scholarly journals Fusion Proteins Consisting of Human Immunodeficiency Virus Type 1 Integrase and the Designed Polydactyl Zinc Finger Protein E2C Direct Integration of Viral DNA into Specific Sites

2004 ◽  
Vol 78 (3) ◽  
pp. 1301-1313 ◽  
Author(s):  
Wenjie Tan ◽  
Kai Zhu ◽  
David J. Segal ◽  
Carlos F. Barbas ◽  
Samson A. Chow
Keyword(s):  
Human Immunodeficiency Virus ◽  
Binding Site ◽  
Zinc Finger ◽  
Fusion Proteins ◽  
Zinc Finger Protein ◽  
Recognition Sequence ◽  
Finger Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT In order to establish a productive infection, a retrovirus must integrate the cDNA of its RNA genome into the host cell chromosome. While this critical process makes retroviruses an attractive vector for gene delivery, the nonspecific nature of integration presents inherent hazards and variations in gene expression. One approach to alleviating the problem involves fusing retroviral integrase to a sequence-specific DNA-binding protein that targets a defined chromosomal site. We prepared proteins consisting of wild-type or truncated human immunodeficiency virus type 1 (HIV-1) integrase fused to the synthetic polydactyl zinc finger protein E2C. The purified fusion proteins bound specifically to the 18-bp E2C recognition sequence as analyzed by DNase I footprinting. The fusion proteins were catalytically active and biased integration of retroviral DNA near the E2C-binding site in vitro. The distribution was asymmetric, and the major integration hot spots were localized within a 20-bp region upstream of the C-rich strand of the E2C recognition sequence. Integration bias was not observed with target plasmids bearing a mutated E2C-binding site or when HIV-1 integrase and E2C were added to the reaction as separate proteins. The results demonstrate that the integrase-E2C fusion proteins offer an efficient approach and a versatile framework for directing the integration of retroviral DNA into a predetermined DNA site.

scholarly journals Human Immunodeficiency Virus Type 1 Incorporated with Fusion Proteins Consisting of Integrase and the Designed Polydactyl Zinc Finger Protein E2C Can Bias Integration of Viral DNA into a Predetermined Chromosomal Region in Human Cells

2006 ◽  
Vol 80 (4) ◽  
pp. 1939-1948 ◽  
Author(s):  
Wenjie Tan ◽  
Zheng Dong ◽  
Thomas A. Wilkinson ◽  
Carlos F. Barbas ◽  
Samson A. Chow
Keyword(s):  
Human Immunodeficiency Virus ◽  
Fusion Protein ◽  
Binding Site ◽  
Zinc Finger ◽  
Fusion Proteins ◽  
Zinc Finger Protein ◽  
Chromosomal Region ◽  
Finger Protein ◽  
Immunodeficiency Virus

ABSTRACT In vitro studies using fusion proteins consisting of human immunodeficiency virus type 1 integrase (IN) and a synthetic polydactyl zinc finger protein E2C, a sequence-specific DNA-binding protein, showed that integration of retroviral DNA can be biased towards a contiguous 18-bp E2C-recognition site. To determine whether the fusion protein strategy can achieve site-specific integration in vivo, viruses were prepared by cotransfection and various IN-E2C fusion proteins were packaged in trans into virions. The resulting viruses incorporated with the IN-E2C fusion proteins were functional and capable of performing integration at a level ranging from 1 to 24% of that of viruses containing wild-type (WT) IN. Two of the more infectious viruses, which contained E2C fused to either the N (E2C/IN) or to the C (IN/E2C) terminus of IN, were tested for their ability to direct integration into a unique E2C-binding site present within the 5′ untranslated region of erbB-2 gene on human chromosome 17. The copy number of proviral DNA was measured using a quantitative real-time nested-PCR assay, and the specificity of directed integration was determined by comparing the number of proviruses within the vicinity of the E2C-binding site to that in the whole genome. Viruses containing IN/E2C fusion proteins had sevenfold higher preference for integrating near the E2C-binding site than those viruses containing WT IN, whereas viruses containing E2C/IN had 10-fold higher preference. The results indicated that the IN-E2C fusion protein strategy is capable of directing integration of retroviral DNA into a predetermined chromosomal region in the human genome.

scholarly journals OTK18, a zinc-finger protein, regulates human immunodeficiency virus type 1 long terminal repeat through two distinct regulatory regions

