Cis-acting sequences may contribute to size variation in the surface glycoprotein of bovine immunodeficiency virus

Yuxing Li1 and Susan Carpenter1

Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50011-1250, USA1

Author for correspondence: Susan Carpenter. Fax +1 515 294 8500. e-mail scarp{at}iastate.edu


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Genetic recombination is an important mechanism of retrovirus variation and diversity. Size variation in the surface (SU) glycoprotein, characterized by duplication and insertion, has been observed during in vivo infection with several lentiviruses, including bovine immunodeficiency virus (BIV), equine infectious anaemia virus (EIAV) and human immunodeficiency virus type 1. These duplication/insertion events are thought to occur through a mechanism of template switching/strand transfer during reverse transcription. Studies of RNA recombination in a number of virus systems indicate that cis-acting sequences can modulate the frequency of template switching/strand transfer. The size variable region of EIAV and BIV SU glycoproteins was examined and an AU-rich region and regions of nucleotide sequence identity that may facilitate template switching/strand transfer were identified. An in vitro strand transfer assay using donor and acceptor templates derived from the size variable region in BIV env detected both precise and imprecise strand transfer products, in addition to full-length products. Sequence analysis of clones obtained from imprecise strand transfer products showed that 87·5% had crossover sites within 10 nt of the crossover site observed in vivo. Mutations in the donor template which altered either the AU-rich region or nucleotide sequence identity dramatically decreased the frequency of imprecise strand transfer. Together, these results suggest that cis-acting elements can modulate non-homologous recombination events during reverse transcription and may contribute to the genetic and biological diversity of lentiviruses in vivo.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The surface (SU) protein of lentiviruses is encoded by the viral env gene and plays an important role in virus–host interactions during the course of infection. The SU protein interacts directly with the host cell receptor and is a primary target for neutralizing antibody. Genetic variation in the SU protein can alter cell tropism and has been shown to be an important mechanism of immune evasion (Coffin, 1992 ; Burns & Desrosiers, 1994 ; Pezo & Wain-Hobson, 1997 ). Several reports have identified size variation in the SU glycoprotein of bovine immunodeficiency virus (BIV) (Suarez & Whetstone, 1995 , 1997 ) and equine infectious anaemia virus (EIAV) (Leroux et al., 1997 ; Zheng et al., 1997a , b ). In both cases, size variation occurred during the course of in vivo infection and was characterized by duplication/insertions in the env gene (see Fig. 1A) (Suarez & Whetstone, 1995 , 1997 ; Zheng et al., 1997a , b ). Size variation was shown to alter biological or immunological properties of BIV and EIAV, respectively (Suarez & Whetstone, 1997 ; Zheng et al., 1997a ), suggesting that recombination may be an important mechanism of lentivirus variation in vivo. Duplication/insertion events have been reported in the human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus SU protein-coding regions (Delassus et al., 1992 ; Groenink et al., 1993 ; Palmer et al., 1996 ; Shioda et al., 1997 ), where they are thought to be associated with disease progression, changes in virus cell tropism and cytopathicity (Groenink et al., 1993 ; Fouchier et al., 1995 ; Palmer et al., 1996 ; Fox et al., 1997 ; Shioda et al., 1997 ; Rudensey et al., 1998 ).



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Fig. 1. Size variation in BIV and EIAV env. (A) Duplications and insertions in the SU protein of BIV FL491 (Suarez & Whetstone, 1997 ) and EIAV V70 (Zheng et al., 1997a , b ) that arise during in vivo infection. Representation of the BIV and EIAV SU and transmembrane (TM) protein regions with the location of the SU size variable region indicated in grey. Patterns of duplicated nucleotide sequences are indicated by repeating (R) patterns. For BIV FL491, the pattern in the original inoculum was R1-R2-R3 and the post-inoculation pattern was R1-R2-R3-R1-R3. EIAV V70 has the original pattern R1-R2-R2-R3-R4-R5 and the three different post-inoculation patterns R1-R2-R2-R3-R1-R2-R2-R3-R4-R5, R1-R2-R2-R3-R4-R2-R2-R3-R4-R5 and R1-R2-R2-R3-R4-R5-R2-R2-R3-R4-R5. (B) Conserved cis-acting elements hypothesized to promote strand transfer in the BIV and EIAV SU proteins. All the switching and landing sites were identified in vivo. The AU-rich regions are shaded. Sequences with identity between switching and landing sites are boxed, with mismatches indicated by an asterisk. Nucleotide sequence numbering is based on the respective env gene, as described previously (Suarez & Whetstone, 1997 ; Zheng et al., 1997b ). (C) A general model of duplication/insertion within the lentivirus SU protein region during reverse transcription of minus-strand DNA. The sequence identity motifs within the switching and landing sites are indicated by black boxes. The duplicated region is indicated in grey and shown as duplicated copies.

