Circular DNA of human immunodeficiency virus: analysis of circle junction nucleotide sequences.

During infection of cells by retroviruses, some of the nonintegrated viral DNA can be found as a circular form containing two tandem, directly repeated long terminal repeats. The nucleotide sequence at the point where the long terminal repeats join (the circle junction) can be used to deduce the terminal nucleotides of the linear form of the viral DNA. Comparison of the termini of linear viral DNA with sequences at the junctions between the integrated provirus and the host chromosome has revealed that for most retroviruses 2 bp are removed from each end of the linear viral DNA during integration. For human immunodeficiency virus type 1 (HIV-1), however, sequence considerations involving primer-binding sites had suggested that only 1 bp is removed during integration. We obtained the nucleotide sequences at the ends of HIV-1 DNA by using the polymerase chain reaction to amplify fragments corresponding to the HIV-1 circle junction. Of 17 clones containing amplified sequences, 10 had identical circle junctions that contained an additional 4 bp (GTAC) relative to the integrated provirus. This indicates that, as for other retroviruses, 2 bp are removed from each end of the linear HIV-1 viral DNA during integration. The remaining seven isolates contained insertions or deletions at the circle junction.     

Human immunodeficiency virus type 1 integration protein: DNA sequence requirements for cleaving and joining reactions.

Using purified integration protein (IN) from human immunodeficiency virus (HIV) type 1 and oligonucleotide mimics of viral and target DNA, we have investigated the DNA sequence specificity of the cleaving and joining reactions that take place during retroviral integration. The first reaction in this process is selective endonucleolytic cleaving of the viral DNA terminus that generates a recessed 3' OH group. This 3' OH group is then joined to a 5' phosphoryl group located at a break in the target DNA. We found that the conserved CA located close to the 3' end of the plus strand of the U5 viral terminus (also present on the minus strand of the U3 terminus) was required for both cleaving and joining reactions. Six bases of HIV U5 or U3 DNA at the ends of model substrates were sufficient for nearly maximal levels of selective endonucleolytic cleaving and joining. However, viral sequence elements upstream of the terminal 6 bases could also affect the efficiencies of the cleaving and joining reactions. The penultimate base (C) on the minus strand of HIV U5 was required for optimal joining activity. A synthetic oligonucleotide mimic of the putative in vivo viral "DNA" substrate for HIV IN, a molecule that contained a terminal adenosine 5'-phosphate (rA) on the minus strand, was indistinguishable in the cleaving and joining reactions from the DNA substrate containing deoxyadenosine instead of adenosine 5'-phosphate at the terminal position. Single-stranded DNA served as an in vitro integration target for HIV IN. The DNA sequence specificity of the joining reaction catalyzed in the reverse direction was also investigated.     

Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase.

The integration of linear retrovirus DNA by the viral integrase (IN) into the host chromosome occurs by a concerted mechanism (full-site reaction). IN purified from avian myeloblastosis virus and using retrovirus-like DNA restriction fragments (487 bp in length) as donors and circular DNA (pGEM-3) as the target can efficiently catalyze that reaction. Nonionic detergent lysates of purified human immunodeficiency virus type 1 (HIV-1) virions were also capable of catalyzing the concerted integration reaction. The donor substrates were restriction fragments (469 bp) containing either U3-U5 (H-2 donor) or U5-U5 (H-5 donor) long terminal repeat sequences at their ends. As was shown previously with bacterially expressed HIV-1 IN, the U5 terminus of H-2 was preferred over the U3 terminus by virion-associated IN. The reactions involving two donors per circular target by HIV-1 IN preferred Mg2+ over Mn2+. Both metal ions were equally effective for the circular half-site reaction involving only one donor molecule. The linear 3.8-kbp recombinant products produced from two donor insertions into pGEM were genetically selected, and the donor-target junctions of individual recombinants were sequenced. A total of 55% of the 87 sequenced recombinants had host site duplications of between 5 and 7 bp, with the HIV-1 5-bp-specific duplication predominating. The other recombinants that migrated at the linear 3.8-kbp position were mainly small deletions that were grouped into four sets of 17, 27, 40, and 47 bp, each having a periodicity mimicking a turn of the DNA helix. Aprotic solvents (dimethyl sulfoxide and 1,4-dioxane) enhanced both the half-site and the linear 3.8-kbp strand transfer reactions which favored low-salt conditions (30 mM NaCl). The order of addition of the donor and target during preincubation with HIV-1 IN on ice did not affect the quantity of linear 3.8-kbp recombinants relative to that of the circular half-site products that were produced; only the quantity of donor-donor versus donor-target recombinants was affected. The presence of Mg2+ in the preincubation mixtures containing donor and target substrates was not necessary for the stability of preintegration complexes on ice or at 22 degrees C. Comparisons of the avian and HIV-1 concerted integration reactions are discussed.     

