while all have apparent deletions. Slightly less statistically significant amino acid matching of two of the regions occurred with macrophage inflammatory protein-2-alpha chemokine, also known as GRO-beta (12). This was not unexpected since, as shown in Table 2, there is a strong homology between this protein and the MGSA/GRO-alpha chemokine. Other members of the superfamily include macrophage inflammatory protein-2-beta (GRO-gamma), neutrophil activating peptide/IL8, platelet factor-4, beta thromboglobulin and interferon-inducible protein-10 (12).
The human MGSA/GRO-alpha chemokine gene has been more extensively studied than the other chemokines because of its possible role in the autocrine stimulation of melanoma cell growth (7-8). The 1,895 nucleotide MGSA/GRO-alpha gene comprises a 5’ non-coding region containing NF-kappaB, HMG(I)Y, IUR, and Sp1 binding sites (13). It has 4 exons and 3 introns (14). The first exon codes a signal peptide (nucleotide 130-229) while the second, third and fourth exons comprise the mature protein (nucleotides 330...451; 565...648, 1180-1195). A long non-coding 3’ end extends from nucleotides 1196 to 1895. cDNA sequences of the MGSA/GRO-beta and MGSA/GRO-gamma genes show extensive homology with the mRNA sequence of the alpha gene. A pseudogene has also been identified with homology covering the 5’ non-coding region, the first and second exon and the first intron of the MGSA/GRO-alpha gene (15).
The absence of intron sequences in the assimilated MGSA/GRO-alpha related regions of clone 3B516 indicates that the putative recombination event occurred at the level of RNA, rather than at the level of genomic DNA (16) The fidelity of replication by reverse transcription is generally less than DNA dependent replication. This may help explain the greater divergence seen when comparing nucleotide sequences than when comparing amino acid sequences. At the same time, the apparent amino acid conservation, especially with regards to the capacity to form disulfide linkages (as indicated in Figure 1), suggests that the proteins are providing some positive selective pressure. It has not been established, however, that any of the chemokine related coding sequences are being transcribed and translated into functional proteins.
The clinical manifestations of stealth virus infections have been primarily neuropsychological (17). This is presumably due to the inability of the brain to readily compensate for localized damage causing a disruption in specific functions (17). The development of cancer provides another example where limited damage, even occurring in a single cell, can have devastating consequences.
The patient from whom stealth virus-1 was isolated has not developed any malignancies. Evidence for stealth viral infection has, however, been obtained from a number of cancer patients and from several animals with various tumors (18,19 and unpublished observations). Useful information should be forthcoming from sequencing studies on stealth virus isolates from cancer patients.
Acknowledgement: Ms. Susie Trang provided valuable assistance with compiling sequence data and preparation of the manuscript.