 
Recombination occurs frequently during reverse transcription (Hu & Temin, 1990a , b ; Stuhlmann & Berg, 1992 ) and approximately 5–10% of the sequenced HIV-1 strains are reported to possess mosaic genomes comprising two or more subtypes of the major group M of HIV-1 (Cornelissen et al., 1996 ; Gao et al., 1998 ; Robertson et al., 1995 ). The majority of retrovirus recombination events are believed to result from a template switching/strand transfer mechanism which occurs during the synthesis of minus-strand proviral DNA (Anderson et al., 1998 ; Coffin, 1979 ; Zhang et al., 2000 ). Early in the retrovirus life cycle, reverse transcriptase (RT) must perform two template switches, also termed strong-stop DNA strand transfers (Telesnitsky & Goff, 1997 ). These strand transfers have been shown to be facilitated by either base pairing between the redundant R regions (Peliska & Benkovic, 1992 ; Topping et al., 1998 ) and/or through some unknown base pairing-independent mechanism (Topping et al., 1998 ). In addition, the viral nucleocapsid (NC) protein can stimulate strong-stop DNA strand transfer in the HIV-1 R region (You & McHenry, 1994 ; Guo et al., 1997 ; Kim et al., 1997 ) and in the murine leukaemia virus (MLV) polypurine tract (Wu et al., 1996 ). Template switching at other sites in the viral genome have been observed during reverse transcription in vivo and in vitro (DeStefano et al., 1994 ; Pathak & Hu, 1997 ). A variety of cis-acting elements have been associated with strand transfer and recombination at these ectopic sites, including runs of identical nucleotides (DeStefano et al., 1992a , b ; Klarmann et al., 1993 ; Wooley et al., 1998 ), short regions of sequence identity between the template switching and landing sites (Zhang & Temin, 1993a , b , 1994 ) and structural features that increase polymerase pausing and/or RNA–RNA interaction (Brian & Spaan, 1997 ; Mikkelsen et al., 1998 , Wu et al., 1995 ). Cis-acting elements have also been shown to contribute to template switching/strand transfer in a number of other RNA viruses in which recombination has been studied more extensively. Factors reported to increase the frequency of homologous and non-homologous recombination include AU-rich regions, RNA secondary structure and regions of identity between the template switching site and landing sites (reviewed by Nagy & Simon, 1997 ). Taken together, these findings indicate that common genetic elements can promote RNA recombination and contribute to genetic diversity in a wide range of RNA viruses.

To explore the mechanism of size variation in the BIV and EIAV SU proteins, we analysed the size variable regions for elements known to contribute to recombination in other viruses. The SU protein-coding region of BIV and EIAV contained two elements that may promote strand transfer and duplication/insertion events consistent with current models of RNA recombination: an AU-rich region and nucleotide sequence identity between the switching site on the donor template and the landing site on the acceptor template. Mutation of either, or both, of these elements decreased the efficiency of imprecise strand transfer in the BIV SU protein. These findings support a model of size variation in the BIV SU protein resulting from duplication/insertion events that occur during minus-strand DNA synthesis and suggest that intrinsic genetic elements may modulate the frequency of recombination in vivo.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Construction of plasmids.
The variable region (nt 180–450) of the BIV SU protein, described originally by Suarez & Whetstone (1995) , was amplified by PCR from FL491 isolate proviral DNA and cloned into pUC19 (New England Biolabs). The resulting plasmid was named pFSU11. The BIV sequence was verified by DNA sequencing and was used as a template for construction of template plasmids as described below. The nucleotide sequence numbering of all constructs is based on that of FL491 SU (GenBank accession no. L43127). Unless otherwise noted, PCRs were carried out in a 50 µl reaction with each primer at 200 nM, 125 µM dNTPs (Gibco-BRL), 2·5 mM MgCl2, 2·5 U of Taq polymerase (Gibco-BRL) and the buffer supplied with the enzyme. Amplification consisted of 35 cycles (1 min at 94 °C, 1 min at 55 °C and 2 min at 72 °C) with 7 min final extension at 72 °C.

The donor template plasmid pBD6, containing the full-length variable region, was made using a forward primer containing an EcoRI site and T7 promoter sequences in addition to the BIV SU protein sequences. The reverse primer comprised nt 432–450 of the BIV SU protein and a HindIII site, which allowed cloning of the amplicon into pUC19 restricted with EcoRI/HindIII. A second donor template lacking the proposed landing site, designated pBD3b, was constructed to avoid possible intramolecular strand transfer between the switching site and landing site within one donor molecule. The pBD3b donor plasmid, containing nt 260–370, was amplified by PCR using primers containing 3'-terminal restriction sites, which allowed insertion into the KpnI and BamHI sites of pBluescript SK(-) (Stratagene) under the control of the T7 promoter. To introduce mutations in the AU-rich region and/or the sequence identity region, a NcoI site was introduced at nt 350 of pFSU11. A double-stranded oligonucleotide cassette, which contained the AU-region mutation (AAUAUUA->CCGCGGC) and/or the sequence identity mutation (AACCC->AGTCG), was synthesized to replace the wild-type XbaI–NcoI fragment in pFSU11. Mutated fragments were amplified and cloned into the KpnI and BamHI sites of pBluescript SK(-) using the primer pairs described above. The wild-type acceptor plasmid, pFA6, was constructed by PCR amplification and cloning of the BIV SU protein nt 220–430 into pUC19 using primers containing T7 promoter sequences and either EcoRI or HindIII sites. Mutations in the acceptor templates were constructed in pUC19 using the same strategy as the wild-type acceptor except that one of the PCR primers contained the desired mutation (AACCC->AGTCG). All constructs were confirmed by DNA sequencing.

{blacksquare} Synthesis and purification of donor and acceptor templates.
The donor and acceptor template plasmids were linearized by restriction enzymes and template RNA was transcribed with T7 RNA polymerase, according to the protocol provided with the RibMAX kit (Promega). The RNA transcripts were electrophoresed on a 6% denaturing polyacrylamide gel, excised as a single full-length transcript band, purified using the RNaid kit (BIO101) and quantified by measuring the A260 with a UV spectrophotometer.

{blacksquare} In vitro strand transfer assay.
An in vitro strand transfer reaction was carried out at 37 °C according to the method described by Guo et al. (1997) . DNA primers BF450BC' (5' ATCCGTGTCCTCCGAGCCC 3') or SK1 (5' GCTCTAGAACTAGTGGATC 3'), complementary to either nt 430–450 of the BIV SU protein or pBluescript SK(-), respectively, were 5' end-labelled with 32P, as described previously (Guo et al., 1997 ). The primer was incubated with 0·2 pmol of donor template at a template:primer ratio of 1:2 at 65 °C for 5 min and gradually cooled to room temperature within 40 min (~1 °C/min). 0·2 pmol of acceptor RNA was added and incubated in a 20 µl reaction mixture containing 0·1U/µl HIV-RT (Worthington), 100 µM dNTPs and 7 mM Mg2+. Reverse transcription was initiated by the addition of dNTPs and Mg2+. The RT enzyme was the heterodimeric form containing RNase H activity. The reactions were incubated at 37 °C for 35 min, or as specified, and stopped by adding EDTA to a final concentration of 50 mM, followed by proteinase K treatment and phenol–chloroform extraction. The sample was mixed at a ratio of 1:1 with denaturing loading buffer (80% formamide, 2 mM EDTA, 0·01% xylene cyanol and 0·01% bromophenol blue) and heated at 90 °C for 5 min. The sample was analysed by PAGE in a 6% gel containing 7 M urea. Following electrophoresis, the gel was dried, exposed to a phosphorimager screen and the reaction products were quantified using the PhosphorImager and ImageQuant (Molecular Dynamics) software. Strand transfer efficiency was calculated as the percentage of the sum of signal intensity of strand transfer bands versus the sum of the signal intensity for all the bands with molecular mass equal to or larger than that of the full-length primer extension product (Palaniappan et al., 1996 ). All assays were done in duplicate and repeated at least three times.