The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration.

The purified integration protein (IN) of avian myeloblastosis virus is shown to nick double-stranded oligodeoxynucleotide substrates that mimic the ends of the linear form of viral DNA. In the presence of Mg2+, nicks are created 2 nucleotides from the 3' OH ends of both the U5 plus strand and the U3 minus strand. Similar cleavage is observed in the presence of Mn2+ but only when the extent of the reaction is limited. Neither the complementary strands nor sequences representing the termini of human immunodeficiency virus type 1 DNA were cleaved at analogous positions. Analysis of a series of substrates containing U5 base substitutions has defined the sequence requirements for site-selective nicking; nucleotides near the cleavage site are most critical for activity. The minimum substrate size required to demonstrate significant activity corresponds to the nearly perfect 15-base terminal inverted repeat. This in vitro activity of IN thus produces viral DNA ends that are joined to host DNA in vivo and corresponds to an expected early step in the integrative recombination reaction. These results provide the first enzymatic support using purified retroviral proteins for a linear DNA precursor to the integrated provirus.     

Coupled integration of human immunodeficiency virus type 1 cDNA ends by purified integrase in vitro: stimulation by the viral nucleocapsid protein.

Integration of retroviral cDNA involves coupled joining of the two ends of the viral genome at precisely spaced positions in the host cell DNA. Correct coupled joining is essential for viral replication, as shown, for example, by the finding that viral mutants defective in coupled joining are defective in integration and replication. To date, reactions with purified human immunodeficiency virus type 1 (HIV-1) integrase protein in vitro have supported mainly uncoupled joining of single cDNA ends. We have analyzed an activity stimulating coupled joining present in HIV-1 virions, which led to the finding that the HIV-1 nucleocapsid (NC) protein can stimulate coupled joining more than 1,000-fold under some conditions. The requirements for stimulating coupled joining were investigated in assays with mutant NC proteins, revealing that mutations in the zinc finger domains can influence stimulation of integration. These findings (i) provide a means for assembling more authentic integrase complexes for mechanistic studies, (ii) reveal a new activity of NC protein in vitro, (iii) indicate a possible role for NC in vivo, and (iv) provide a possible method for identifying a new class of inhibitors that disrupt coupled joining.     

Human immunodeficiency virus type 1 nucleocapsid protein specifically stimulates Mg2+-dependent DNA integration in vitro.

The integrase (IN) protein of the human immunodeficiency virus mediates integration of the viral DNA into the cellular genome. In vitro, this reaction can be mimicked by using purified recombinant IN and model DNA substrates. IN mediates two reactions: an endonucleolytic cleavage at each 3' end of the proviral DNA (terminal cleavage) and the joining of the linear viral DNA to 5' phosphates in the target DNA (strand transfer). Previous investigators have shown that purified IN requires Mn2+ or Mg2+ to promote strand transfer in vitro, although Mg2+ is the likely metal cofactor in vivo. IN activity in the presence of Mg2+ in vitro requires high IN concentrations and low concentrations of salt. Here, we show that the viral nucleocapsid protein NCp7 allows efficient IN-mediated strand transfer in the presence of Mg2+ at low enzyme concentrations. This potentiating effect appears to be unique to NCp7, as other small DNA-binding proteins, while capable of stimulating integration in the presence of Mn2+, all failed to stimulate strand transfer in the presence of Mg2+.     

Analysis of envelope sequence variants suggests multiple mechanisms of mother-to-child transmission of human immunodeficiency virus type 1.

