Bryan Cullen

Overview:

My laboratory has for sometime been interested in understanding the molecular biology of the replication cycle of the pathogenic retrovirus HIV-1. Because HIV-1 gene expression is primarily regulated by specific RNA:protein interactions, my laboratory has also become interested in the more general area of RNA sequence mediated gene regulation, including nuclear mRNA export and the phenomenon of RNA interference.

In the past, my laboratory has worked extensively on Tat, the transcriptional regulator encoded by HIV-1, and on Rev, a virally encoded nuclear mRNA export factor. Our major focus at present is a third HIV-1 regulatory protein termed Vif. In the absence of Vif, HIV-1 virions are produced normally but are largely non-infectious. It has now been demonstrated that Vif functions to block an innate human antiretroviral defense pathway that relies on a factor called APOBEC3G or CEM15. In the absence of Vif, APOBEC3G is packaged into virions and induces degradation of the HIV-1 genome during reverse transcription in target cells. Vif directly interacts with APOBEC3G and thereby allows reverse transcription to proceed unimpeded. Among other issues, we are currently interested in how APOBEC3G is packaged into virions and in how Vif blocks APOBEC3G function. The role of APOBEC3G in cellular defense against other retroviruses and retrotransposons is also an area of interest.

A second major research area in my group relates to how microRNA precursors are processed to yield mature microRNAs and how microRNAs, and the closely related small interfering RNAs, function in human cells. We were the first group to demonstrate overexpression of human microRNAs and therefore have a system in place which should allow us to make rapid progress in this area. We also remain interested in using RNA interference to determine the role of specific cellular factors in different processes, including HIV-1 replication and nuclear mRNA export.

Positions:

James B. Duke Distinguished Professor of Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Director, Center for Virology

Molecular Genetics and Microbiology
School of Medicine

Professor of Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Professor in Medicine

Medicine, Rheumatology and Immunology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1984

University of Medicine and Dentistry of New Jersey

Grants:

TM-SU Interaction in the Native HIV/SIV Env

Administered By
Surgery, Surgical Sciences
Awarded By
National Institutes of Health
Role
Collaborator
Start Date
End Date

Epitranscriptomic modification of HIV-1 transcripts: Effects of drugs of abuse

Administered By
Molecular Genetics and Microbiology
Awarded By
National Institutes of Health
Role
Principal Investigator
Start Date
End Date

Disruption of latent HIV-1 proviruses using CRISPR/Cas endonucleases

Administered By
Molecular Genetics and Microbiology
Awarded By
National Institutes of Health
Role
Principal Investigator
Start Date
End Date

The Center for HIV/AIDS-Related Structural Biology

Administered By
Molecular Genetics and Microbiology
Awarded By
University of Michigan
Role
Principal Investigator
Start Date
End Date

Effect of m6A methylation of HIV-1 transcripts on viral replication

Administered By
Molecular Genetics and Microbiology
Awarded By
National Institutes of Health
Role
Principal Investigator
Start Date
End Date

Publications:

Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique

<jats:title>Abstract</jats:title><jats:p>Pseudouridine (Ψ) is the most common non-canonical ribonucleoside present on mammalian non-coding RNAs (ncRNAs), including rRNAs, tRNAs and snRNAs, where it contributes ∼7% of the total uridine level. However, Ψ constitutes only ∼0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. Ψ residues have been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of Ψ residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based Ψ mapping technique called photo-crosslinking assisted Ψ sequencing (PA-Ψ-seq) and use it to map Ψ residues on not only multiple cellular RNAs but also on the mRNAs and genomic RNA encoded by HIV-1. We describe several 293T-derived cell lines in which human PUS enzymes previously reported to add Ψ residues to human mRNAs, specifically PUS1, PUS7 and TRUB1/PUS4, were inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of Ψ addition on cellular mRNAs to each of these three PUS enzymes, the Ψ sites present on HIV-1 transcripts remained unaffected. Moreover, loss of PUS1, PUS7 or TRUB1 function did not significantly reduce the level of Ψ residues detected on total human mRNA below the ∼0.1% level seen in wild type cells, thus implying that the PUS enzyme(s) that adds the bulk of Ψ residues to human mRNAs remains to be defined.</jats:p>
Authors
Campos, CM; Tsai, K; Courtney, DG; Bogerd, HP; Holley, CL; Cullen, BR
MLA Citation
Campos, Cecilia Martinez, et al. Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique. Cold Spring Harbor Laboratory. Crossref, doi:10.1101/2021.05.01.442255.
URI
https://scholars.duke.edu/individual/pub1481742
Source
crossref
DOI
10.1101/2021.05.01.442255

Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique.

Pseudouridine (Ψ) is the most common noncanonical ribonucleoside present on mammalian noncoding RNAs (ncRNAs), including rRNAs, tRNAs, and snRNAs, where it contributes ∼7% of the total uridine level. However, Ψ constitutes only ∼0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. Ψ residues have been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of Ψ residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based Ψ mapping technique called photo-crosslinking-assisted Ψ sequencing (PA-Ψ-seq) and use it to map Ψ residues on not only multiple cellular RNAs but also on the mRNAs and genomic RNA encoded by HIV-1. We describe 293T-derived cell lines in which human PUS enzymes previously reported to add Ψ residues to human mRNAs, specifically PUS1, PUS7, and TRUB1/PUS4, were inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of Ψ addition on cellular mRNAs to each of these three PUS enzymes, Ψ sites present on HIV-1 transcripts remained unaffected. Moreover, loss of PUS1, PUS7, or TRUB1 function did not significantly reduce the level of Ψ residues detected on total human mRNA below the ∼0.1% level seen in wild-type cells, thus implying that the PUS enzyme(s) that adds the bulk of Ψ residues to human mRNAs remains to be defined.
Authors
Martinez Campos, C; Tsai, K; Courtney, DG; Bogerd, HP; Holley, CL; Cullen, BR
MLA Citation
Martinez Campos, Cecilia, et al. “Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique.Rna, vol. 27, no. 11, Nov. 2021, pp. 1400–11. Pubmed, doi:10.1261/rna.078940.121.
URI
https://scholars.duke.edu/individual/pub1492972
PMID
34376564
Source
pubmed
Published In
Rna
Volume
27
Published Date
Start Page
1400
End Page
1411
DOI
10.1261/rna.078940.121

Epitranscriptomic addition of m6A regulates HIV-1 RNA stability and alternative splicing.

Previous work has demonstrated that the epitranscriptomic addition of m6A to viral transcripts can promote the replication and pathogenicity of a wide range of DNA and RNA viruses, including HIV-1, yet the underlying mechanisms responsible for this effect have remained unclear. It is known that m6A function is largely mediated by cellular m6A binding proteins or readers, yet how these regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m6A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is largely mediated by the nuclear m6A reader YTHDC1 and the cytoplasmic m6A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs. Unexpectedly, while YTHDF2 binding to m6A residues present on cellular mRNAs resulted in their destabilization as previously reported, YTHDF2 binding to m6A sites on HIV-1 transcripts resulted in a marked increase in the stability of these viral RNAs. Thus, YTHDF2 binding can exert diametrically opposite effects on RNA stability, depending on RNA sequence context.
Authors
Tsai, K; Bogerd, HP; Kennedy, EM; Emery, A; Swanstrom, R; Cullen, BR
MLA Citation
Tsai, Kevin, et al. “Epitranscriptomic addition of m6A regulates HIV-1 RNA stability and alternative splicing.Genes Dev, vol. 35, no. 13–14, July 2021, pp. 992–1004. Pubmed, doi:10.1101/gad.348508.121.
URI
https://scholars.duke.edu/individual/pub1485707
PMID
34140354
Source
pubmed
Published In
Genes Dev
Volume
35
Published Date
Start Page
992
End Page
1004
DOI
10.1101/gad.348508.121

Mapping RNA Modifications Using Photo-Crosslinking-Assisted Modification Sequencing.