{blacksquare} Sequence analysis of strand transfer products.
Bands representing strand transfer products were excised from the polyacrylamide gel and the single-stranded DNA within the band was purified as described previously (Maniatis et al., 1982 ). The DNA was amplified either by PCR using the template-specific primers BF376 (5' TCCAGTCGTGGTGTTGCAC 3') and SK1 or by 5' RACE (Gibco-BRL). For reactions using template-specific primers, PCR amplification started with 1 cycle of denaturation at 95 °C for 1 min, annealing at 57 °C for 1 min and extension at 72 °C for 1 min, followed by 9 cycles with the same denaturing and extension conditions but with the annealing temperatures decreasing from 57 to 52 °C at 0·5 °C change per cycle. This was followed by 30 cycles of 95 °C for 1 min, 52 °C for 1 min and 72 °C for 1 min and a final extension for 7 min at 72 °C. In some cases, 5' RACE was used to ensure amplification of products with altered or missing 3' ends. A homopolymeric tail containing dC was added to the purified minus-strand DNA product by terminal deoxynucleotidyl transferase. This modified minus-stand DNA was used as the template in a PCR using a primer containing homopolymer dG and either BF450bC' (for pBD6 product) or SK1 (for pBD3b). In all cases, the PCR products were ligated to the pGEM-T easy vector (Promega) and transformed into Escherichia coli SureCell (recB recJ) (Stratagene). Individual clones were picked and sequenced.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Common sequence motifs are found in the size variable region of lentivirus SU proteins
Size variation in the BIV and EIAV SU proteins is characterized by duplication and insertion of 33–54 nt in the SU protein-coding region (Fig. 1A) (Suarez & Whetstone, 1997 ; Zheng et al., 1997a , b ). To understand better the mechanism of size variation, we analysed the hypervariable region of the BIV and EIAV SU proteins for elements known to contribute to RNA recombination in other virus systems. Two cis-acting structural elements were identified within the size variable region in the SU protein-coding region that could promote strand transfer and duplication/insertion events consistent with current models of RNA recombination (Fig. 1B). In both viruses, a 7–20 nt AU-rich region was found just downstream from the reported template switching site in the SU protein-coding region. In some other RNA viruses, AU-rich regions are thought to promote template switching by increasing polymerase slippage along the donor template (Nagy & Bujarski, 1996 , 1997 ). In addition, there may be weaker annealing between the donor template and the nascent minus-strand DNA at AU-rich regions, facilitating dissociation of the nascent minus-strand DNA from the donor template. The second feature common to the variable region of the BIV and EIAV SU proteins were small regions of nucleotide sequence identity between the switching and the landing sites (Fig. 1B). The presence of characteristic cis-acting elements known to promote recombination in other RNA viruses supported the hypothesis that the duplication/insertion events observed in vivo within BIV and EIAV SU proteins occurred as a result of template switching during minus-strand DNA synthesis (Fig. 1C).

Strand transfer occurs in the hypervariable region of the BIV SU protein
To examine the role of strand transfer in BIV size variation, we used an in vitro strand transfer assay in which donor and acceptor templates were of varying length and a unique primer-binding site was present on the donor template (Fig. 2). A 32P end-labelled primer was used to initiate reverse transcription along the donor RNA template and subsequent strand transfer events resulted in a final minus-strand DNA product which was increased in length due to the additional sequences at the 3' end of the acceptor template. Both precise and imprecise strand transfer products were identifiable by their size following electrophoresis in denaturing polyacrylamide gels.



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Fig. 2. General steps of the in vitro strand transfer assay. Fragments of the BIV SU protein hypervariable region were cloned under the control of the T7 promoter to generate donor and acceptor RNA templates of varying lengths. A 32P end-labelled primer was used to initiate reverse transcription along a donor RNA template and subsequent strand transfer events result in a final minus-strand DNA product with an increased length due to the additional sequences at the 3' end of the acceptor template. Both precise and imprecise strand transfer products are identifiable by their size following electrophoresis in denaturing polyacrylamide gels.

 
Fragments of the hypervariable region of the BIV SU protein were cloned under the control of the T7 promoter to generate donor and acceptor RNA templates of varying length (Fig. 3). Initial experiments were conducted using pBD6 (nt 260–450) or pBD3b as the donor templates (Fig. 3A) and pBA6 (nt 220–430) as the acceptor template (Fig. 3C). In vitro reverse transcription in the absence of pBA6 acceptor RNA resulted in only the full-length (F) products and smaller premature termination products (Fig. 4A, B, lanes 6–10 and 2, respectively). The number of incomplete products is similar to that observed in other cell-free systems that lack the NC protein (Rodriguez-Rodriguez et al., 1995 ; Kim et al., 1997 ), although it is possible that the use of HIV-1 RT with a BIV template also contributed to the incomplete primer extension observed in our studies. When both donor and acceptor RNAs were present, the full-length synthesis product F was observed as well as an additional, higher molecular mass product of the correct size as a strand transfer product resulting from precise or imprecise recombination. These strand transfer products are referred to as precise (PT) (Fig. 4A) or imprecise (IT) (Fig. 4B), respectively. The efficiency of strand transfer, defined as the quantity of transfer products (PT+IT) divided by the quantity of full-length plus transfer products (F+PT+IT), was approximately 17%, comparable to levels reported previously in cell-free systems in the absence of the NC protein (Rodriguez-Rodriguez et al., 1995 ; Palaniappan et al., 1996 ). The fact that no strand transfer products were detectable in the absence of acceptor RNA indicated that minimal intramolecular strand transfer occurred under the reaction conditions used. Therefore, the higher molecular mass products were probably the result of intermolecular strand transfer. To confirm this, primers specific for sequences unique to either the acceptor or the donor template were used to amplify, clone and sequence the strand transfer products. The 231 nt product, PT in Fig. 4(A), was verified by DNA sequencing to result from precise strand transfer (data not shown). These higher molecular mass products appeared to migrate differently in the gel and it is possible that this heterogeneity in migration resulted from partial denaturation of the reaction products during electrophoresis. However, we were not able to attribute these differences to the presence of differently sized strand transfer products.



