Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
Add more filters










Database
Type of study
Language
Publication year range
1.
Acta Virol ; 63(2): 129-138, 2019.
Article in English | MEDLINE | ID: mdl-31230441

ABSTRACT

The integrated proviral genome is the major barrier to a cure for HIV-1 infection. Genome editing technologies, such as CRISPR/Cas9, may disable or remove the HIV-1 provirus by introducing DNA double strand breaks at sequence specific sites in the viral genome. Host DNA repair by the error-prone non-homologous end joining pathway generates mutagenic insertions or deletions at the break. CRISPR/Cas9 editing has been shown to reduce replication competent viral genomes in cell culture, but only a minority of possible genome editing targets have been assayed. Currently there is no map of double strand break genetic fitness for HIV-1 to inform the choice of editing targets. However, CRISPR/Cas9 genome editing makes it possible to target double strand breaks along the length of the provirus to generate a double strand break genetic fitness map. We identified all possible HIV-1 targets with different bacterial species of CRISPR/Cas9. This library of guide RNAs was evaluated for GC content and potential off-target sites in the human genome. Complexity of the library was reduced by eliminating duplicate guide RNA targets in the HIV-1 long terminal repeats and targets in the env gene. Although the HIV-1 genome is AT-rich, the S. pyogenes CRISPR/Cas9 with the proto-spacer adjacent motif NGG offers the most HIV-1 guide RNAs. This library of HIV-1 guide RNAs may be used to generate a double strand break genetic fragility map to be further applied to any genome editing technology designed for the HIV-1 provirus. Keywords: HIV-1; genome editing; CRISPR; genetic fitness; guide RNAs.


Subject(s)
Genetic Fitness , Genomic Library , HIV-1 , CRISPR-Cas Systems , Genome, Viral , HIV-1/genetics , Humans , Proviruses/genetics
2.
Mol Gen Microbiol Virol ; 34(2): 81-83, 2019.
Article in English | MEDLINE | ID: mdl-33867663

ABSTRACT

In spite of effective anti-retroviral therapy, HIV-1 infection may still lead to neurological impairment in patients. The underlying mechanism of neurodegeneration remains mysterious. HIV-1 does not infect neurons, but does infect microglia cells in the brain. It is controversial whether HIV-1 productively infects astrocytes, an abundant glial cell type in the brain. Thirty years of investigation have led to conflicting reports concerning the entry, infection, and production of progeny virions from astrocytes. New models from studies in primary human fetal astrocytes suggest phagocytosis of HIV-1 with little productive infection. The retroviral life cycle requires integration of the viral genome to the host genome. The host protein LEDGF/p75 is required for efficient HIV-1 integration. In the absence of LEDGF/p75, HIV-1 integration and infection efficiency is reduced ten fold. Differentiated astrocytes do not appear to express LEDGF/p75, which suggests these cells are disabled for efficient integration. Phagocytosis of HIV-1 virions and the lack of LEDGF/p75 expression in astrocytes suggest that this cell type is not efficiently infected in vivo.

3.
Biochem Biophys Res Commun ; 503(3): 1241-1246, 2018 09 10.
Article in English | MEDLINE | ID: mdl-30017200

ABSTRACT

Retroviruses have two essential activities: reverse transcription and integration. The viral protein integrase (IN) covalently joins the viral cDNA genome to the host DNA. Prototype foamy virus (PFV) IN has become a model of retroviral intasome structure. However, this retroviral IN has not been well-characterized biochemically. Here we compare PFV IN to previously reported HIV-1 IN activities and discover significant differences. PFV IN is able to utilize the divalent cation calcium during strand transfer while HIV-1 IN is not. HIV-1 IN was shown to completely commit to a target DNA within 1 min, while PFV IN is not fully committed after 60 min. These results suggest that PFV IN is more promiscuous compared to HIV-1 IN in terms of divalent cation and target commitment.


Subject(s)
DNA, Viral/metabolism , Integrases/metabolism , Spumavirus/enzymology , DNA, Viral/genetics , HIV-1/enzymology , Integrases/isolation & purification , Substrate Specificity
4.
EMBO J ; 20(12): 3272-81, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11406603

ABSTRACT

Early after infection, the retroviral RNA genome is reverse transcribed to generate a linear cDNA copy, then that copy is integrated into a chromosome of the host cell. We report that unintegrated viral cDNA is a substrate for the host cell non-homologous DNA end joining (NHEJ) pathway, which normally repairs cellular double-strand breaks by end ligation. NHEJ activity was found to be required for an end-ligation reaction that circularizes a portion of the unintegrated viral cDNA in infected cells. Consistent with this, the NHEJ proteins Ku70 and Ku80 were found to be bound to purified retroviral replication intermediates. Cells defective in NHEJ are known to undergo apoptosis in response to retroviral infection, a response that we show requires reverse transcription to form the cDNA genome but not subsequent integration. We propose that the double-strand ends present in unintegrated cDNA promote apoptosis, as is known to be the case for chromosomal double-strand breaks, and cDNA circularization removes the pro-apoptotic signal.


Subject(s)
Antigens, Nuclear , DNA Helicases , DNA, Viral/physiology , HIV-1/physiology , Moloney murine leukemia virus/physiology , 3T3 Cells , Animals , Apoptosis , CHO Cells , Cell Line, Transformed , Cricetinae , DNA-Binding Proteins/metabolism , HIV-1/genetics , Humans , Ku Autoantigen , Mice , Moloney murine leukemia virus/genetics , Nuclear Proteins/metabolism , Terminal Repeat Sequences , Transcription, Genetic , Virus Integration
5.
J Virol ; 74(23): 11191-200, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11070016

ABSTRACT

Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3' DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5' two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex.


Subject(s)
DNA Repair , DNA-Binding Proteins , Retroviridae/genetics , Virus Integration , Base Sequence , DNA Ligase ATP , DNA Ligases/pharmacology , DNA-Activated Protein Kinase , Integrases/pharmacology , Molecular Sequence Data , Protein Serine-Threonine Kinases/physiology , RNA-Directed DNA Polymerase/pharmacology
6.
J Infect Dis ; 180(4): 1386-9, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10479179

ABSTRACT

Bartonella henselae and B. quintana induce an unusual vascular proliferative tissue response known as bacillary angiomatosis (BA) and bacillary peliosis (BP) in some human hosts. The mechanisms of Bartonella-associated vascular proliferation remain unclear. Although host factors probably play a role, microbial coinfection has not been ruled out. Because of the vascular proliferative characteristics noted in both Kaposi's sarcoma (KS) and BA and occasional colocalization of KS and BA, the possibility was explored that KS-associated herpesvirus (KSHV) might be associated with BA lesions. Tissues with BA and positive and negative control tissues were tested for the presence of KSHV DNA by a sensitive polymerase chain reaction assay. Only 1 of 10 BA tissues, a splenic biopsy, was positive in this assay; this tissue was from a patient with concomitant KS of the skin. Thus, KSHV is probably not involved in the vascular proliferative response seen in BA-BP.


Subject(s)
Angiomatosis, Bacillary/pathology , DNA, Viral/analysis , Herpesvirus 8, Human/isolation & purification , Peliosis Hepatis/pathology , Adult , Angiomatosis, Bacillary/virology , Bartonella/isolation & purification , DNA, Bacterial/analysis , DNA, Ribosomal/analysis , Homosexuality, Male , Humans , Male , Peliosis Hepatis/microbiology , Peliosis Hepatis/virology , Polymerase Chain Reaction , RNA, Ribosomal, 18S/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...