Epitranscriptomic RNA modifications function as an important layer of gene regulation that modulates the function of RNA transcripts. A key step in understanding how RNA modifications regulate biological processes is the mapping of their locations, which is most commonly done by RNA immunoprecipitation (RIP) using modification-specific antibodies. Here, we describe the use of a photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) method, in conjunction with RNA modification-specific antibodies, to map modification sites. First described as photo-crosslinking-assisted m6A sequencing (PA-m6A-seq), this method allows the mapping of RNA modifications at a higher resolution, with lower background than traditional RIP, and can be adapted to any RNA modification for which a specific antibody is available.
Authors
Cullen, BR; Tsai, K
MLA Citation
Cullen, Bryan R., and Kevin Tsai. “Mapping RNA Modifications Using Photo-Crosslinking-Assisted Modification Sequencing.Methods Mol Biol, vol. 2298, 2021, pp. 123–34. Pubmed, doi:10.1007/978-1-0716-1374-0_8.
URI
https://scholars.duke.edu/individual/pub1484402
PMID
34085242
Source
pubmed
Published In
Methods Mol Biol
Volume
2298
Published Date
Start Page
123
End Page
134
DOI
10.1007/978-1-0716-1374-0_8

Tax Induces the Recruitment of NF-κB to Unintegrated HIV-1 DNA To Rescue Viral Gene Expression and Replication.

We previously reported that the normally essential step of integration of the HIV-1 proviral DNA intermediate into the host cell genome becomes dispensable in T cells that express the human T cell leukemia virus 1 (HTLV-1) Tax protein, a known activator of cellular NF-κB. The rescue of integrase (IN)-deficient HIV-1 replication by Tax results from the strong activation of transcription from the long terminal repeat (LTR) promoter on episomal HIV-1 DNA, an effect that is closely correlated with the recruitment of activating epigenetic marks, such as H3Ac, and depletion of repressive epigenetic marks, such as H3K9me3, from chromatinized unintegrated proviruses. In addition, activation of transcription from unintegrated HIV-1 DNA coincides with the recruitment of NF-κB to the two NF-κB binding sites found in the HIV-1 LTR enhancer. Here, we report that the recruitment of NF-κB to unintegrated viral DNA precedes, and is a prerequisite for, Tax-induced changes in epigenetic marks, so that an IN- HIV-1 mutant lacking both LTR NF-κB sites is entirely nonresponsive to Tax and fails to undergo the epigenetic changes listed above. Interestingly, we found that induction of Tax expression at 24 h postinfection, when unintegrated HIV-1 DNA is already fully repressed by inhibitory chromatin modifications, is able to effectively reverse the epigenetic silencing of that DNA and rescue viral gene expression. Finally, we report that heterologous promoters introduced into IN-deficient HIV-1-based vectors are transcriptionally active even in the absence of Tax and do not increase their activity when the HIV-1 promoter and enhancer, located in the LTR U3 region, are deleted, as has been recently proposed. IMPORTANCE Integrase-deficient expression vectors based on HIV-1 are becoming increasingly popular as tools for gene therapy in vivo due to their inability to cause insertional mutagenesis. However, many IN- lentiviral vectors are able to achieve only low levels of gene expression, and methods to increase this low level have not been extensively explored. Here, we analyzed how the HTLV-1 Tax protein is able to rescue the replication of IN- HIV-1 in T cells, and we describe IN- lentiviral vectors, lacking any inserted origin of replication, that are able to express a heterologous gene effectively.
Authors
Irwan, ID; Cullen, BR
MLA Citation
Irwan, Ishak D., and Bryan R. Cullen. “Tax Induces the Recruitment of NF-κB to Unintegrated HIV-1 DNA To Rescue Viral Gene Expression and Replication.J Virol, vol. 95, no. 13, June 2021, p. e0028521. Pubmed, doi:10.1128/JVI.00285-21.
URI
https://scholars.duke.edu/individual/pub1480326
PMID
33883218
Source
pubmed
Published In
J Virol
Volume
95
Published Date
Start Page
e0028521
DOI
10.1128/JVI.00285-21