Hal P. Bogerd

A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins

The HIV‐1 Vif protein suppresses the inhibition of viral replication caused by the human antiretroviral factor APOBEC3G. As a result, HIV‐1 mutants that do not express the Vif protein are replication incompetent in ‘nonpermissive’ cells, such as primary T cells and the T‐cell line CEM, that express APOBEC3G. In contrast, Vif‐defective HIV‐1 replicates effectively in ‘permissive’ cell lines, such as a derivative of CEM termed CEM‐SS, that do not express APOBEC3G. Here, we show that a second human protein, APOBEC3F, is also specifically packaged into HIV‐1 virions and inhibits their infectivity. APOBEC3F binds the HIV‐1 Vif protein specifically and Vif suppresses both the inhibition of virus infectivity caused by APOBEC3F and virion incorporation of APOBEC3F. Surprisingly, APOBEC3F and APOBEC3G are extensively coexpressed in nonpermissive human cells, including primary lymphocytes and the cell line CEM, where they form heterodimers. In contrast, both genes are quiescent in the permissive CEM derivative CEM‐SS. Together, these data argue that HIV‐1 Vif has evolved to suppress at least two distinct but related human antiretroviral DNA‐editing enzymes.

Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay.

The Rex protein of human T-cell leukemia virus type 1, like the functionally equivalent Rev protein of human immunodeficiency virus type 1, contains a leucine-rich activation domain that specifically interacts with the human nucleoporin-like Rab/hRIP cofactor. Here, this Rex sequence is shown to function also as a protein nuclear export signal (NES). Rex sequence libraries containing randomized forms of the activation domain/NES were screened for retention of the ability to bind Rab/hRIP by using the yeast two-hybrid assay. While the selected sequences differed widely in primary sequence, all were functional as Rex activation domains. In contrast, randomized sequences that failed to bind Rab/hRIP lacked Rex activity. The selected sequences included one with homology to the Rev activation domain/NES and a second that was similar to the NES found in the cellular protein kinase inhibitor alpha. A highly variant, yet fully active, activation domain sequence selected on the basis of Rab/hRIP binding retained full NES function even though this sequence preserved only a single leucine residue. In contrast, nonfunctional activation domain mutants that were unable to bind Rab/hRIP had also lost NES function. These data demonstrate that NES activity is a defining characteristic of the activation domains found in the Rev/Rex class of retroviral regulatory proteins and strongly support the hypothesis that the Rab/hRIP cofactor plays a critical role in mediating the biological activity of these NESs. In addition, these data suggest a consensus sequence for NESs of the Rev/Rex class.

Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins

HIV-1 Rev is the prototype of a class of retroviral regulatory proteins that induce the sequence-specific nuclear export of target RNAs. This function requires the Rev activation domain, which is believed to bind an essential cellular cofactor. We report the identification of a novel human gene product that binds to not only the HIV-1 Rev activation domain in vitro and in vivo but also to functionally equivalent domains in other Rev and Rex proteins. The Rev/Rex activation domain-binding (Rab) protein occupies a binding site on HIV-1 Rev that precisely matches that predicted by genetic analysis. Rab binds the Rev activation domain when Rev is assembled onto its RNA target and can significantly enhance Rev activity when overexpressed. We conclude that Rab is the predicted activation domain-specific cofactor for the Rev/Rex class of RNA export factors.

Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species specificity of HIV-1 Tat.

Human cyclin T1 (hCycT1), a major subunit of the essential elongation factor P‐TEFb, has been proposed to act as a cofactor for human immunodeficiency virus type 1 (HIV‐1) Tat. Here, we show that murine cyclin T1 (mCycT1) binds the activation domain of HIV‐1 Tat but, unlike hCycT1, cannot mediate Tat function because it cannot be recruited efficiently to TAR. In fact, overexpression of mCycT1, but not hCycT1, specifically inhibits Tat–TAR function in human cells. This discordant phenotype results from a single amino acid difference between hCycT1 and mCycT1, a tyrosine in place of a cysteine at residue 261. These data indicate that the ability of Tat to recruit CycT1/P‐TEFb to TAR determines the species restriction of HIV‐1 Tat function in murine cells and therefore demonstrate that this recruitment is a critical function of the Tat protein.