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Fig. 3. Constructs used in the in vitro strand transfer assay and the expected product sizes resulting from full extension of donor template (F), precise (PT) and imprecise (IT) strand transfer. (A) The wild-type donor templates pBD6 and pBD3b. Note that pBD3b has a donor-specific primer-binding site derived from pBluescript SK(-) (indicated in grey). (B) Donor templates containing specific mutations in cis-acting elements. {Delta}SS (switching site sequence identity mutant) contains a mutation at nt 342–346 of AACCC->AGTCG, {Delta}AU (AU-rich region mutant) contains mutations at nt 333–339 of AAUAUUA->CCGCGGC, {Delta}SS/{Delta}AU, combined mutation of both {Delta}SS and {Delta}AU. The sequence identical to the landing site is boxed and the AU-rich region is shaded. The mutated regions are underlined. (C) The wild-type and mutant acceptor templates. pBA6, wild-type acceptor containing nt 220–430. {Delta}LS contains mutations in the landing site at nt 414–418 of AACCC->AGTCG. This sequence is identical to the {Delta}SS mutation.

 


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Fig. 4. Strand transfer assay results using wild-type acceptor pBA6. Reaction products include those resulting from full extension (F) on the donor template and strand transfer with precise (PT) and imprecise (IT) recombination. (A) Wild-type pBD6 as donor. A+D, acceptor+donor; A, acceptor; D, donor. Incubation times were 0 min (lanes 1 and 6), 1 min (lanes 2 and 7), 5 min (lanes 3 and 8), 20 min (lanes 4 and 9) and 30 min (lanes 5, 10 and 11). (B) Wild-type pBD3b as the donor template in the presence (lane 1) and absence (lane 2) of pBA6 acceptor RNA. STE (strand transfer efficiency) is calculated as the sum of signal intensity of strand transfer bands versus the sum of the signal intensity for all the bands with molecular mass equal to or larger than that of the full-length primer extension productx100 (Palaniappan et al., 1996 ).

 
Reactions using pBD3b donor RNA resulted in strand transfer products of a higher molecular mass than were predicted for precise strand transfer, indicating that they are the result of imprecise strand transfer (Fig. 4B, lane 1). The increase in the efficiency of imprecise strand transfer following the deletion of nt 370–450 suggests that this region may be important for correct positioning of the nascent strand following dissociation from the donor template. This increase in the observed imprecise strand transfer may be due to removal either of the 80 nt region or of specific sequences within that region. In either case, these findings indicate that the efficiency of imprecise strand transfer can be altered by changes in the donor RNA template.

Identification of the switching and landing sites in the imprecise strand transfer products
To confirm the identity of the higher molecular mass products observed in Fig. 4(B), the IT products from reactions containing pBD3b donor RNA were excised from the gel and amplified by PCR using primers specific for strand transfer products. Amplicons were cloned and 16 individual clones were sequenced to identify crossover sites. Three different in vitro crossover sites, designated A, B and C, were found among the 16 clones sequenced (Fig. 5A). The switching sites were distributed along a 53 nt region in the donor template, whereas the landing sites were clustered in a specific 20 nt region. In each pair of switching and landing sites, there was 3–5 nt sequence identity that appeared to facilitate re-association between the nascent minus-strand, single-stranded DNA and the acceptor template. Although we cannot rule out the possibility that these recombination events could also occur during the synthesis of plus-strand proviral DNA, these findings are consistent with a model of size variation in the BIV SU protein that results from imprecise strand transfer during minus-strand DNA synthesis.



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Fig. 5. Imprecise recombination results in duplication/insertion in the SU protein of BIV strain FL491 during in vitro strand transfer and in vivo recombination. (A) Location of the crossover sites during imprecise strand transfer. The crossover sites resulting from the in vitro strand transfer assay are indicated by single-headed arrows and are labelled A, B or C. The in vivo crossover site is indicated with a double-headed arrow and is boxed. (B) The amino acid sequence of BIV SU protein size variants. The amino acid sequence of BIV-FL491-I is the sequence of the original inoculum and BIV-FL491-V is the size variant identified by Suarez & Whetstone (1997) . The three patterns of insertions present in the clones amplified from the in vitro strand transfer products are indicated by A, B and C. The frequency indicates the number of times each pattern was present among the 16 clones sequenced. The potential N-glycosylation site ({Psi}) and the translation stop site (*) are indicated. The duplicated amino acid sequences in each group are indicated by underlining and shading.

 
All of the insertion sequences duplicated a predicted N-linked glycosylation site that could confer a selective advantage for virus replication in vivo. The most frequent (9/16) crossover site observed in vitro was pattern B (Fig. 5), with a switching site at nt 320–322 and a landing site at nt 420–422 (Fig. 5B). This 100 nt insertion results in a frame shift and a premature stop codon in the SU protein, which would be deleterious for virus replication in vivo. However, in-frame insertions were present in the remaining seven clones: five clones contained a 72 nt in-frame insertion (Fig. 5, pattern C) and two clones had a 129 nt in-frame insertion (Fig. 5, pattern A). The fact that approximately half of all strand transfer products are predicted to be defective for virus replication underscores the importance of selection in determining the pattern of crossover sites observed in vivo. Thus, the finding that 14/16 clones exhibited in vitro crossover sites located within 10 nt of the crossover site observed in vivo indicates that the in vitro assay reproduced strand transfer events similar to those that occur in vivo.

