Simian virus 40-based replication of catalytically inactive human immunodeficiency virus type 1 integrase mutants in nonpermissive T cells and monocyte-derived macrophages

Integrase function is required for retroviral replication in most instances. Although certain permissive T-cell lines support human immunodeficiency virus type 1 (HIV-1) replication in the absence of functional integrase, most cell lines and primary human cells are nonpermissive for integrase mutant growth. Since unintegrated retroviral DNA is lost from cells following cell division, we investigated whether incorporating a functional origin of DNA replication into integrase mutant HIV-1 might overcome the block to efficient gene expression and replication in nonpermissive T-cell lines and primary cells. Whereas the Epstein-Barr virus (EBV) origin (oriP) did little to augment expression from an integrase mutant reporter virus in EBV nuclear antigen 1-expressing cells, simian virus 40 (SV40) oriT dramatically enhanced integrase mutant infectivity in T-antigen (Tag)-expressing cells. Incorporating oriT into the nef position of a full-length, integrase-defective virus strain yielded efficient replication in Tag-expressing nonpermissive Jurkat T cells without reversion to an integration-competent genotype. Adding Tag to integrase mutant-oriT viruses yielded 11.3-kb SV40-HIV chimeras that replicated in Jurkat cells and primary monocyte-derived macrophages. Real-time quantitative PCR analyses of Jurkat cell infections revealed that amplified copies of unintegrated DNA likely contributed to SV40-HIV integrase mutant replication. SV40-based HIV-1 integrase mutant replication in otherwise nonpermissive cells suggests alternative approaches to standard integrase-mediated retroviral gene transfer strategies.     

Human immunodeficiency virus type 1 replication in the absence of integrase-mediated dna recombination: definition of permissive and nonpermissive T-cell lines

Functional retroviral integrase protein is thought to be essential for productive viral replication. Yet, previous studies differed on the extent to which integrase mutant viruses expressed human immunodeficiency virus type 1 (HIV-1) genes from unintegrated DNA. Although one reason for this difference was that class II integrase mutations pleiotropically affected the viral life cycle, another reason apparently depended on the identity of the infected cell. Here, we analyzed integrase mutant viral infectivities in a variety of cell types. Single-round infectivity of class I integration-specific mutant HIV-1 ranged from <0.03 to 0.3% of that of the wild type (WT) across four different T-cell lines. Based on this approximately 10-fold influence of cell type on mutant gene expression, we examined class I and class II mutant replication kinetics in seven different cell lines and two primary cell types. Unexpectedly, some cell lines supported productive class I mutant viral replication under conditions that restricted class II mutant growth. Cells were defined as permissive, semipermissive, or nonpermissive based on their ability to support the continual passage of class I integration-defective HIV-1. Mutant infectivity in semipermissive and permissive cells as quantified by 50% tissue culture infectious doses, however, was only 0.0006 to 0.005% of that of WT. Since the frequencies of mutant DNA recombination in these lines ranged from 0.023 to <0.093% of the WT, we conclude that productive replication in the absence of integrase function most likely required the illegitimate integration of HIV-1 into host chromosomes by cellular DNA recombination enzymes.     

Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells.

Integrase is the only viral protein necessary for integration of retroviral DNA into chromosomal DNA of the host cell. Biochemical analysis of human immunodeficiency virus type 1 (HIV-1) integrase with purified protein and synthetic DNA substrates has revealed extensive information regarding the mechanism of action of the enzyme, as well as identification of critical residues and functional domains. Since in vitro reactions are carried out in the absence of other viral proteins and they analyze strand transfer of only one end of the donor substrate, they do not define completely the process of integration as it occurs during the course of viral infection. In an effort to further understand the role of integrase during viral infection, we initially constructed a panel of 24 HIV-1 mutants with specific alanine substitutions throughout the integrase coding region and analyzed them in a human T-cell line infection. Of these mutant viruses, 12 were capable of sustained viral replication, 11 were replication defective, and 1 was temperature sensitive for viral growth. The replication defective viruses express and correctly process the integrase and Gag proteins. Using this panel of mutants and an additional set of 18 mutant viruses, we identified nine amino acids which, when replaced with alanine, destroy integrase activity. Although none of the replication-defective mutants are able to integrate into the host genome, a subset of them with alterations in the catalytic triad are capable of Tat-mediated transactivation of an indicator gene linked to the viral long terminal repeat promoter. We present evidence that integration of the HIV-1 provirus is essential not only for productive infection of T cells but also for virus passage in both cultured peripheral blood lymphocytes and macrophage cells.     

vif-negative human immunodeficiency virus type 1 persistently replicates in primary macrophages, producing attenuated progeny virus.

