Relatedness of baculovirus and gypsy retrotransposon envelope proteins
© Rohrmann and Karplus; licensee BioMed Central Ltd. 2001
Received: 2 January 2001
Accepted: 19 February 2001
Published: 19 February 2001
Current evidence suggests that lepidopteran baculoviruses may be divided into two phylogenetic groups based on their envelope fusion proteins. One group utilizes gp64, a low pH-dependent envelope fusion protein, whereas the other employs a protein family (e.g. LD130 in the Lymantria dispar nucleopolyhedrovirus) unrelated to gp64, but that is also low pH-dependent. Database searches with members of the LD130 protein family often record significant levels of homology to envelope proteins from a number of insect retrovirus-like transposable elements of the gypsy class. In this report, the significance of the homology between these two types of envelope proteins is analyzed.
The significance of the alignment scores was evaluated using Z-scores that were calculated by comparing the observed alignment score to the distribution of scores obtained for alignments after one of the sequences was subjected to 100 random shuffles of its sequence. These analyses resulted in Z-scores of >9 for members of the LD130 family when compared to most gypsy envelope proteins. Furthermore, in addition to significant levels of sequence homology and the presence of predicted signal sequences and transmembrane domains, members of this family contain a possible a furin cleavage motif, a conserved motif downstream of this site, predicted coiled-coil domains, and a pattern of conserved cysteine residues.
These analyses provide a link between envelope proteins from a group of insect retrovirus-like elements and a baculovirus protein family that includes low-pH-dependent envelope fusion proteins. The ability of gypsy retroelements to transpose from insect into baculovirus genomes suggests a pathway for the exchange of this protein between these viral families.
Baculoviruses are a diverse family of insect viruses that are pathogenic for insects particularly members of the Lepidoptera, Diptera and Hymenoptera. They have large double-stranded, circular, supercoiled, DNA genomes of 100-180 kb and are characterized by the occlusion of their virions in crystalline protein matrices. There are two genera of baculoviruses, the nucleopolyhedroviruses (NPVs) in which many virions are occluded in large polyhedron-shaped occlusion bodies, and the granuloviruses (GVs) which normally occlude a single nucleocapsid per small granular occlusion body. A novel feature of these viruses is the production of two types of virions . One, the occlusion derived virion (ODV) type, is present in occlusion bodies and spreads the infection between insects, whereas the other, the budded virion (BV) type, spreads the infection between cells within insects or in cell culture. A major difference between these two types of virions is their envelope proteins. The envelope of ODV is not well characterized; and although it may be composed of multiple proteins, the mechanism by which it facilitates the initiation of infection of insect midgut cells is unclear [reviewed in ]. In contrast, BV envelopes contain an envelope fusion protein that causes the merging of the virion envelope and the membrane of cellular endocytic vesicles when exposed to low pH.
Current evidence suggests that lepidopteran baculoviruses may be divided into two phylogenetic groups based on the envelope fusion proteins of budded virions . One group that includes Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) and Orgyia pseudotsugata MNPV (OpMNPV), contain GP64 in their budded virion envelopes [4, 5]. Homologs of gp64 are also found in a genus of orthomyxoviruses, the thogotoviruses . Recently a number of complete baculovirus genome sequences have been described and were found to lack an open reading frame with homology to gp64. Analysis of the genome of the Lymantria dispar MNPV (LdMNPV), revealed a single orf (ld130) with predicted signal and transmembrane domains. Characterization of LD130, indicated that it localizes to the membrane of infected cells, is a component of budded virions, and is N-glycosylated. Uninfected cells transiently transfected with a plasmid encoding LD130, showed localization of the protein to the cell membrane and low-pH mediated cell fusion suggesting that LD130 is the envelope fusion protein of LdMNPV . Similar results have been reported for the LD130 homolog in SeMNPV .
Homologs of ld130 are found in the genomes of all the sequenced gp64-minus viruses including LdMNPV, SeMNPV , Plutella xylostella GV (PxGV)  and Xestia c-nigrum GV (XcGV) . In addition, homologs of ld130 are also found in gp64-containing viruses, although they do not appear to be capable of mediating low-pH-dependent membrane fusion (Pearson et al, unpublished). In contrast to the close relatedness of baculovirus gp64 homologs, the LD130 homologs are highly variable suggesting that gp64 was recently incorporated into a baculovirus genome where it displaced the envelope fusion function of the ld130 homologs .