Cis-acting elements can increase the frequency of imprecise recombination
The size variable region of BIV contains cis-acting elements that contribute to recombination in other RNA viruses. Therefore, it was of interest to consider whether these elements contribute to imprecise strand transfer in our in vitro assay. A series of mutations designed to reduce AU content and/or switching/landing site sequence identity were introduced into the donor and acceptor RNAs (Fig. 3B, C) and tested in our strand transfer assay (Fig. 6). Mutation of the AU-rich region on the donor template ({Delta}AU) resulted in a decrease in the efficiency of imprecise strand transfer of more than eightfold as compared to the wild-type donor RNA. The efficiency of imprecise strand transfer was also reduced by disruption of sequence identity between the switching and landing sites, as indicated by mutation of either the switching on the donor RNA ({Delta}SS) or the landing site on the acceptor RNA ({Delta}LS). A similar reduction in imprecise strand transfer was observed when the donor RNA contained both {Delta}SS and {Delta}AU mutations. These data indicated that both the AU content and the sequence identity between the switching and landing sites can contribute to the rate of imprecise strand transfer.



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Fig. 6. Effect of mutations in donor and acceptor templates on strand transfer efficiency. Donor templates are as follows: pBD3b (wild-type); {Delta}AU mutant (nt 333–339, AAUAUUA->CCGCGGC); {Delta}SS donor (nt 342–346, AACCC->AGTCG); and {Delta}SS/{Delta}AU, template containing both {Delta}AU and {Delta}SS mutations. Acceptor templates are as follows: pBA6 (wild-type); {Delta}LS, landing site mutant (nt 414–418, AACCC->AGTCG). STE, calculated as [IT/(IT+F)]x100 (Palaniappan et al., 1996 ), results represent the mean of four independent experiments ±SD.

 
The model for size variation in the BIV SU protein proposes that the sequence identity between the switching and landing sites plays a role in promoting imprecise strand transfer by providing base pairing between the nascent minus-strand DNA and the acceptor RNA. If only the base pairing is necessary, then compensatory mutations in the acceptor RNA ({Delta}LS) should be able to increase the frequency of strand transfer of the {Delta}SS donor mutation to that observed with wild-type sequences. However, no strand transfer products were detected in reactions containing {Delta}SS and {Delta}LS mutations (Fig. 6), indicating that base pairing alone is not sufficient to promote imprecise strand transfer. Increased concentrations of {Delta}LS could restore imprecise strand transfer in reactions containing the wild-type donor RNA (data not shown), consistent with other studies indicating that accessibility and/or availability of the acceptor template is an important factor in imprecise strand transfer (White & Morris, 1995 ). We did not observe a similar increase in strand transfer efficiency in reactions containing {Delta}SS donor RNA, even in the presence of a tenfold excess of acceptor {Delta}LS RNA (data not shown). This suggested that primary sequence identity between switching and landing sites may be necessary, but not sufficient, to promote imprecise strand transfer. Further studies discerning the secondary structure of donor and acceptor RNAs are needed to elucidate fully the role of structural elements in precise and imprecise strand transfer during reverse transcription. However, the results presented here provide evidence that primary sequence elements can modulate the frequency of imprecise strand transfer during reverse transcription of lentiviral RNAs.


   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Size variation in the SU protein has been shown to occur during the course of in vivo infection with BIV and EIAV (Suarez & Whetstone, 1995 , 1997 ; Zheng et al., 1997a , b ). Moreover, size variable viruses differed in replication efficiency and antigenicity, indicating that insertions/deletions in the SU protein can confer a selective advantage in vivo. To delineate the mechanism of size variation in lentiviruses, we examined the role of cis-acting elements in promoting strand transfer/recombination in the SU protein-coding region. Our analysis of the size variable region in BIV and EIAV identified common cis-acting elements that have been shown to facilitate RNA recombination in a number of virus systems (reviewed by Nagy & Simon, 1997 ). Both precise and imprecise strand transfer products were detected following reverse transcription of BIV env in vitro. Sequence analysis of the imprecise strand transfer products showed that 87·5% had crossover sites within 10 nt of the crossover site observed in vivo. Mutation of cis-acting elements in the size variable region significantly decreased the efficiency of imprecise strand transfer. The results of these in vitro studies suggest that intrinsic genetic elements can modulate the rate of recombination and contribute to genetic and biological diversity of lentivirus infections in vivo.

An in vitro strand transfer assay was used to examine cis-acting elements that promote template switching/strand transfer in the size variable region of the BIV SU protein. We observed both precise strand transfer, which does not produce size variation, and imprecise strand transfer, which leads to duplication/insertion. In the model proposed for size variation in the BIV SU protein (Fig. 1C), the nucleotide sequence identity between the switching site on the donor and the landing site on the acceptor facilitates incorrect alignment of the nascent minus-strand DNA with acceptor template. Indeed, disruption of sequence identity by mutating the donor switching site motif from AACCC to AGTCG drastically reduced the efficiency of imprecise strand transfer. However, sequence identity alone appears to be insufficient for determining the site of re-association, as demonstrated by the fact that the use of an acceptor template containing the compensatory mutation did not restore levels of imprecise strand transfer to that observed with wild-type templates. Therefore, factors in addition to base pairing are required for association of the nascent minus-strand DNA with the acceptor template. The likelihood that a stretch of nucleotides can act as the landing site for non-homologous recombination may depend on its accessibility (e.g. single-strandedness or the spatial relationship with the switching site or nascent strand). It has been reported that the landing site on the acceptor template is preferentially located in single-stranded regions, ready to base pair with the detached nascent strand during template switching (White & Morris, 1995 ). The most straightforward explanation for the failure of the compensatory acceptor to restore imprecise strand transfer is that the mutation changed the folding of the acceptor template in such a way that the landing site was no longer an ideal landing site. Also, the landing site may require a spatial configuration that brings appropriate switching and landing sites into close proximity with one another. For example, a kissing-loop dimerization region within the MLV 5' leader sequence is thought to be a hot spot for recombination because of the formation of an RNA heteroduplex within this region (Mikkelsen et al., 1998 ). Detailed RNA secondary structure analysis of the BIV SU protein donor and acceptor templates will help to delineate the mechanism of how the switching site is guided to the landing site.