The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived macrophages (MDM). Viruses carrying missense or deletion mutations in vif were constructed on the background of the monocytotropic recombinant NLHXADA-GP. Using MDM from multiple donors, we found that vif mutants produced in complementing or partially complementing cell lines were approximately 10% as infectious as wild-type virus when assayed for incomplete, complete, and circularized viral DNA molecules by quantitative PCR amplification or for viral core antigen p24 production by enzyme-linked immunosorbent assay. We then determined the structure and infectivity of vif mutant HIV-1 by using MDM exclusively both for virus production and as targets for infection. Biosynthetic labeling and immunoprecipitation analysis of sucrose cushion-purified vif-negative HIV-1 made in MDM revealed that the virus had reduced p24 content compared with wild-type HIV-1. Cell-free MDM-derived vif mutant HIV-1 was infectious in macrophages as determined by the synthesis and maintenance of full-length viral DNA and by the produc- tion of particle-associated viral RNA, but its infectivity was approximately 2,500-fold lower than that of wild-type virus whose titer was determined in parallel by measurement of the viral DNA burden. MDM infected with MDM-derived vif-negative HIV-1 were able to transmit the virus to uninfected MDM by cocultivation, confirming the infectiousness of this virus. We conclude that mutations in vif significantly reduce but do not eliminate the capacity of HIV-1 to replicate and produce infectious progeny virus in primary human macrophages.     

Inhibition of early steps of HIV-1 replication by SNF5/Ini1

To replicate, human immunodeficiency virus, type 1 (HIV-1) needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNF5/Ini1, a subunit of the SWI/SNF chromatin remodeling complex, was the first cofactor identified to interact with IN. We report here that SNF5/Ini1 interferes with early steps of HIV-1 replication. Inhibition of SNF5/Ini1 expression by RNA interference increases HIV-1 replication. Using quantitative PCR, we show that both the 2-long terminal repeat circle and integrated DNA forms accumulate upon SNF5/Ini1 knock down. By yeast two-hybrid assay, we screened a library of HIV-1 IN random mutants obtained by PCR random mutagenesis using SNF5/Ini1 as prey. Two different mutants of interaction, IN E69G and IN K71R, were impaired for SNF5/Ini1 interaction. The E69G substitution completely abolished integrase catalytic activity, leading to a replication-defective virus. On the contrary, IN K71R retained in vitro integrase activity. K71R substitution stimulates viral replication and results in higher infectious titers. Taken together, these results suggest that, by interacting with IN, SNF5/Ini1 interferes with early steps of HIV-1 infection.     

Human immunodeficiency virus type 1 2-LTR circles reside in a nucleoprotein complex which is different from the preintegration complex.

The preintegration complex of human immunodeficiency virus type 1 (HIV-1) is a large nucleoprotein complex containing viral nucleic acids in association with products of the viral gag and pol genes. One of these proteins, integrase, is absolutely required for the integration and formation of the provirus. Although HIV-1-specific 2-LTR circles from nuclei of HIV-1-infected cells were found to be associated within a high-molecular-weight nucleoprotein complex, antibodies to HIV-1 integrase failed to precipitate this form of viral DNA. This result indicates that circular forms of HIV-1 DNA are not associated with integrase. These viral DNA forms seem to exist in a context of a nucleoprotein complex that is different from a preintegration complex of HIV-1.     