2007 ◽  
Vol 88 (1) ◽  
pp. 236-241 ◽  
Author(s):  
Masahide Horiba ◽  
Lindsey B. Martinez ◽  
James L. Buescher ◽  
Shinji Sato ◽  
Jenae Limoges ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Long Terminal Repeat ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Regulatory Elements ◽  
Terminal Repeat ◽  
Human Macrophages ◽  
Finger Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1

It has previously been shown by our laboratory that OTK18, a human immunodeficiency virus (HIV)-inducible zinc-finger protein, reduces progeny-virion production in infected human macrophages. OTK18 antiviral activity is mediated through suppression of Tat-induced HIV-1 long terminal repeat (LTR) promoter activity. Through the use of LTR-scanning mutant vectors, the specific regions responsible for OTK18-mediated LTR suppression have been defined. Two different LTR regions were identified as potential OTK18-binding sites by an enhanced DNA–transcription factor ELISA system; the negative-regulatory element (NRE) at −255/−238 and the Ets-binding site (EBS) at −150/−139 in the LTR. In addition, deletion of the EBS in the LTR blocked OTK18-mediated LTR suppression. These data indicate that OTK18 suppresses LTR activity through two distinct regulatory elements. Spontaneous mutations in these regions might enable HIV-1 to escape from OTK18 antiretroviral activity in human macrophages.

scholarly journals Designed zinc finger protein interacting with the HIV-1 integrase recognition sequence at 2-LTR-circle junctions

2009 ◽  
Vol 18 (11) ◽  
pp. 2219-2230 ◽  
Author(s):  
Supachai Sakkhachornphop ◽  
Supat Jiranusornkul ◽  
Kanchanok Kodchakorn ◽  
Sawitree Nangola ◽  
Thira Sirisanthana ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Recognition Sequence ◽  
Finger Protein ◽  
Hiv 1

scholarly journals Trim-Cyclophilin A Fusion Proteins Can Restrict Human Immunodeficiency Virus Type 1 Infection at Two Distinct Phases in the Viral Life Cycle

2006 ◽  
Vol 80 (8) ◽  
pp. 4061-4067 ◽  
Author(s):  
Melvyn W. Yap ◽  
Mark P. Dodding ◽  
Jonathan P. Stoye
Keyword(s):  
Human Immunodeficiency Virus ◽  
Life Cycle ◽  
Fusion Protein ◽  
Human Immunodeficiency Virus Type ◽  
Fusion Proteins ◽  
Cyclophilin A ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
B30.2 Domain

ABSTRACT The Trim5α protein from several primates restricts retroviruses in a capsid (CA)-dependent manner. In owl monkeys, the B30.2 domain of Trim5 has been replaced by cyclophilin A (CypA) following a retrotransposition. Restriction of human immunodeficiency virus type 1 (HIV-1) by the resulting Trim5-CypA fusion protein depends on CA binding to CypA, suggesting both that the B30.2 domain might be involved in CA binding and that the tripartite RING motif, B-BOX, and coiled coil (RBCC) motif domain can function independently of the B30.2 domain in restriction. To investigate the potential of RBCCs from other Trims to participate in restricting HIV-1, CypA was fused to the RBCC of Trim1, Trim18, and Trim19 and tested for restriction. Despite low identity within the RBCC domain, all fusion proteins were found to restrict HIV-1 but not the nonbinding G89V mutant, indicating that the overall structure of RBCC and not its primary sequence was important for the restriction function. The critical interaction between CA and Trim-CypA appears to take place soon after viral entry. Quantitative PCR analysis of viral reverse transcriptase products revealed that the different fusion proteins block HIV-1 at two distinct stages of its life cycle, either prior to reverse transcription or just before integration. With Trim1 and Trim18, this timing is dependent on the length of the Trim component of the fusion protein. These observations suggest that restriction factor binding can have different mechanistic consequences.

scholarly journals Mutations in the U5 Region Adjacent to the Primer Binding Site Affect tRNA Cleavage by Human Immunodeficiency Virus Type 1 Reverse Transcriptase In Vivo

2007 ◽  
Vol 82 (2) ◽  
pp. 719-727 ◽  
Author(s):  
Jangsuk Oh ◽  
Mary Jane McWilliams ◽  
John G. Julias ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Rnase H ◽  
Primer Binding Site ◽  
Cleavage Specificity ◽  
Immunodeficiency Virus ◽  
Trna Primer ◽  
Hiv 1