Genetic variation, including point mutations and recombination, plays an important role in enabling lentiviruses to adapt to the changing host environment. During BIV replication in vivo, SU protein variants containing duplications/insertions appeared at 28 days post-infection, a few days after the onset of BIV-specific antibodies, and became dominant 1 year after infection (Suarez & Whetstone, 1995 ). Thus, the larger SU protein variants had a selective advantage in vivo. Approximately one-half of the recombinants generated in vitro contained in-frame insertions and each insertion duplicated a putative N-linked glycosylation site. The complex factors important in variant selection in vivo may explain why we did not get the exact duplication/insertion sequence as was observed in vivo. However, the similarity between the in vitro and in vivo recombinants with respect to the crossover site and nature of insertion indicates that all variants probably arose through a similar mechanism of recombination. Thus, both virus and host factors may contribute to the generation and selection of recombinant viruses in vivo.

The in vitro studies reported here are the first to explore the mechanism of lentivirus recombination using virus sequences that are known to undergo size variation in vivo. The finding that cis-acting elements can influence strand transfer efficiency during reverse transcription supports other studies demonstrating ‘hot spots’ of retrovirus recombination (DeStefano et al., 1992a ; Klarmann et al., 1993 ; Wooley et al., 1998 ; Mikkelsen et al., 1998 ). However, our in vitro assay system cannot fully duplicate the events of strand transfer/recombination in vivo and additional studies are needed to elucidate fully the mechanisms of genetic recombination during lentivirus infections. Notably, the only protein in our in vitro system was the heterodimeric RT. Numerous studies have demonstrated that the NC protein plays a critical role in the initiation of reverse transcription (Prats et al., 1988 ; Barat et al., 1989 ; Li et al., 1996 ; Lapadat-Tapolsky et al., 1993 , 1997 ) and can stimulate strand transfer by acting as an RNA chaperon (Allain et al., 1998 ; Rodriguez-Rodriguez et al., 1995 ; Guo et al., 1997 ; Kim et al., 1997 ; Wu et al., 1996 ). Also, we used heterologous HIV-1 RT in the assay system. Although lentivirus RT proteins share some degree of identity, the inclusion of the NC protein in a homologous system will mimic virus replication in vivo more precisely. Further elucidation of genetic mechanisms of lentivirus recombination and their role in persistence and pathogenesis will aid in the understanding of virus evolution and the design of effective vaccines and antiviral therapies.


   Acknowledgments
 
We thank Dr David Suarez for providing BIV FL491 proviral DNA, Yvonne Wannemuehler for technical support and Drs Michael Belshan and Gennadiy Koev for critical review of the manuscript. This work was supported in part by USDA CSRS IOWV-411-23-9805 and funds from the Iowa State University Healthy Livestock Initiative.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
Allain, B., Rascle, J. B., de Rocquigny, H., Roques, B. & Darlix, J.-L. (1998). Cis elements and trans-acting factors required for minus strand DNA transfer during reverse transcription of the genomic RNA of murine leukemia virus. Journal of Molecular Biology 277, 225-235.[Medline]

Anderson, J. A., Teufel, R. J.II, Yin, P. D. & Hu, W.-S. (1998). Correlated template-switching events during minus-strand DNA synthesis: a mechanism for high negative interference during retroviral recombination. Journal of Virology 72, 1186-1194.[Abstract/Free Full Text]

Barat, C., Lullien, V., Schatz, O., Keith, G., Nugeyre, M. T., Gruninger-Leitch, F., Barre-Sinoussi, F., LeGrice, S. F. & Darlix, J.-L. (1989). HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO Journal 8, 3279-3285.[Abstract]

Brian, D. A. & Spaan, W. J. M. (1997). Recombination and coronavirus defective interfering RNAs. Seminars in Virology 8, 101-111.

Burns, D. P. W. & Desrosiers, R. C. (1994). Envelope sequence variation, neutralizing antibodies, and primate lentivirus persistence. Current Topics in Microbiology and Immunology 188, 185-219.[Medline]

Coffin, J. M. (1979). Structure, replication, and recombination of retrovirus genomes: some unifying hypotheses. Journal of General Virology 42, 1-26.[Medline]

Coffin, J. M. (1992). Genetic diversity and evolution of retroviruses. Current Topics in Microbiology and Immunology 176, 143-164.[Medline]

Cornelissen, M., Kampinga, G., Zorgdrager, F. & Goudsmit, J. (1996). Human immunodeficiency virus type 1 subtypes defined by env show high frequency of recombinant gag genes. The UN-AIDS network for HIV isolation and characterization. Journal of Virology 70, 8209-8212.[Abstract]

Delassus, S., Cheynier, R. & Wain-Hobson, S. (1992). Nonhomogeneous distribution of human immunodeficiency virus type 1 proviruses in the spleen. Journal of Virology 66, 5642-5645.[Abstract]

DeStefano, J. J., Buiser, R. G., Mallaber, L. M., Fay, P. J. & Bambara, R. A. (1992a). Parameters that influence processive synthesis and site-specific termination by human immunodeficiency virus reverse transcriptase on RNA and DNA templates. Biochimica et Biophysica Acta 1131, 270-280.[Medline]

DeStefano, J. J., Mallaber, L. M., Rodriguez-Rodriguez, L., Fay, P. J. & Bambara, R. A. (1992b). Requirements for strand transfer between internal regions of heteropolymer templates by human immunodeficiency virus reverse transcriptase. Journal of Virology 66, 6370-6378.[Abstract]

DeStefano, J. J., Bambara, R. A. & Fay, P. J. (1994). The mechanism of human immunodeficiency virus reverse transcriptase-catalyzed strand transfer from internal regions of heteropolymeric RNA templates. Journal of Biological Chemistry 269, 161-168.[Abstract/Free Full Text]

Fouchier, R. A. M., Broersen, S. M., Brouwer, M., Tersmette, M., van’t Wout, A. B., Groenink, M. & Schuitemaker, H. (1995). Temporal relationship between elongation of the HIV type 1 glycoprotein 120 V2 domain and the conversion toward a syncytium-inducing phenotype. AIDS Research and Human Retroviruses 12, 1473-1478.