APOBEC3A and APOBEC3B are potent inhibitors of LTR-retrotransposon function in human cells

While the ability of APOBEC3G to reduce the replication of a range of exogenous retroviruses is now well established, recent evidence has suggested that APOBEC3G can also inhibit the replication of endogenous retrotransposons that bear long terminal repeats. Here, we extend this earlier work by showing that two other members of the human APOBEC3 protein family, APOBEC3B and APOBEC3A, can reduce retrotransposition by the intracisternal A-particle (IAP) retrotransposon in human cells by 20-fold to up to 100-fold, respectively. This compares to an ∼4-fold inhibition in IAP retrotransposition induced by APOBEC3G. While both APOBEC3G and APOBEC3B specifically interact with the IAP Gag protein in co-expressing cells, and induce extensive editing of IAP reverse transcripts, APOBEC3A fails to package detectably into IAP virus-like particles and does not edit IAP reverse transcripts. These data, which identify human APOBEC3A as a highly potent inhibitor of LTR-retrotransposon function, are the first to ascribe a biological activity to APOBEC3A. Moreover, these results argue that APOBEC3A inhibits IAP retrotransposition via a novel mechanism that is distinct from, and in this case more effective than, the DNA editing mechanism characteristic of APOBEC3G and APOBEC3B.

Differential Sensitivity of Murine Leukemia Virus to APOBEC3-Mediated Inhibition Is Governed by Virion Exclusion

ABSTRACT While members of the APOBEC3 family of human intrinsic resistance factors are able to restrict the replication of Vif-deficient forms of human immunodeficiency virus type 1 (HIV-1), they are unable to block replication of wild-type HIV-1 due to the action of Vif, which induces their degradation. In contrast, HIV-1 Vif is unable to block inhibition mediated by APOBEC3 proteins expressed by several heterologous species, including mice. Here, we have asked whether the simple retrovirus murine leukemia virus (MLV) is sensitive to restriction by the cognate murine or heterologous, human APOBEC3 proteins. We demonstrate that MLV is highly sensitive to inhibition by human APOBEC3G and APOBEC3B but resistant to inhibition by murine APOBEC3 or by other human APOBEC3 proteins, including APOBEC3F. This sensitivity fully correlates with the ability of these proteins to be packaged into MLV virion particles: i.e., human APOBEC3G and APOBEC3B are packaged while murine APOBEC3 and human APOBEC3F are excluded. Moreover, this packaging in turn correlates with the differential ability of these APOBEC3 proteins to bind MLV Gag. Together, these data suggest that MLV Gag has evolved to avoid binding, and hence virion packaging, of the cognate murine APOBEC3 protein but that MLV infectivity is still restricted by certain heterologous APOBEC3 proteins that retain this ability. Moreover, these results suggest that APOBEC3 proteins may help prevent the zoonotic infection of humans by simple retroviruses and provide a mechanism for how simple retroviruses can avoid inhibition by APOBEC3 family members.

Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease

Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in infected hepatocytes. Using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs (sgRNAs) specific for HBV, we observed effective inhibition of HBV DNA production in in vitro models of both chronic and de novo HBV infection. Cas9/sgRNA combinations specific for HBV reduced total viral DNA levels by up to ~1000-fold and HBV cccDNA levels by up to ~10-fold and also mutationally inactivated the majority of the residual viral DNA. Together, these data provide proof of principle for the hypothesis that CRISPR/Cas systems have the potential to serve as effective tools for the depletion of the cccDNA pool in chronically HBV infected individuals.

A nuclear role for the Fragile X mental retardation protein.