Gag-processing defect of human immunodeficiency virus type 1 integrase E246 and G247 mutants is caused by activation of an overlapping 5' splice site

We have previously described several human immunodeficiency virus type 1 (HIV-1) mutants that are characterized by an excessive-RNA-splicing phenotype and reduced virus particle production. In one of these mutants (NLD2up), the sequence of 5′ splice site D2 was changed to a consensus splice donor site. This splice site overlaps the HIV-1 integrase reading frame, and thus, the NLD2up mutant also bears a G-to-W change at amino acid 247 of the integrase. A previously described E-to-K mutant at position 246 of the C-terminal domain of the integrase, which resulted in a G-to-A mutation at the +3 position of overlapping splice donor D2 (NLD2A3), was also shown to affect virus particle production and Gag protein processing. By using second-site mutations to revert the excessive-splicing phenotype, we show that the effects on Gag protein processing and virus particle production of both the NLD2up and NLD2A3 mutants are caused by excessive viral RNA splicing due to the activation of the overlapping 5′ splice site and not to the changes in the integrase protein. Both integrase protein mutations, however, are lethal for virus infectivity. These studies suggest that changes in the usage of overlapping splice sites may be a possible alternative explanation for a defective virus phenotype resulting from changes in protein-coding sequences or in the nucleotide sequence during codon optimization.     

Irreversible inhibition of human immunodeficiency virus type 1 integrase by dicaffeoylquinic acids

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses require integration of a double-stranded DNA copy of the RNA genome into the host cell chromosome for productive infection. The viral enzyme, integrase, catalyzes the integration of retroviral DNA and represents an attractive target for developing antiretroviral agents. We identified several derivatives of dicaffeoylquinic acids (DCQAs) that inhibit HIV-1 replication in tissue culture and catalytic activities of HIV-1 integrase in vitro. The specific step at which DCQAs inhibit the integration in vitro and the mechanism of inhibition were examined in the present study. Titration experiments with different concentrations of HIV-1 integrase or DNA substrate found that the effect of DCQAs was exerted on the enzyme and not the DNA. In addition to HIV-1, DCQAs also inhibited the in vitro activities of MLV integrase and truncated variants of feline immunodeficiency virus integrase, suggesting that these compounds interacted with the central core domain of integrase. The inhibition on retroviral integrases was relatively specific, and DCQAs had no effect on several other DNA-modifying enzymes and phosphoryltransferases. Kinetic analysis and dialysis experiments showed that the inhibition of integrase by DCQAs was irreversible. The inhibition did not require the presence of a divalent cation and was unaffected by preassembling integrase onto viral DNA. The results suggest that the irreversible inhibition by DCQAs on integrase is directed toward conserved amino acid residues in the central core domain during catalysis.     

Unintegrated human immunodeficiency virus type 1 DNA in chronically infected cell lines is not correlated with surface CD4 expression.

Using a polymerase chain reaction-based assay on total cell lysates, we have detected unintegrated human immunodeficiency virus type 1 (HIV-1) DNA in chronically infected T-lymphocytic (ACH-2, J1) and promyelocytic (OM-10.1) cell lines. Treatment with 3'-azido-3'-deoxythymidine (AZT) or soluble CD4 inhibited accumulation of unintegrated viral DNA about 10-fold within 72 h; removal of AZT permitted recovery to pretreatment levels within 72 h. Our results indicate that unintegrated HIV-1 DNA is unstable in these cell lines and originates from a continuous process of reinfection. OM-10.1 cells had relatively high levels of surface CD4 by flow cytometry and high levels of unintegrated viral DNA by polymerase chain reaction. ACH-2 cells had very low levels of both surface CD4 and unintegrated viral DNA. However, J1 cells, with surface CD4 below the level of detection of flow cytometry had a high level of unintegrated viral DNA similar to that of OM-10.1 cells. This implies that the number of CD4 receptors is not rate limiting for reinfection.     

Characterization of the functional domains of human foamy virus integrase using chimeric integrases

Retroviral integrases insert viral DNA into target DNA. In this process they recognize their own DNA specifically via functional domains. In order to analyze these functional domains, we constructed six chimeric integrases by swapping domains between HIV-1 and HFV integrases, and two point mutants of HFV integrase. Chimeric integrases with the central domain of HIV-1 integrase had strand transfer and disintegration activities, in agreement with the idea that the central domain determines viral DNA specificity and has catalytic activity. On the other hand, chimeric integrases with the central domain of HFV integrase did not have any enzymatic activity apart from FFH that had weak disintegration activity, suggesting that the central domain of HFV integrase was defective catalytically or structurally. However, these inactive chimeras were efficiently complemented by the point mutants (D164A and E200A) of HFV integrase, indicating that the central domain of HFV integrase possesses potential enzymatic activity but is not able to recognize viral or target DNA without the help of its homologous N-terminal and C-terminal domains.