In order to elucidate the molecular mechanisms involved in human immunodeficiency virus type 1 (HIV-1) mother-to-child transmission, we have analyzed the genetic variation within the V3 hypervariable domain and flanking regions of the HIV-1 envelope gene in four mother-child transmission pairs. Phylogenetic analysis and amino acid sequence comparison were performed on cell-associated viral sequences derived from maternal samples collected at different time points during pregnancy, after delivery, and from child samples collected from the time of birth until the child was approximately 1 year of age. Heterogeneous sequence populations were observed to be present in all maternal samples collected during pregnancy and postdelivery. In three newborns, viral sequence populations obtained within 2 weeks after birth revealed a high level of V3 sequence variability. In contrast, V3 sequences obtained from the fourth child (diagnosed at the age of 1 month) displayed a more restricted heterogeneity. The phylogenetic analysis performed for each mother-child sequence set suggested that several mechanisms may potentially be involved in HIV-1 vertical transmission. For one pair, child sequences were homogeneous and clustered in a single branch within the phylogenetic tree, consistent with selective transmission of a single maternal variant. For the other three pairs, the child sequences were more heterogeneous and clustered in several separate branches within the tree. In these cases, it appeared likely that more than one maternal variant was responsible for infection of the child. In conclusion, no single mechanism can account for mother-to-child HIV-1 transmission; both the selective transmission of a single maternal variant and multiple transmission events may occur.     

HMG protein family members stimulate human immunodeficiency virus type 1 and avian sarcoma virus concerted DNA integration in vitro

We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration in vitro with specially designed mini-donor HIV-1 DNA, a supercoiled plasmid acceptor, purified bacterium-derived HIV-1 integrase (IN), and host HMG protein family members. This system is comparable to one previously described for avian sarcoma virus (ASV) (A. Aiyar et al., J. Virol. 70:3571-3580, 1996) that was stimulated by the presence of HMG-1. Sequence analyses of individual HIV-1 integrants showed loss of 2 bp from the ends of the donor DNA and almost exclusive 5-bp duplications of the acceptor DNA at the site of integration. All of the integrants sequenced were inserted into different sites in the acceptor. These are the features associated with integration of viral DNA in vivo. We have used the ASV and HIV-1 reconstituted systems to compare the mechanism of concerted DNA integration and examine the role of different HMG proteins in the reaction. Of the three HMG proteins examined, HMG-1, HMG-2, and HMG-I(Y), the products formed in the presence of HMG-I(Y) for both systems most closely match those observed in vivo. Further analysis of HMG-I(Y) mutants demonstrates that the stimulation of integration requires an HMG-I(Y) domain involved in DNA binding. While complexes containing HMG-I(Y), ASV IN, and donor DNA can be detected in gel shift experiments, coprecipitation experiments failed to demonstrate stable interactions between HMG-I(Y) and ASV IN or between HMG-I(Y) and HIV-1 IN.     

Different roles of bases within the integration signal sequence of human immunodeficiency virus type 1 in vitro.

To investigate the roles of bases near the tips of each strand of the long terminal repeat of the human immunodeficiency virus type 1 in the integration reaction, we examined the efficiencies of both binding and integration activities of staggered-ended substrates and mismatched mutant substrates by the integration assay and the UV cross-linking assay. Our results suggest that some bases of the human immunodeficiency virus type 1 long terminal repeat are required primarily for binding, whereas others are more critical for later reaction steps in vitro.     

Structural and functional analysis of the human immunodeficiency virus type 2 Rev protein.

The Rev proteins of the human immunodeficiency virus (HIV) are necessary for expression of viral structural gene products. Site-directed mutations were made within the HIV-2 rev gene to identify functional domains. We observed that similar to HIV-1 Rev, the HIV-2 Rev protein was phosphorylated, albeit to a much lesser extent than was HIV-1 Rev. We also found that like HIV-1 Rev, HIV-2 Rev localized to the nucleus, with a marked accumulation in the nucleolus. Mutations within a stretch of basic residues prevented both nuclear and nucleolar localization. Furthermore, mutant Rev proteins able to localize in the nucleus but unable to localize in the nucleolus were nonfunctional.     

V3 loop of the human immunodeficiency virus type 1 Env protein: interpreting sequence variability.