Fragile X syndrome results from lack of expression of a functional form of Fragile X mental retardation protein (FMRP), a cytoplasmic RNA‐binding protein of uncertain function. Here, we report that FMRP contains a nuclear export signal (NES) that is similar to the NES recently identified in the Rev regulatory protein of human immunodeficiency virus type 1 (HIV‐1). Mutation of this FMRP NES results in mis‐localization of FMRP to the cell nucleus. The FMRP NES is encoded within exon 14 of the FMR1 gene, thus explaining the aberrant nuclear localization of a natural isoform of FMRP that lacks this exon. The NES of FMRP can substitute fully for the Rev NES in mediating Rev‐dependent nuclear RNA export and specifically binds a nucleoporin‐like cellular cofactor that has been shown to mediate Rev NES function. Together, these findings demonstrate that the normal function of FMRP involves entry into the nucleus followed by export via a pathway that is identical to the one utilized by HIV‐1 Rev. In addition, these data raise the possibility that FMRP could play a role in mediating the nuclear export of its currently undefined cellular RNA target(s).

Formation of Tap/NXT1 Heterodimers Activates Tap-Dependent Nuclear mRNA Export by Enhancing Recruitment to Nuclear Pore Complexes

ABSTRACT The Tap protein has been shown to activate the nuclear export of mRNA species bearing retroviral constitutive transport elements and is also believed to play an essential role in the sequence nonspecific export of cellular mRNAs. However, it has remained unclear how Tap activity is regulated in vivo. Here, we report that the small NXT1/p15-1 protein functions as a critical cofactor for Tap-mediated mRNA export in both human and invertebrate cells. In the absence of NXT1 binding, the Tap protein is unable to effectively interact with components of the nuclear pore complex and both Tap nucleocytoplasmic shuttling and the nuclear export of mRNA molecules tethered to Tap are therefore severely attenuated. Formation of a Tap/NXT1 heterodimer enhances nucleoporin binding both in vitro and in vivo and induces the formation of a Tap/NXT1/nucleoporin ternary complex that is likely to be a key intermediate in the process of nuclear mRNA export. The critical importance of NXT1 for the nuclear export of poly(A)+ RNA is emphasized by the finding that specific inhibition of the expression of the Drosophila homolog of human NXT1, by using RNA interference, results in the nuclear accumulation of poly(A)+ RNA in cultured insect cells. These data suggest that NXT1 may act as a molecular switch that regulates the ability of Tap to mediate nuclear mRNA export by controlling the interaction of Tap with components of the nuclear pore.

Mutational analysis of the transcription activation domain of RelA: identification of a highly synergistic minimal acidic activation module.

The potent C-terminal activation domain of the RelA (p65) subunit of the cellular transcription factor NF-kappa B is shown to contain several discrete acidic activation modules. These short, approximately 11-amino-acid modules were able to give rise to only a low level of transcription activation when fused to the GAL4 DNA-binding domain as monomers. However, dimers and higher-order multimers activated the transcription of minimal promoter elements as effectively as the full-length RelA or VP16 activation domain. Therefore, this 11-amino-acid RelA-derived acidic module appears to contain all of the sequence information required to fully activate a target promoter element as long as it is presented in a form that permits functional synergy. Critical primary sequence requirements for acidic activation module function included a core phenylalanine residue and flanking bulky hydrophobic residues. Overall negative charge was necessary but not sufficient for function. While dimeric forms of the 11-amino-acid acidic activation module bound to either TFIIB or TATA-binding protein efficiently in vitro, a similarly charged peptide lacking the core phenylalanine residue failed to interact. Overall, these data demonstrate that the biological activity of the RelA activation domain is dependent on acidic activator sequences that are closely comparable to those detected in the activation domain of the viral VP16 regulatory protein. We hypothesize that the ability of these acidic activators to specifically interact with multiple components of the transcription initiation complex likely underlies the dramatic functional synergy exhibited by this class of activation domains in vivo.