ABSTRACT In retroviruses, the first nucleotide added to the tRNA primer defines the end of the U5 region in the right long terminal repeat, and the subsequent removal of this tRNA primer by RNase H exactly defines the U5 end of the linear double-stranded DNA. In most retroviruses, the entire tRNA is removed by RNase H cleavage at the RNA/DNA junction. However, the RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase cleaves the tRNA 1 nucleotide from the RNA/DNA junction at the U5/primer binding site (PBS) junction, which leaves an rA residue at the U5 terminus. We made sequence changes at the end of the U5 region adjacent to the PBS in HIV-1 to determine whether such changes affect the specificity of tRNA primer cleavage by RNase H. In some of the mutants, RNase H usually removed the entire tRNA, showing that the cleavage specificity was shifted by 1 nucleotide. This result suggests that the tRNA cleavage specificity of the HIV-1 RNase domain H depends on sequences in U5.

scholarly journals Potent Rescue of Human Immunodeficiency Virus Type 1 Late Domain Mutants by ALIX/AIP1 Depends on Its CHMP4 Binding Site

2007 ◽  
Vol 81 (12) ◽  
pp. 6614-6622 ◽  
Author(s):  
Yoshiko Usami ◽  
Sergei Popov ◽  
Heinrich G. Göttlinger
Keyword(s):  
Human Immunodeficiency Virus ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Budding ◽  
Rich Domain ◽  
Cellular Expression ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The release of human immunodeficiency virus type 1 (HIV-1) and of other retroviruses from certain cells requires the presence of distinct regions in Gag that have been termed late assembly (L) domains. HIV-1 harbors a PTAP-type L domain in the p6 region of Gag that engages an endosomal budding machinery through Tsg101. In addition, an auxiliary L domain near the C terminus of p6 binds to ALIX/AIP1, which functions in the same endosomal sorting pathway as Tsg101. In the present study, we show that the profound release defect of HIV-1 L domain mutants can be completely rescued by increasing the cellular expression levels of ALIX and that this rescue depends on an intact ALIX binding site in p6. Furthermore, the ability of ALIX to rescue viral budding in this system depended on two putative surface-exposed hydrophobic patches on its N-terminal Bro1 domain. One of these patches mediates the interaction between ALIX and the ESCRT-III component CHMP4B, and mutations which disrupt the interaction also abolish the activity of ALIX in viral budding. The ability of ALIX to rescue a PTAP mutant also depends on its C-terminal proline-rich domain (PRD), but not on the binding sites for Tsg101, endophilin, CIN85, or for the newly identified binding partner, CMS, within the PRD. Our data establish that ALIX can have a dramatic effect on HIV-1 release and suggest that the ability to use ALIX may allow HIV-1 to replicate in cells that express only low levels of Tsg101.

scholarly journals Evolution of the Human Immunodeficiency Virus Type 1 Long Terminal Repeat Promoter by Conversion of an NF-κB Enhancer Element into a GABP Binding Site

1999 ◽  
Vol 73 (2) ◽  
pp. 1331-1340 ◽  
Author(s):  
Koen Verhoef ◽  
Rogier W. Sanders ◽  
Veronique Fontaine ◽  
Shigetaka Kitajima ◽  
Ben Berkhout
Keyword(s):  
Human Immunodeficiency Virus ◽  
Transcription Factor ◽  
Long Terminal Repeat ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Binding Sites ◽  
Immunodeficiency Virus ◽  
Ltr Promoter ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) transcription is regulated by the viral Tat protein and cellular factors, of which the concentration and activity may depend on the cell type. Viral long terminal repeat (LTR) promoter sequences are therefore optimized to suit the specific nuclear environment of the target host cell. In long-term cultures of a Tat-defective, poorly replicating HIV-1 mutant, we selected for a faster-replicating virus with a 1-nucleotide deletion in the upstream copy of two highly conserved NF-κB binding sites. The variant enhancer sequence demonstrated a severe loss of NF-κB binding in protein binding assays. Interestingly, we observed a new binding activity that is specific for the variant NF-κB sequence and is present in the nuclear extract of unstimulated cells that lack NF-κB. These results suggest that inactivation of the NF-κB site coincides with binding of another transcription factor. Fine mapping of the sequence requirements for binding of this factor revealed a core sequence similar to that of Ets binding sites, and supershift assays with antibodies demonstrated the involvement of the GABP transcription factor. Transient transfection experiments with LTR-chloramphenicol acetyltransferase constructs indicated that the variant LTR promoter is specifically inhibited by GABP in the absence of Tat, but this promoter was dramatically more responsive to Tat than the wild-type LTR. Introduction of this GABP site into the LAI virus yielded a specific gain of fitness in SupT1 cells, which contain little NF-κB protein. These results suggest that GABP potentiates Tat-mediated activation of LTR transcription and viral replication in some cell types. Conversion of an NF-κB into a GABP binding site is likely to have occurred also during the worldwide spread of HIV-1, as we noticed the same LTR modification in subtype E isolates from Thailand. This typical LTR promoter configuration may provide these viruses with unique biological properties.

