Heather L. Wiegand

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.

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.

Exon junction complexes mediate the enhancing effect of splicing on mRNA expression

Intron-containing genes are generally expressed more effectively in human cells than are intronless versions of the same gene. We have asked whether this effect is due directly to splicing or instead reflects the action of components of the exon junction complex (EJC) that is assembled at splice junctions after splicing is completed. Here, we show that intron removal does not enhance gene expression if EJC formation is blocked. Conversely, RNA tethering of the EJC components SRm160 or RNPS1 boosts the expression of intronless mRNAs but not of spliced mRNAs. Splicing and RNPS1 tethering are shown to enhance the same steps in mRNA biogenesis and function, including mRNA 3′ end processing and translation. Together, these data argue that the EJC is primarily responsible for the positive effect of splicing on gene expression.

Foamy Virus Bet Proteins Function as Novel Inhibitors of the APOBEC3 Family of Innate Antiretroviral Defense Factors

ABSTRACT Foamy viruses are a family of complex retroviruses that establish common, productive infections in a wide range of nonhuman primates. In contrast, humans appear nonpermissive for foamy virus replication, although zoonotic infections do occur. Here we have analyzed the ability of primate and mouse APOBEC3G proteins to inhibit the infectivity of primate foamy virus (PFV) virions produced in their presence. We demonstrate that several APOBEC3 proteins can potently inhibit the infectivity of a PFV-based viral vector. This inhibition correlated with the packaging of inhibitory APOBEC3 proteins into PFV virions, due to a specific PFV Gag/APOBEC3 interaction, and resulted in the G to A hypermutation of PFV reverse transcripts. While inhibition of PFV virion infectivity by primate APOBEC3 proteins was largely relieved by coexpression of the PFV Bet protein, a cytoplasmic auxiliary protein of previously uncertain function, Bet failed to relieve inhibition caused by murine APOBEC3. PFV Bet bound to human, but not mouse, APOBEC3 proteins in coexpressing cells, and this binding correlated with the specific inhibition of their incorporation into PFV virions. Of note, both PFV Bet and a second Bet protein, derived from an African green monkey foamy virus, rescued the infectivity of Vif-deficient human immunodeficiency virus type 1 (HIV-1) virions produced in the presence of African green monkey APOBEC3G and blocked the incorporation of this host factor into HIV-1 virion particles. However, neither foamy virus Bet protein reduced APOBEC3 protein expression levels in virion producer cells. While these data identify the foamy virus Bet protein as a functional ortholog of the HIV-1 Vif auxiliary protein, they also indicate that Vif and Bet block APOBEC3 protein function by distinct mechanisms.

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.

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.

Inhibition of Alpharetrovirus Replication by a Range of Human APOBEC3 Proteins

ABSTRACT The mammalian APOBEC3 family of cytidine deaminases includes members that can act as potent inhibitors of retroviral infectivity and retrotransposon mobility. Here, we have examined whether the alpharetrovirus Rous sarcoma virus (RSV) is susceptible to inhibition by a range of human APOBEC3 proteins. We report that RSV is highly susceptible to inhibition by human APOBEC3G, APOBEC3F, and APOBEC3B and moderately susceptible to inhibition by human APOBEC3C and APOBEC3A. For all five proteins, inhibition of RSV infectivity was associated with selective virion incorporation and with C-to-T editing of the proviral DNA minus strand. In the case of APOBEC3G, editing appeared to be critical for effective inhibition. These data represent the first report of inhibition of retroviral infectivity and induction of proviral DNA editing by human APOBEC3A and reveal that alpharetroviruses, which do not normally encounter APOBEC3 proteins in their avian hosts, are susceptible to inhibition by all human APOBEC3 proteins tested. These data further suggest that the resistance of mammalian retroviruses to inhibition by the APOBEC3 proteins expressed in their normal host species is likely to have evolved subsequent to the appearance of this family of mammalian antiretroviral proteins some 35 million years ago; i.e., the base state of a naïve retrovirus is susceptibility to inhibition.