Database searches with LD130 and its homologs routinely identify, not only homologous baculovirus proteins, but some members also showed significant levels of homology with the predicted envelope proteins of a number of insect retrovirus-like elements from Drosophila and Lepidoptera , (also see genbank documentation for AcMNPV orf23 in the AcMNPV sequence ). Evidence suggests that at least some of these retrovirus-like elements are infectious and have been classified as insect retroviruses [13,14,15] or errantiviruses . In this report, we provide evidence for an evolutionary link between this newly characterized family of baculovirus envelope proteins and the envelope proteins predicted for a variety of insect retrovirus-like retrotransposons. These relationships suggest a possible common origin of the envelope fusion proteins for these two groups of viruses.
Database searches with members of the LD130 family
Sequences used for analysis
0.26* and 0.63
0.69* and 0.17
With one exception, all the non-baculovirus sequences showing the best scores are derived from retrovirus-like retrotransposons from insects that fall in the T3-gypsy group . The exception is a Drosophila orf called cg4715 (Table 1). The orfs adjacent to cg4715 (two upstream and two downstream) appear to be non-homologous to retrovirus orfs (data not shown) suggesting that it may either be an insect gene or a remnant of an integrated retroelement.
Significance of the relatedness of the members of the Ld130 family
Furthermore, guided by the assumption that sequence features conserved over long evolutionary distances should also be conserved over short distances, a secondary criteria used for the more distant relationships was that Z-scores derived from comparing families of sequences should show enhanced significance over that of the individual sequences. The Z-scores revealed strong evidence for the homology of the seventeen sequences (Fig. 1). Members of the LD130 family showed Z-scores of 9 and 10 to two groups containing eight gypsy proteins. These are similar to scores determined for comparisons between different gypsy envelope proteins of from 9 to12. In addition, only one other protein [called rhoptry, from Plasmodium yoelii (accession # T28677)], fell within our 10-3 BLAST search cutoff in two instances (with T13994 and p20829 [Table 1]). However, when aligned to each of the 17 sequences, only one alignment resulted in a Z-score of 4 and all the other z-scores were ≤2. Also when members of each subfamily were grouped together and compared with this protein, the scores were not enhanced (data not shown). A matrix comparing the levels of sequence identities among these sequences is shown in Fig. 2, and reveals that in contrast to Z-scores, sequence identity is not a reliable indicator of evolutionary relatedness at the longer distances. For example, although the proteins with the groups composed of #1-4, #7-10 and #11-14 (Table 1) are clearly related to one another, their relationship with other members of the data set are unclear.
Features of the LD130 protein family
In this report we have investigated the evolutionary relatedness between three categories of proteins, the retrovirus-like gypsy envelope proteins, the baculovirus LD130 group of envelope-associated proteins, and an insect cellular protein. Globally optimal sequence alignments in combination with assessment of the significance of the alignment scores resulted in striking patterns of relatedness. Not only did our analyses link the gypsy-derived envelope proteins together with a high level of significance as would be predicted from studies of other gypsy genes , but it also linked two of the major categories of the gypsy sequences to the baculovirus ld130 group with similar levels of significance. In addition, a cell-encoded protein from Drosophila showed relatedness at a high level of significance to the baculovirus ld130 protein group. The parameters we used as a basis for these analyses were conservative with cut-off Z-score values representative of at least 6 standard deviations above the value for the randomized sequences. In addition to the overall homology, a number of other features common to members of this class of proteins were identified. These included the predicted signal sequences and transmembrane domains and a striking conservation surrounding a possible protease cleavage site in the proteins that likely serve as envelope fusion proteins . In addition, predicted coiled-coil domains in similar locations relative to the cleavage site in a number of the sequences further suggest both functional and structural similarities between members of this protein group.
Concurrent with the preparation of this research for publication, a similar study by Malik et al was reported . They also describe a relationship between the envelope protein of gypsy retrovirus-like elements and the ld130 group of baculovirus envelope proteins. They identified the predicted signal and transmembrane domains that we describe in Fig. 3 and the conserved region that we compare in Fig. 5. Their alignments also highlight the conservation of the cysteine residues that we describe in Fig. 4.