scholarly journals Inhibition of multiple phases of human immunodeficiency virus type 1 replication by a dithiane compound that attacks the conserved zinc fingers of retroviral nucleocapsid proteins.

1997 ◽  
Vol 41 (2) ◽  
pp. 419-426 ◽  
Author(s):  
W G Rice ◽  
D C Baker ◽  
C A Schaeffer ◽  
L Graham ◽  
M Bu ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Zinc Finger ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Zinc Fingers ◽  
Immunodeficiency Virus ◽  
Zinc Finger Domains ◽  
Hiv 1

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid p7 protein contains two retrovirus-type zinc finger domains that are required for multiple phases of viral replication. Chelating residues (three Cys residues and one His residue) of the domains are absolutely conserved among all strains of HIV-1 and other retroviruses, and mutations in these residues in noninfectious virions. These properties establish the zinc finger domains as logical targets for antiviral chemotherapy. Selected dithiobis benzamide (R-SS-R) compounds were previously found to inhibit HIV-1 replication by mediating an electrophilic attack on the zinc fingers. Unfortunately, reaction of these disulfide-based benzamides with reducing agents yields two monomeric structures (two R-SH structures) that can dissociated and no longer react with the zinc fingers, suggesting that in vivo reduction would inactivate the compounds. Through an extensive drug discovery program of the National Cancer Institute, a nondissociable tethered dithiane compound (1,2-dithiane-4,5-diol, 1,1-dioxide, cis; NSC 624151) has been identified. This compound specifically attacks the retroviral zinc fingers, but not other antiviral targets. The lead compound demonstrated broad antiretroviral activity, ranging from field isolates and drug-resistant strains of HIV-1 to HIV-2 and simian immunodeficiency virus. The compound directly inactivated HIV-1 virions and blocked production of infectious virus from cells harboring integrated proviral DNA. NSC 624151 provides a scaffold from which medicinal chemists can develop novel compounds for the therapeutic treatment of HIV infection.

scholarly journals The N-Terminal V3 Loop Glycan Modulates the Interaction of Clade A and B Human Immunodeficiency Virus Type 1 Envelopes with CD4 and Chemokine Receptors

2000 ◽  
Vol 74 (23) ◽  
pp. 11008-11016 ◽  
Author(s):  
Susan E. Malenbaum ◽  
David Yang ◽  
Lisa Cavacini ◽  
Marshall Posner ◽  
James Robinson ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Chemokine Receptors ◽  
V3 Loop ◽  
Clade B ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We investigated the underlying mechanism by which the highly conserved N-terminal V3 loop glycan of gp120 conferred resistance to neutralization of human immunodeficiency virus type 1 (HIV-1). We find that the presence or absence of this V3 glycan on clade A and B viruses accorded various degrees of susceptibility to neutralization by antibodies to the CD4 binding site, CD4-induced epitopes, and chemokine receptors. Our data suggest that this carbohydrate moiety on gp120 blocks access to the binding site for CD4 and modulates the chemokine receptor binding site of phenotypically diverse clade A and clade B isolates. Its presence also contributes to the masking of CD4-induced epitopes on clade B envelopes. These findings reveal a common mechanism by which diverse HIV-1 isolates escape immune recognition. Furthermore, the observation that conserved functional epitopes of HIV-1 are more exposed on V3 glycan-deficient envelope glycoproteins provides a basis for exploring the use of these envelopes as vaccine components.

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