Eri Miyagi

The Human Immunodeficiency Virus Type 1 Vif Protein Reduces Intracellular Expression and Inhibits Packaging of APOBEC3G (CEM15), a Cellular Inhibitor of Virus Infectivity

ABSTRACT Replication of human immunodeficiency virus type 1 (HIV-1) in most primary cells and some immortalized T-cell lines depends on the activity of the viral infectivity factor (Vif). Vif has the ability to counteract a cellular inhibitor, recently identified as CEM15, that blocks infectivity of Vif-defective HIV-1 variants. CEM15 is identical to APOBEC3G and belongs to a family of proteins involved in RNA and DNA deamination. We cloned APOBEC3G from a human kidney cDNA library and confirmed that the protein acts as a potent inhibitor of HIV replication and is sensitive to the activity of Vif. We found that wild-type Vif inhibits packaging of APOBEC3G into virus particles in a dose-dependent manner. In contrast, biologically inactive variants carrying in-frame deletions in various regions of Vif or mutation of two highly conserved cysteine residues did not inhibit packaging of APOBEC3G. Interestingly, expression of APOBEC3G in the presence of wild-type Vif not only affected viral packaging but also reduced its intracellular expression level. This effect was not seen in the presence of biologically inactive Vif variants. Pulse-chase analyses did not reveal a significant difference in the stability of APOBEC3G in the presence or absence of Vif. However, in the presence of Vif, the rate of synthesis of APOBEC3G was slightly reduced. The reduction of intracellular APOBEC3G in the presence of Vif does not fully account for the Vif-induced reduction of virus-associated APOBEC3G, suggesting that Vif may function at several levels to prevent packaging of APOBEC3G into virus particles.

Vpu enhances HIV-1 virus release in the absence of Bst-2 cell surface down-modulation and intracellular depletion

HIV-1 Vpu enhances the release of virions from infected cells. Recent work identified Bst-2/CD317/tetherin as a host factor whose inhibitory activity on viral release is counteracted by Vpu. A current working model proposes that Bst-2 inhibits virus release by tethering viral particles to the cell surface. Here, we analyzed endogenous Bst-2 with respect to its effect on virus release from HeLa cells, T cells, and macrophages. We noted significant cell type-dependent variation in Bst-2 expression. Vpu caused a reduction in Bst-2 expression in transfected HeLa cells and long-term infected macrophages. However, Vpu expression did not result in cell surface down-modulation of Bst-2 or a reduction in intracellular Bst-2 expression in CEMx174 or H9 cells, yet virus replication in these cells was Vpu-responsive. Surprisingly, Bst-2 was undetectable in cell-free virions that were recovered from the surface of HeLa cells by physical shearing, suggesting that a tethering model may not explain all of the functional properties of Bst-2. Taken together we conclude that enhancement of virus release by Vpu does not, at least in CEMx174 and H9 cells, require cell surface down-modulation or intracellular depletion of Bst-2, nor does it entail exclusion of Bst-2 from viral particles.

Viral RNA is required for the association of APOBEC3G with human immunodeficiency virus type 1 nucleoprotein complexes.

ABSTRACT APOBEC3G (APO3G) is a host cytidine deaminase that is incorporated into human immunodeficiency virus type 1 (HIV-1) particles. We report here that viral RNA promotes stable association of APO3G with HIV-1 nucleoprotein complexes (NPC). A target sequence located within the 5′-untranslated region of the HIV-1 RNA was identified to be necessary and sufficient for efficient APO3G packaging. Fine mapping revealed a sequence normally involved in viral genomic RNA dimerization and Gag binding to be important for APO3G packaging and association with viral NPC. Our data suggest that packaging of APO3G into HIV-1 NPC is enhanced by viral RNA.

Enzymatically Active APOBEC3G Is Required for Efficient Inhibition of Human Immunodeficiency Virus Type 1

ABSTRACT APOBEC3G (APO3G) is a cellular cytidine deaminase with potent antiviral activity. Initial studies of the function of APO3G demonstrated extensive mutation of the viral genome, suggesting a model in which APO3Gs antiviral activity is due to hypermutation of the viral genome. Recent studies, however, found that deaminase-defective APO3G mutants transiently expressed in virus-producing cells exhibited significant antiviral activity, suggesting that the antiviral activity of APO3G could be dissociated from its deaminase activity. To directly compare the antiviral activities of wild-type (wt) and deaminase-defective APO3G, we used two approaches: (i) we titrated wt and deaminase-defective APO3G in transient-transfection studies to achieve similar levels of virus-associated APO3G and (ii) we constructed stable cell lines and selected clones expressing comparable amounts of wt and deaminase-defective APO3G. Viruses produced under these conditions were tested for viral infectivity. The results from the two approaches were consistent and suggested that the antiviral activity of deaminase-defective APO3G was significantly lower than that of wt APO3G. We conclude that efficient inhibition of vif-defective human immunodeficiency virus type 1 requires catalytically active APO3G.