Two different states of human immunodeficiency virus type 1 are apparent in the asymptomatic and late stages of infection. Important determinants associated with these two states have been found within the V3 loop of the viral Env protein. In this study, two large data sets of published V3 sequences were analyzed to identify patterns of sequence variability that would correspond to these two states of the virus. We were especially interested in the pattern of basic amino acid substitutions, since the presence of basic amino acids in V3 has been shown to change virus tropism in cell culture. Four features of the sequence heterogeneity in V3 were observed: (i) approximately 70% of all nonconservative basic substitutions occur at four positions in V3, and V3 sequences with a basic substitution in at least one of these four positions contain approximately 95% of all nonconservative basic substitutions; (ii) substitution patterns within V3 are influenced by the identity of the amino acid at position 25; (iii) sequence polymorphisms account for a significant fraction of uncharged amino acid substitutions at several positions in V3, and sequence heterogeneity other than these polymorphisms is most significant at two positions near the tip of V3; and (iv) sequence heterogeneity in V3 (in addition to the basic amino acid substitutions) is approximately twofold greater in V3 sequences that contain basic amino acid substitutions. By using this sequence analysis, we were able to identify distinct groups of V3 sequences in infected patients that appear to correspond to these two virus states. The identification of these discrete sequence patterns in vivo demonstrates how the V3 sequence can be used as a genetic marker for studying the two states of human immunodeficiency virus type 1.     

Analysis of long terminal repeat circle junctions of human immunodeficiency virus type 1.

Circle junctions of unintegrated human immunodeficiency virus type 1 strain IIIB were analyzed after polymerase chain reaction amplification. Among the 28 colonies sequenced, eight unique circle junction species were detected. Five of the eight species resulted in circle junctions with larger inserts than predicted. A majority of these could result from heterogeneity in generating the U5' long terminal repeat terminus.     

DNA substrate requirements for different activities of the human immunodeficiency virus type 1 integrase protein.

The integrase protein (IN) of human immunodeficiency virus type 1 removes two nucleotides from both 3' ends of the viral DNA (donor cleavage) and subsequently couples the newly generated 3' OH groups to phosphates in the target DNA (integration). The sequence requirements of IN for cleavage as well as for integration of viral DNA substrates have previously been studied by mutational analyses and by adduct interference assays. We extended these studies by analysis of heteroduplex oligonucleotide substrates and by missing-base analysis. We found for some base pairs that mutation of only one of the two bases and not the other affected IN activity. These base pairs center around the cleavage site. Besides donor cleavage and integration, IN can also perform "intermolecular disintegration," which has been described as the reversal of the integration reaction. We found that this reaction is independent of viral DNA sequences. In addition, the optimum spacing between the integration sites in intermolecular disintegration does not reflect the spacing found in vivo. These results indicate that this reaction is not the exact reversal of integration but rather is a sequence-independent phosphoryl transfer reaction between gapped DNA duplex molecules.     

DNA strand exchange and selective DNA annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein.

Nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) was expressed in Escherichia coli and purified. The protein displayed a variety of activities on DNA structure, all reflecting an ability to promote transition between double-helical and single-stranded conformations. We found that, in addition to its previously described ability to accelerate renaturation of complementary DNA strands, the HIV-1 NC protein could substantially lower the melting temperature of duplex DNA and could promote strand exchange between double-stranded and single-stranded DNA molecules. Moreover, in the presence of HIV-1 NC, annealing of a single-stranded DNA molecule to a complementary DNA strand that would yield a more stable double-stranded product was favored over annealing to alternative complementary DNA strands that would form less stable duplex products (selective annealing). NC thus appears to lower the kinetic barrier so that double-strand <==> single-strand equilibrium is rapidly reached to favor the lowest free-energy nucleic acid conformation. This activity of NC may be important for correct folding of viral genomic RNA and may have practical applications.     

Tethering human immunodeficiency virus type 1 preintegration complexes to target DNA promotes integration at nearby sites.

Integration of retroviral cDNA in vivo is normally not sequence specific with respect to the integration target DNA. We have been investigating methods for directing the integration of retroviral DNA to predetermined sites, with the dual goal of understanding potential mechanisms governing normal site selection and developing possible methods for gene therapy. To this end, we have fused retroviral integrase enzymes to sequence-specific DNA-binding domains and investigated target site selection by the resulting proteins. In a previous study, we purified and analyzed a fusion protein composed of human immunodeficiency virus integrase linked to the DNA-binding domain of lambda repressor. This fusion could direct selective integration in vitro into target DNA containing lambda repressor binding sites. Here we investigate the properties of a fusion integrase in the context of a human immunodeficiency virus provirus. We used a fusion of integrase to the DNA binding domain of the zinc finger protein zif268 (IN-zif). Initially we found that the fusion was highly detrimental to replication as measured by the multinuclear activation of a galactosidase indicator (MAGI) assay for infected centers. However, we found that viruses containing mixtures of wild-type integrase and IN-zif were infectious. We prepared preintegration complexes from cells infected with these viruses and found that such complexes directed increased integration near zif268 recognition sites.