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The efficiency of a virus to establish its infection in host cells varies broadly among viruses. It remains unclear if there is a key step in this process that controls viral infectivity. To address this question, we use single-particle tracking and Brownian dynamics simulation to examine human immunodeficiency virus type 1 (HIV-1) infection in cell culture. We find that the frequency of viral-cell encounters is consistent with diffusion-limited interactions. However, even under the most favorable conditions, only 1% of the viruses can become immobilized on cell surface and subsequently enter the cell. This is a result of weak interaction between viral surface gp120 and CD4 receptor, which is insufficient to form a stable complex the majority of the time. We provide the first direct quantitation for efficiencies of these events relevant to measured HIV-1 infectivity and demonstrate that immobilization on host cell surface post-virion-diffusion is the key step in viral infection. Variation of its probability controls the efficiency of a virus to infect its host cells. These results explain the low infectivity of cell-free HIV-1 in vitro and offer a potential rationale for the pervasive high efficiency of cell-to-cell transmission of animal viruses.

Wan-Xiang Zheng,* Fei Yan,* Qin Xue, Guo-Jun Wu, Wei-Jun Qin, Fu-Li Wang, Jun Qin, Chun-Juan Tian, Jian-Lin Yuan Department of Urology, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China *These authors contributed equally tothis work Background: We analyzed the expression of heme oxygenase-1 (HO-1) in patients undergoing radical nephrectomy for advanced clear cell renal cell carcinoma (CC-RCC) and evaluated the effects of the targeted therapies treated with sorafenib and sunitinib. Methods: Expression of HO-1 in cancer tissue from 66 patients was measured by immunohistochemical staining. The patients received either oral sorafenib (n=40) or oral sunitinib (n=26) within 4 weeks after nephrectomy and were followed up long term to determine the tumor response and prognosis. Results: Our current study revealed a high HO-1 expression level in 57.6% (38/66) of patients and a low HO-1 expression level in 42.4% (28/66) of patients with CC-RCC. The study also revealed that patients with high HO-1 expression did not have a higher objective response rate (2.6% versus 53.6%, P

Although radical nephrectomy alone is widely accepted as the standard of care in localized treatment for renal cell carcinoma (RCC), it is not sufficient for the treatment of metastatic RCC (mRCC), which invariably leads to an unfavorable outcome despite the use of multiple therapies. Currently, sequential targeted agents are recommended for the management of mRCC, but the optimal drug sequence is still debated. This case was a 57-year-old man with clear-cell mRCC who received multiple therapies following his first operation in 2003 and has survived for over 10 years with a satisfactory quality of life. The treatments given included several surgeries, immunotherapy, and sequentially administered sorafenib, sunitinib, and everolimus regimens. In the course of mRCC treatment, well-planned surgeries, effective sequential targeted therapies and close follow-up are all of great importance for optimal management and a satisfactory outcome.

Although Ashkin and Dziedzic first demonstrated optical trapping of individual tobacco mosaic viruses in suspension as early as 1987, this pioneering work has not been followed up only until recently. Using human immunodeficiency virus type 1 (HIV-1) as a model virus, we have recently demonstrated that a single HIV-1 virion can be stabled trapped, manipulated and measured in physiological media with high precision. The capability to optically trap a single virion in suspension not only allows us to determine, for the first time, the refractive index of a single virus with high precision, but also quantitate the heterogeneity among individual virions with single-molecule resolution, the results of which shed light on the molecular mechanisms of virion infectivity. Here we report the further development of a set of microscopic techniques to physically deliver a single HIV-1 virion to a single host cell in solution. Combined with simultaneous epifluorescence imaging, the attachment and dissociation events of individual manipulated virions on host cell surface can be measured and the results help us understand the role of diffusion in mediating viral attachment to host cells. The establishment of these techniques opens up new ways for investigation of a wide range of virion-cell interactions, and should be applicable for study of B cell interactions with particulate antigens such as viruses.

Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) proteins have been classified as either Z1- or Z2-type cytidine deaminases on the basis of phylogenetic analysis of their catalytic domains. Despite the identification of a number of Z1-type domain-containing cytidine deaminases, only one copy of Z2-type cytidine deaminase has been detected in each of the mammalian species evaluated thus far. Z1-type human APOBEC3 proteins are known to exhibit broad activities against diverse retroelements. However, the potential role of the only human Z2-type cytidine deaminase, APOBEC3H (A3H), in the restriction of retroelements has not yet been fully characterized. Here, we demonstrate that human A3H is a potent inhibitor of non-LTR LINE-1 transposition. Interestingly, it was also as efficient as A3G in inhibiting Alu retrotransposition, despite its poor association with Alu RNA. We have further demonstrated, for the first time, that human APOBEC3DE is also a potent inhibitor of Alu retrotransposition. Variants of A3H have divergent antiviral activities against HIV-1-Vif-deficient viruses. Unlike the anti-HIV-1 cytidine deaminases A3G and A3F, A3H is moderately regulated by interferons. These observations suggest that human Z2-type cytidine deaminase A3H variants have varying intrinsic abilities to restrict retroelements and that various APOBEC3 proteins may have evolved distinct inhibitory mechanisms against retroelements.—Tan, L., Sarkis, P. T. N., Wang, T., Tian, C., Yu, X.-F. Sole copy of Z2-type human cytidine deaminase APOBEC3H has inhibitory activity against retrotransposons and HIV-1.