Fox, D. G., Balfe, P., Palmer, C. P., May, J. C., Arnold, C. & McKeating, J. A. (1997). Length polymorphism within the second variable region of the human immunodeficiency virus type 1 envelope glycoprotein affects accessibility of the receptor binding site. Journal of Virology 71, 759-765.[Abstract]

Gao, F., Robertson, D. L., Carruthers, C. D., Morrison, S. G., Jian, B., Chen, Y., Barre-Sinoussi, F., Girard, M., Srinivasan, A., Abimiku, A. G., Shaw, G. M., Sharp, P. M. & Hahn, B. H. (1998). A comprehensive panel of near-full-length clones and reference sequences for non-subtype B isolates of human immunodeficiency virus type 1. Journal of Virology 72, 5680-5698.[Abstract/Free Full Text]

Groenink, M., Fouchier, R. A. M., Broersen, S., Baker, C. H., Koot, M., van’t Wout, A. B., Huisman, H. G., Miedema, F., Tersmette, M. & Schuitemaker, H. (1993). Relation of phenotype evolution of HIV-1 to envelope V2 configuration. Science 260, 1513-1516.[Medline]

Guo, J., Henderson, L. E., Bess, J., Kane, B. & Levin, J. G. (1997). Human immunodeficiency virus type 1 nucleocapsid protein promotes efficient strand transfer and specific viral DNA synthesis by inhibiting TAR-dependent self-priming from minus-strand strong-stop DNA. Journal of Virology 71, 5178-5188.[Abstract]

Hu, W.-S. & Temin, H. M. (1990a). Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination. Proceedings of the National Academy of Sciences, USA 87, 1556-1560.[Abstract]

Hu, W.-S. & Temin, H. M. (1990b). Retroviral recombination and reverse transcription. Science 250, 1227-1233.[Medline]

Kim, J. K., Palaniappan, C., Wu, W., Fay, P. J. & Bambara, R. A. (1997). Evidence for a unique mechanism of strand transfer from the transactivation response region of HIV-1. Journal of Biological Chemistry 272, 16769-16777.[Abstract/Free Full Text]

Klarmann, G. J., Schauber, C. A. & Preston, B. D. (1993). Template-directed pausing of DNA synthesis by HIV-1 reverse transcriptase during polymerization of HIV-1 sequences in vitro. Journal of Biological Chemistry 268, 9793-9802.[Abstract/Free Full Text]

Lapadat-Tapolsky, M., de Rocquigny, H., Van Gent, D., Roques, B., Plasterk, R. & Darlix, J.-L. (1993). Interactions between HIV-1 nucleocapsid protein and viral DNA may have important functions in the viral life cycle. Nucleic Acids Research 21, 831-839.[Abstract]

Lapadat-Tapolsky, M., Gabus, C., Rau, M. & Darlix, J.-L. (1997). Possible roles of HIV-1 nucleocapsid in the specificity of proviral DNA synthesis and in its variability. Journal of Molecular Biology 268, 250-260.[Medline]

Leroux, C., Issel, C. J. & Montelaro, R. C. (1997). Novel and dynamic evolution of equine infectious anemia virus genomic quasispecies associated with sequential disease cycles in an experimentally infected pony. Journal of Virology 71, 9627-9639.[Abstract]

Li, X., Quan, Y., Arts, E. J., Li, Z., Preston, B. D., de Rocquigny, H., Roques, B. P., Darlix, J.-L., Kleiman, L., Parniak, M. A. & Wainberg, M. A. (1996). Human immunodeficiency virus Type 1 nucleocapsid protein (NCp7) directs specific initiation of minus-strand DNA synthesis primed by human tRNA(Lys3) in vitro: studies of viral RNA molecules mutated in regions that flank the primer binding site. Journal of Virology 70, 4996-5004.[Abstract]

Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Mikkelsen, J. G., Lund, A. H., Duch, M. & Pedersen, F. S. (1998). Recombination in the 5' leader of murine leukemia virus is accurate and influenced by sequence identity with a strong bias toward the kissing-loop dimerization region. Journal of Virology 72, 6967-6978.[Abstract/Free Full Text]

Nagy, P. D. & Bujarski, J. J. (1996). Homologous RNA recombination in brome mosaic virus: AU-rich sequences decrease the accuracy of crossovers. Journal of Virology 70, 415-426.[Abstract]

Nagy, P. & Bujarski, J. J. (1997). Engineering of homologous recombination hotspots with AU-rich sequences in brome mosaic virus. Journal of Virology 71, 3799-3810.[Abstract]

Nagy, P. & Simon, A. E. (1997). New insights into the mechanisms of RNA recombination. Virology 235, 1-9.[Medline]

Palaniappan, C., Wisniewski, M., Wu, W., Fay, P. J. & Bambara, R. A. (1996). Misincorporation by HIV-1 reverse transcriptase promotes recombination via strand transfer synthesis. Journal of Biological Chemistry 271, 22331-22338.[Abstract/Free Full Text]

Palmer, C., Balfe, P., Fox, D., May, J. C., Frederiksson, R., Fenyo, E.-M. & McKeating, J. A. (1996). Functional characterization of the V1V2 region of human immunodeficiency virus type 1. Virology 220, 436-449.[Medline]

Pathak, V. K. & Hu, W.-S. (1997). ‘Might as well jump!’ template switching by retroviral reverse transcriptase, defective genome formation, and recombination. Seminars in Virology 8, 141-150.