The formation of cysteine-linked dimers of BST-2/tetherin is important for inhibition of HIV-1 virus release but not for sensitivity to Vpu

BackgroundThe Human Immunodeficiency virus type 1 (HIV-1) Vpu protein enhances virus release from infected cells and induces proteasomal degradation of CD4. Recent work identified BST-2/CD317 as a host factor that inhibits HIV-1 virus release in a Vpu sensitive manner. A current working model proposes that BST-2 inhibits virus release by tethering viral particles to the cell surface thereby triggering their subsequent endocytosis.ResultsHere we defined structural properties of BST-2 required for inhibition of virus release and for sensitivity to Vpu. We found that BST-2 is modified by N-linked glycosylation at two sites in the extracellular domain. However, N-linked glycosylation was not important for inhibition of HIV-1 virus release nor did it affect surface expression or sensitivity to Vpu. Rodent BST-2 was previously found to form cysteine-linked dimers. Analysis of single, double, or triple cysteine mutants revealed that any one of three cysteine residues present in the BST-2 extracellular domain was sufficient for BST-2 dimerization, for inhibition of virus release, and sensitivity to Vpu. In contrast, BST-2 lacking all three cysteines in its ectodomain was unable to inhibit release of wild type or Vpu-deficient HIV-1 virions. This defect was not caused by a gross defect in BST-2 trafficking as the mutant protein was expressed at the cell surface of transfected 293T cells and was down-modulated by Vpu similar to wild type BST-2.ConclusionWhile BST-2 glycosylation was functionally irrelevant, formation of cysteine-linked dimers appeared to be important for inhibition of virus release. However lack of dimerization did not prevent surface expression or Vpu sensitivity of BST-2, suggesting Vpu sensitivity and inhibition of virus release are separable properties of BST-2.

Expression of thyroid transcription factor-1 in normal and neoplastic lung tissues.

The expression of thyroid transcription factor-1 in normal and neoplastic tissues and cell lines of the human lung was investigated using immunohistochemistry and in situ hybridization in conjunction with reverse transcription polymerase chain reaction. In normal lung tissues, immunoproducts of thyroid transcription factor-1 were observed in the nuclei of alveolar cells and bronchiolar cells. Interestingly, in distal bronchioles, immunohistochemistry and in situ hybridization revealed that thyroid transcription factor-1 was present not only in nonciliated cells (Clara cells) but also in ciliated cells and basal cells. In neoplastic tissues, thyroid transcription factor-1 was demonstrated in adenocarcinomas and small cell lung carcinomas with high frequency: 96% and 89% of cases, respectively. Thyroid transcription factor-1 was not detected in squamous cell carcinomas and large cell carcinomas. The strong immunoreactivity of thyroid transcription factor-1 or simultaneous expressions of thyroid transcription factor-1 and surfactant protein A tended to correlate with the differentiation phenotypes in adenocarcinomas; they were more frequently present in the well-differentiated type than were moderately and/or poorly differentiated types. By reverse transcription polymerase chain reaction, expression of thyroid transcription factor-1 messenger RNA was observed in squamous cell carcinomas in addition to in adenocarcinomas and small cell lung carcinomas, and this finding was confirmed in the cell lines from squamous cell carcinomas. Only one case of 99 adenocarcinomas that originated in various organs other than lung and thyroid immunohistochemically expressed thyroid transcription factor-1. Our results suggest that thyroid transcription factor-1 can play an important role for the maintenance and/or differentiation process in bronchiolar and alveolar cells. Thyroid transcription factor-1 expression associates with histologic types and/or differentiation of lung cancers and can be a valuable marker for the better understanding of their biological nature and pathological behavior.

Human Immunodeficiency Virus Type 1 Vif Inhibits Packaging and Antiviral Activity of a Degradation-Resistant APOBEC3G Variant

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Vif counteracts the antiviral activity of the human cytidine deaminase APOBEC3G (APO3G) by inhibiting its incorporation into virions. This has been attributed to the Vif-induced degradation of APO3G by cytoplasmic proteasomes. We recently demonstrated that although APO3G has a natural tendency to form RNA-dependent homo-multimers, multimerization was not essential for encapsidation into HIV-1 virions or antiviral activity. We now demonstrate that a multimerization-defective APO3G variant (APO3G C97A) is able to assemble into RNase-sensitive high-molecular-mass (HMM) complexes, suggesting that homo-multimerization of APO3G and assembly into HMM complexes are unrelated RNA-dependent processes. Interestingly, APO3G C97A was highly resistant to Vif-induced degradation even though the two proteins were found to interact in coimmunoprecipitation experiments and exhibited partial colocalization in transfected HeLa cells. Surprisingly, encapsidation and antiviral activity of APO3G C97A were both inhibited by Vif despite resistance to degradation. These results demonstrate that targeting of APO3G to proteasome degradation and interference with viral encapsidation are distinct functional properties of Vif.