The Betaretrovirus Mason-Pfizer Monkey Virus Selectively Excludes Simian APOBEC3G from Virion Particles

ABSTRACT The APOBEC3 protein family can constitute a potent barrier to the successful infection of mammalian species by retroviruses. Therefore, any retrovirus that has evolved the ability to replicate in a given animal must have developed mechanisms that allow it to avoid or inhibit the APOBEC3 proteins expressed in that animal. Here, we demonstrate that Mason-Pfizer monkey virus (MPMV) is resistant to inhibition by the APOBEC3G protein expressed in its normal host, the rhesus macaque, but highly susceptible to inhibition by murine APOBEC3 (mA3). MPMV virion particles fail to package rhesus APOBEC3G (rA3G), and MPMV Gag binds rA3G poorly in coexpressing cells. In contrast, MPMV virions package mA3 efficiently and MPMV Gag-mA3 complexes are readily detected. Moreover, mA3, but not rA3G, partially colocalizes with MPMV Gag in the cytoplasm of coexpressing cells. Previously, we have demonstrated that murine leukemia virus also escapes inhibition by APOBEC3 proteins by avoiding virion incorporation of its cognate APOBEC3 protein, mA3, yet is inhibited by primate APOBEC3G proteins, which it packages effectively (B. P. Doehle, A. Schäfer, H. L. Wiegand, H. P. Bogerd, and B. R. Cullen, J. Virol. 79:8201-8207, 2005). The finding that two essentially unrelated beta- and gammaretroviruses use similar mechanisms to escape inhibition by the APOBEC3 proteins found in their normal host species suggests that the selective exclusion of APOBEC3 proteins from virion particles may be a general mechanism used by simple mammalian retroviruses.

Functional Differences between Human and Bovine Immunodeficiency Virus Tat Transcription Factors

ABSTRACT Transcriptional transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter element by the essential viral Tat protein requires recruitment of positive transcription elongation factor b (P-TEFb) to the viral TAR RNA target. The recruitment of P-TEFb, which has been proposed to be necessary and sufficient for activation of viral gene expression, is mediated by the highly cooperative interaction of Tat and cyclin T1, an essential component of P-TEFb, with the HIV-1 TAR element. Species, such as rodents, that encode cyclin T1 variants that are unable to support TAR binding by the Tat-cyclin T1 heterodimer are also unable to support HIV-1 Tat function. In contrast, we here demonstrate that the bovine immunodeficiency virus (BIV) Tat protein is fully able to bind to BIV TAR both in vivo and in vitro in the absence of any cellular cofactor. Nevertheless, BIV Tat can specifically recruit cyclin T1 to the BIV TAR element, and this recruitment is as essential for BIV Tat function as it is for HIV-1 Tat activity. However, because the cyclin T1 protein does not contribute to TAR binding, BIV Tat is able to function effectively in cells from several species that do not support HIV-1 Tat function. Thus, BIV Tat, while apparently dependent on the same cellular cofactor as the Tat proteins encoded by other lentiviruses, is nevertheless unique in terms of the mechanism used to recruit the BIV Tat-cyclin T1 complex to the viral LTR promoter.

Both Ran and importins have the ability to function as nuclear mRNA export factors

The Ran protein regulates nucleocytoplasmic transport mediated by the karyopherin family of nuclear transport factors. Ran is converted to the active, GTP bound form in the nucleus and then binds to a conserved domain found in all karyopherins. This interaction induces cargo binding for exportins and cargo release for importins. In either case, the Ran.GTP is then transported to the cytoplasm by the karyopherin, where it is hydrolyzed to Ran.GDP. To ask whether Ran could function as a nuclear mRNA export factor, we fused Ran to the MS2 coat protein and inserted MS2 RNA-binding sites into an unspliced cat mRNA that is normally sequestered in the nucleus. Coexpression of MS2-Ran induced cat mRNA export and CAT enzyme expression as effectively as, for example, an MS2-Rev fusion protein. MS2-Ran dependent nuclear mRNA export was reduced by inhibitors specific for Crm1, but not blocked as was seen with MS2-Rev. Consistent with the hypothesis that Crm1 is not the only karyopherin cofactor for MS2-Ran mediated mRNA export, we show that not only Crm1 but also CAS, transportin, importin beta and exportin t can all export mRNA from the nucleus when tethered via the MS2 RNA-binding domain. In contrast, two shuttling hnRNPs, hnRNP A1 and hnRNP K, proved unable to function as nuclear RNA export factors when expressed as MS2 fusions. Together, these data argue that karyopherins that normally function to transport proteins into or out of the nucleus are also capable of exporting tethered mRNA molecules.