Although our analyses convincingly indicate a relationship between the insect cellular, gypsy, and baculovirus membrane proteins, we can only speculate on the evolutionary pathway leading to the presence of members of this set of proteins in all three types of organisms. If homologs of the cg4715 gene are conserved among Diptera and Lepidoptera, the interrelationship between members of the baculoviridae and TED, the lepidopteran gypsy element capable of integrating into both a host insect and a baculovirus genome, provides a clear pathway via DNA recombination for the exchange of members of the ld130 gene family between host cells, baculovirus genomes, and the transposable element. Indeed, a phylogenetic analysis of the envelope protein of these elements reported by Malik et al  suggests that they are derived from a monophyletic event that resulted from their incorporation of a baculovirus envelope protein gene in an ancestral member of these retrovirus-like elements.
TED has features that suggest that it may have specifically evolved to exploit a relationship with a baculovirus and this may have facilitated its acquisition of a baculovirus envelope protein gene. It is present as a mid-level repeated element in its host insect genome (about 50 copies/genome) [19, 24], it encodes a set of proteins (gag, pol, env) with features similar to those of well-characterized retrovirus proteins  and it is capable of producing virus-like particles . A specific example of TED's adaptation to baculovirus biology is the presence of palindromes containing the baculovirus late promoter element in its long terminal repeat [17, 24]. Evidence suggests that this results in the high level transcription of the TED genome by the viral RNA polymerase  and could lead to the production of TED virus-like particles concomitant with the baculovirus late gene expression. This would provide a novel two-pronged escape mechanism from insects suffering from a fatal baculovirus infection; TED might be passed to other insects as an integrated transposable element in an infectious baculovirus, or as an infectious retrovirus. Oral transmission is the normal route for baculovirus infection and has been reported for gypsy retrovirus-like retrotransposons . This strategy could elevate the retrotransposon's potential to exchange genetic material with the genomes of its viral and cellular hosts.
Members of the Ld130 family of baculovirus envelope proteins have been demonstrated to mediate low-pH membrane fusion . In contrast, retroviruses normally enter cells by receptor-mediated fusion at the cell surface (reviewed in ) or by a combination of both receptor and low-pH mediated events . Despite the differing mechanisms of cell entry, members of these two groups have been related by a number of structural criteria . The link that we have described in this report and that of Malik et al  suggests the possibility of a genetic relationship as well.
The relatedness of a class of low pH-dependent baculovirus envelope fusion proteins to envelope proteins of gypsy retrovirus-like elements was demonstrated to be highly significant. Transposon mediated exchange provides a documented pathway for the movement of this gene between insect cells, and two different types of viruses.
Materials and Methods
Blast searches were performed using the Gap-Blast search engine . Sequence alignments were performed with the program SEQUOIA by C.M Bruns which is available on the web at http://www.scripps.edu/∼bruns/sequoia.html. SEQUOIA uses a conventional dynamic programming algorithm based on the work of Needleman and Wunsch  to find the globally optimal alignment given a particular residue comparison matrix and gapping model. The statistical significance of the optimal alignment was assessed by calculation of their Z-score. The Z-score is derived by comparison of an observed alignment score with the distribution of scores obtained for alignments after one of the sequences was shuffled to create a random sequence with the same amino acid composition. 100 shuffles were carried out for each sequence comparison and the significance was measured relative to the mean and standard deviation of scores from the shuffled sequences. Quantitatively, the Z-score is defined as the number of standard deviations of the observed score above the mean of the shuffled scores and is determined by the following equation:
Z-score = Observed score - mean shuffled score/Standard deviation of shuffled scores
In the work we describe here, alignments were done using a gap penalty of 10 and the BLOSUM62 scoring matrix . An additional important feature of SEQUOIA is that a "sequence" need not be just a single sequence, but can be a prealigned family of sequences that are treated as a unit.
We thank Doug Leisy and Margot Pearson for critically reading this manuscript, and Y. Hashimoto for access to the PxGV sequence prior to its publication. This research was supported by NSF grant MCB 9982536 to G.R. and is Technical Report No. 11758 from the Oregon State University Agricultural Experiment Station.
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