Abstract:
:Previous studies in our laboratory showed that the RNA debranching enzyme (DBR1) is not required for early steps in HIV cDNA formation but is necessary for synthesis of intermediate and late cDNA products. To further characterize this effect, we evaluated the topology of the 5' end of the HIV-1 RNA genome during early infection with and without inhibition of DBR1 synthesis. Cells were transfected with DBR1 short hairpin RNA (shRNA) followed 48 h later by infection with an HIV-1-derived vector containing an RNase H-deficient reverse transcriptase (RT). RNA was isolated at several times postinfection and treated with various RNA-modifying enzymes prior to rapid amplification of 5' cDNA ends (5' RACE) for HIV-1 RNA and quantitative reverse transcriptase PCR (qRT-PCR). In infected cells, DBR1 knockdown inhibited detection of free HIV-1 RNA 5' ends at all time points. The difference in detection of free HIV-1 RNA 5' ends in infected DBR1 knockdown versus control cells was eliminated by in vitro incubation of infected cell RNAs with yeast or human DBR1 enzyme prior to 5' RACE and qRT-PCR. This was dependent on the 2'-5' phosphatase activity of DBR1, since it did not occur when we used the catalytically inactive DBR1(N85A) mutant. Finally, HIV-1 RNA from infected DBR1 knockdown cells was resistant to RNase R that degrades linear RNAs but not RNAs in circular or lariat-like conformations. These results provide evidence for formation of a lariat-like structure involving the 5' end of HIV-1 RNA during an early step in infection and the involvement of DBR1 in resolving it.IMPORTANCE Our findings support a new view of the early steps in HIV genome replication. We show that the HIV genomic RNA is rapidly decapped and forms a lariat-like structure after entering a cell. The lariat-like structure is subsequently resolved by the cellular enzyme DBR1, leaving a 5' phosphate. This pathway is similar to the formation and resolution of pre-mRNA intron lariats and therefore suggests that similar mechanisms may be used by HIV. Our work therefore opens a new area of investigation in HIV replication and may ultimately uncover new targets for inhibiting HIV replication and for preventing the development of AIDS.
journal_name
J Viroljournal_title
Journal of virologyauthors
Galvis AE,Fisher HE,Fan H,Camerini Ddoi
10.1128/JVI.01377-17subject
Has Abstractpub_date
2017-11-14 00:00:00issue
23eissn
0022-538Xissn
1098-5514pii
JVI.01377-17journal_volume
91pub_type
杂志文章abstract::Cytotoxic T lymphocytes (CTLs) play an important role in the immune response against respiratory syncytial virus (RSV) infection. The cell surface molecule lymphocyte function-associated antigen 1 (LFA-1) is an important contributor to CTL activation, CTL-mediated direct cell lysis, and lymphocyte migration. In an att...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/jvi.78.6.3014-3023.2004
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pub_type: 杂志文章
doi:10.1128/JVI.75.21.10015-10023.2001
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journal_title:Journal of virology
pub_type: 杂志文章
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journal_title:Journal of virology
pub_type: 杂志文章
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journal_title:Journal of virology
pub_type: 杂志文章
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journal_title:Journal of virology
pub_type: 杂志文章
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更新日期:1980-06-01 00:00:00
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pub_type: 杂志文章
doi:10.1128/JVI.70.12.8993-8996.1996
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.41.1.78-87.1982
更新日期:1982-01-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
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更新日期:1975-07-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.63.11.4913-4918.1989
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/jvi.76.15.7868-7873.2002
更新日期:2002-08-01 00:00:00
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pub_type: 杂志文章
doi:10.1128/JVI.4.5.567-573.1969
更新日期:1969-11-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.70.12.9041-9045.1996
更新日期:1996-12-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
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journal_title:Journal of virology
pub_type: 杂志文章
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.72.3.1725-1730.1998
更新日期:1998-03-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.32.3.1047-1050.1979
更新日期:1979-12-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.31.1.1-7.1979
更新日期:1979-07-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.02337-10
更新日期:2011-07-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.49.1.205-213.1984
更新日期:1984-01-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.66.7.4399-4406.1992
更新日期:1992-07-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.67.4.2083-2090.1993
更新日期:1993-04-01 00:00:00
abstract::Efforts to develop a vaccine against human immunodeficiency virus type 1 (HIV-1) are complicated by resistance of virus to neutralization. The neutralization resistance phenotype of HIV-1 has been linked to high infectivity. We studied the mechanisms determining this phenotype using clones of the T-cell-line-adapted (...
journal_title:Journal of virology
pub_type: 杂志文章
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更新日期:2003-01-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.71.1.34-41.1997
更新日期:1997-01-01 00:00:00
abstract::As nonacademic careers in science have become less and less "alternative," one field that has consistently attracted early-career virologists is public health research. The desire to make tangible contributions to public health needs and better protect the public from infectious disease often motivates the transition....
journal_title:Journal of virology
pub_type: 杂志文章,评审
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更新日期:2017-04-13 00:00:00
abstract::Adeno-associated virus (AAV) is a human parvovirus currently being developed as a vector for gene therapy applications. Because the gene transfer vector commonly retains only the AAV terminal repeats, propagation of recombinant AAV (rAAV) requires that the viral replication (Rep) and capsid (Cap) proteins be supplied ...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.71.3.1897-1905.1997
更新日期:1997-03-01 00:00:00