Peliska, J. A. & Benkovic, S. J. (1992). Mechanism of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. Science 258, 1112-1118.[Medline]

Pezo, V. & Wain-Hobson, S. (1997). HIV genetic variation: life at the edge. Journal of Infection 34, 201-203.[Medline]

Prats, A. C., Sarih, L., Gabus, C., Litvak, S., Keith, G. & Darlix, J.-L. (1988). Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA. EMBO Journal 7, 1777-1783.[Abstract]

Robertson, D. L., Sharp, P. M., McCutchan, F. E. & Hahn, B. H. (1995). Recombination in HIV-1. Nature 374, 124-126.[Medline]

Rodriguez-Rodriguez, L., Tsuchihashi, Z., Fuentes, G. M., Bambara, R. A. & Fay, P. J. (1995). Influence of human immunodeficiency virus nucleocapsid protein on synthesis and strand transfer by the reverse transcriptase in vitro. Journal of Biological Chemistry 270, 15005-15011.[Abstract/Free Full Text]

Rudensey, L. M., Kimata, J. T., Long, E. M., Chackerian, B. & Overbaugh, J. (1998). Changes in the extracellular envelope glycoprotein of variants that evolve during the course of simian immunodeficiency virus infection SIVMne affect neutralizing antibody recognition, syncytium formation, and macrophage tropism but not replication, cytopathicity, or CCR-5 coreceptor recognition. Journal of Virology 72, 209-217.[Abstract/Free Full Text]

Shioda, T., Oka, S., Xin, X., Liu, H., Harukuni, R., Kurotani, A., Fukushima, M., Hasan, M. K., Shiino, T., Takebe, Y., Iwamoto, A. & Nagai, Y. (1997). In vivo sequencing variability of human immunodeficiency virus type 1 envelope gp120: association of V2 extension with slow disease progression. Journal of Virology 71, 4871-4881.[Abstract]

Stuhlmann, H. & Berg, P. (1992). Homologous recombination of copackaged retrovirus RNAs during reverse transcription. Journal of Virology 66, 2378-2388.[Abstract]

Suarez, D. L. & Whetstone, C. A. (1995). Identification of hypervariable and conserved regions in the surface envelope gene in the bovine lentivirus. Virology 212, 728-733.[Medline]

Suarez, D. L. & Whetstone, C. A. (1997). Size variation within the second hypervariable region of the surface envelope gene of the bovine lentivirus BIV in experimentally and naturally infected cattle. Journal of Virology 71, 2482-2486.[Abstract]

Telesnitsky, A. & Goff, S. P. (1997). Reverse transcriptase and the generation of retroviral DNA. In Retroviruses , pp. 121-160. Edited by J. M. Coffin, S. H. Hughes & H. E. Varmus. Cold Spring Harbor, NY:Cold Spring Harbor Laboratory.

Topping, R., Demoitie, M.-A., Shin, N. H. & Telesnitsky, A. (1998). Cis-acting elements required for strong stop acceptor template selection during Moloney murine leukemia virus reverse transcription. Journal of Molecular Biology 281, 1-15.[Medline]

White, K. A. & Morris, T. J. (1995). RNA determinants of junction site selection in RNA virus recombinants and defective interfering RNAs. RNA 1, 1029-1040.[Abstract]

Wooley, D. P., Bircher, L. A. & Smith, R. A. (1998). Retroviral recombination is nonrandom and sequence dependent. Virology 243, 229-234.[Medline]

Wu, W., Blumberg, B. M., Fay, P. J. & Bambara, R. A. (1995). Strand transfer mediated by human immunodeficiency virus reverse transcriptase in vitro is promoted by pausing and results in misincorporation. Journal of Biological Chemistry 270, 325-332.[Abstract/Free Full Text]

Wu, W., Henderson, L. E., Copeland, T. D., Gorelick, R. J., Bosche, W. J., Rein, A. & Levin, J. G. (1996). Human immunodeficiency virus type 1 nucleocapsid protein reduces reverse transcriptase pausing at a secondary structure near the murine leukemia virus polypurine tract. Journal of Virology 70, 7132-7142.[Abstract]

You, J. C. & McHenry, C. S. (1994). Human immunodeficiency virus nucleocapsid protein accelerates strand transfer of the terminally redundant sequences involved in reverse transcription. Journal of Biological Chemistry 269, 31491-31495.[Abstract/Free Full Text]

Zhang, J. & Temin, H. M. (1993a). 3' junctions of oncogene-virus sequences and the mechanisms for formation of highly oncogenic retroviruses. Journal of Virology 67, 1747-1751.[Medline]

Zhang, J. & Temin, H. M. (1993b). Rate and mechanism of nonhomologous recombination during a single cycle of retroviral replication. Science 259, 234-238.[Medline]

Zhang, J. & Temin, H. M. (1994). Retrovirus recombination depends on the length of sequence identity and is not error prone. Journal of Virology 68, 2409-2414.[Abstract]

Zhang, J., Tang, L.-Y., Li, T., Ma, Y. & Sapp, C. M. (2000). Most retroviral recombinations occur during minus-strand DNA synthesis. Journal of Virology 74, 2313-2322.[Abstract/Free Full Text]

Zheng, Y.-H., Nakaya, T., Sentsui, H., Kameoka, M., Kishi, M., Hagiwara, K., Takahashi, H., Kono, Y. & Ikuta, K. (1997a). Insertions, duplications and substitutions in restricted gp90 regions of equine infectious anaemia virus during febrile episodes in an experimentally infected horse. Journal of General Virology 78, 807-820.[Abstract]

Zheng, Y.-H., Sentsui, H., Nakaya, T., Kono, Y. & Ikuta, K. (1997b). In vivo dynamics of equine infectious anemia viruses emerging during febrile episodes: insertions/duplications at the principle neutralizing domain. Journal of Virology 71, 5031-5039.[Abstract]

Received 11 April 2001; accepted 28 August 2001.



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