Production of infectious human immunodeficiency virus type 1 does not require depletion of APOBEC3G from virus-producing cells

BackgroundThe human immunodeficiency virus Vif protein overcomes the inhibitory activity of the APOBEC3G cytidine deaminase by prohibiting its packaging into virions. Inhibition of APOBEC3G encapsidation is paralleled by a reduction of its intracellular level presumably caused by the Vif-induced proteasome-dependent degradation of APOBEC3G.ResultsIn this report we employed confocal microscopy to study the effects of Vif on the expression of APOBEC3G on a single cell level. HeLa cells dually transfected with Vif and APOBEC3G expression vectors revealed efficient co-expression of the two proteins. Under optimal staining conditions approximately 80% of the transfected cells scored double-positive for Vif and APOBEC3G. However, the proportion of double-positive cells observed in identical cultures varied dependent on the fixation protocol and on the choice of antibodies used ranging from as low as 40% to as high as 80% of transfected cells. Importantly, single-positive cells expressing either Vif or APOBEC3G were observed both with wild type Vif and a biologically inactive Vif variant. Thus, the lack of APOBEC3G in some Vif-expressing cells cannot be attributed to Vif-induced degradation of APOBEC3G. These findings are consistent with our results from immunoblot analyses that revealed only moderate effects of Vif on the APOBEC3G steady state levels. Of note, viruses produced under such conditions were fully infectious demonstrating that the Vif protein used in our analyses was both functional and expressed at saturating levels.ConclusionsOur results suggest that Vif and APOBEC3G can be efficiently co-expressed. Thus, depletion of APOBEC3G from Vif expressing cells as suggested previously is not a universal property of Vif and thus is not imperative for the production of infectious virions.

Expression of thyroid transcription factor-1 (TTF-1) in human C cells and medullary thyroid carcinomas

Thyroid transcription factor-1 (TTF-1) has been known to regulate the transcriptional activity of thyroid-specific genes in thyroid follicular cells. We recently identified TTF-1 mRNA expression in rat thyroid C cells. The current study was undertaken to elucidate how TTF-1 is expressed in human C cells and medullary thyroid carcinomas (MTCs), and how this expression influences the functions and clinical behavior of these cells. By immunohistochemistry, the nuclei of normal and hyperplastic C cells distinctively reacted with antibody against TTF-1, whereas the immunostaining intensity in C cells was rather weak and heterogeneous in comparison with that in follicular cells. Identical TTF-1 immunoreactivity was observed in all 15 MTC specimens examined. The reaction intensity did not depend on tumor patterns or cell features. In nonisotopic in situ hybridization, an antisense riboprobe clearly hybridized the cytoplasms of C cells and MTC cells, which concurrently showed immunohistochemical positivity for calcitonin. Northern blot analysis indicated a marked hybridization with TTF-1 mRNA of approximately 2.3 kb in an MTC specimen. Furthermore, the presence of TTF-1 mRNA was confirmed by reverse transcription polymerase chain reaction (RT-PCR) in the human MTC cell line, TT. Our results suggest that human thyroid C cells and MTC cells express TTF-1 in connection with their functional ability. Therefore, TTF-1 expression can be a functional marker not only for follicular cells and follicular cell tumors but also for C cells and medullary C cell carcinomas.

Monomeric APOBEC3G Is Catalytically Active and Has Antiviral Activity

ABSTRACT APOBEC3G (APO3G) is a cytidine deaminase that restricts replication of vif-defective human immunodeficiency virus type 1 (HIV-1). Like other members of the cellular deaminase family, APO3G has the propensity to form homo-multimers. In the current study, we investigated the functional determinants for multimerization of human APO3G and studied the role of APO3G multimerization for catalytic activity, virus encapsidation, and antiviral activity. We found that human APO3G is capable of forming multimeric complexes in transfected HeLa cells. Interestingly, multimerization of APO3G was exquisitely sensitive to RNase treatment, suggesting that interaction of APO3G subunits is facilitated or stabilized by an RNA bridge. Mutation of a conserved cysteine residue (C97) that is part of an N-terminal zinc-finger motif in APO3G abolished multimerization of APO3G; however, the C97 mutation inhibited neither in vitro deaminase activity nor antiviral function of APO3G. These results suggest that monomeric APO3G is both catalytically active and has antiviral activity. Interference studies employing either catalytically inactive or packaging-incompetent APO3G variants suggest that wild-type APO3G is packaged into HIV-1 particles in monomeric form. These results provide novel insights into the catalytic function and antiviral property of APO3G and demonstrate an important role for C97 in the RNA-dependent multimerization of this protein.