Human APOBEC3G and other APOBEC3 cytidine deaminases inhibit a variety of retroviruses, including Vif-deficient HIV-1. These host proteins are packaged into viral particles and inhibit the replication of virus in new target cells. A3G and A3F are known to be efficiently packaged into HIV-1 virions by binding to 7SL RNA through the Gag NC domain; however, the packaging mechanisms of other APOBEC3 proteins are poorly defined. We have now demonstrated that APOBEC3C (A3C) can be efficiently packaged into HIV-1 virions that are deficient for viral genomic RNA. Inhibition of the encapsidation of 7SL RNA into HIV-1 virions blocked the packaging of A3G, but not A3C. While the NC domain is required for efficient packaging of A3G, deletion of this domain had little effect on A3C packaging into HIV-1 Gag particles. A3C interacted with HIV-1 Gag which was MA domain-dependent and RNA-dependent. Deletion of the MA domain of HIV-1 Gag inhibited A3C but not A3G packaging into HIV-1 Gag particles. Thus, A3G and A3C have evolved to use distinct mechanisms for targeting retroviruses.

Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3F (APOBEC3F, or A3F), like APOBEC3G, has broad antiviral activity against diverse retroelements, including Vif-deficient human immunodeficiency virus (HIV)-1. Its antiviral functions are known to rely on its virion encapsidation and be suppressed by HIV-1 Vif, which recruits Cullin5-based E3 ubiquitin ligases. However, the factors that mediate A3F virion packaging have not yet been identified. In this study, we demonstrate that A3F specifically interacts with cellular signal recognition particle (SRP) RNA (7SL RNA), which is selectively packaged into HIV-1 virions. Efficient packaging of 7SL RNA as well as A3F was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. Reducing 7SL RNA packaging by overexpression of SRP19 protein inhibited A3F virion packaging. Although A3F has been shown to interact with P bodies and viral genomic RNA, our data indicated that P bodies and HIV-1 genomic RNA were not required for A3F packaging. Thus, in addition to its well-known function in SRPs, 7SL RNA, which is encapsidated into diverse retroviruses, also participates in the innate antiviral function of host cytidine deaminases.

Diverse retroviruses have been shown to package host SRP (7SL) RNA. However, little is known about the viral determinants of 7SL RNA packaging. Here we demonstrate that 7SL RNA is more selectively packaged into HIV-1 virions than are other abundant Pol-III-transcribed RNAs, including Y RNAs, 7SK RNA, U6 snRNA and cellular mRNAs. The majority of the virion-packaged 7SL RNAs were associated with the viral core structures and could be reverse-transcribed in HIV-1 virions and in virus-infected cells. Viral Pol proteins influenced tRNAlys,3 packaging but had little influence on virion packaging of 7SL RNA. The N-terminal basic region and the basic linker region of HIV-1 NCp7 were found to be important for efficient 7SL RNA packaging. Although Alu RNAs are derived from 7SL RNA and share the Alu RNA domain with 7SL RNA, the packaging of Alu RNAs was at least 50-fold less efficient than that of 7SL RNA. Thus, 7SL RNAs are selectively packaged into HIV-1 virions through mechanisms distinct from those for viral genomic RNA or primer tRNAlys,3. Virion packaging of both human cytidine deaminase APOBEC3G and cellular 7SL RNA are mapped to the same regions in HIV-1 NC domain.

APOBEC3G (A3G) and related cytidine deaminases are potent inhibitors of retroviruses. HIV-1 Vif hijacks the cellular Cul5-E3 ubiquitin ligase to degrade APOBEC3 proteins and render them ineffective against these viruses. Here, we report that HIV-1 Vif is a novel zinc-binding protein containing an H-x5-C-x17–18-C-x3–5-H motif that is distinct from other recognized classes of zinc fingers. Zinc-binding stabilized a conserved hydrophobic interface within the HCCH motif that is critical for Vif-Cul5 E3 assembly and Vif function. An N-terminal region in the first Cullin repeat of Cul5, which is dispensable for adaptor ElonginC binding, was required for interaction with Vif. This region is the most divergent sequence between Cul2 and Cul5, a factor that may contribute to the selection of Cul5 and not Cul2 by Vif. This is the first example of a zinc-binding substrate receptor responsible for the assembly of a Cullin-RING ligase, representing a new target for antiviral development.

Cellular immune responses against human immunodeficiency virus type 1 (HIV-1) and the related simian immunodeficiency virus (SIV) have been shown to play an important role in controlling HIV-1 and SIV infection and in delaying disease progression. Containment of primary HIV-1 infection in infected individuals correlates with the emergence of virus-specific cytotoxic T-lymphocyte (CTL) responses (4, 14, 26). In chronically infected individuals, a high-frequency CTL response against HIV-1 is also correlated with a low viral load and slow disease progression (24, 25). An HIV-1-specific CTL response has also been demonstrated in certain highly exposed seronegative individuals (2, 15, 32). Also, strong HIV-specific proliferative responses, which may be critical for the maintenance of CTL responses, have been identified in long-term nonprogressors (31, 35). HIV-1 Gag is one of the most conserved viral proteins. Broad, cross-clade CTL responses recognizing conserved epitopes in HIV-1 Gag have been detected in HIV-1-infected people (11, 21), and the development of a safe and effective HIV-1 vaccine may depend on the induction of effective CTL and/or T-helper responses against conserved HIV-1 proteins such as Gag. DNA vaccines have been shown to induce efficient cellular immune responses and protection against a variety of viral, bacterial, and parasitic pathogens in animal models. However, DNA vaccines that could induce potent cellular immune responses against HIV-1 Gag are not yet available. We have recently demonstrated that by destroying inhibitory sequences in the coding region of HIV-1 gag, we could significantly increase Gag protein expression in primate as well as in mouse cells (27, 34, 36, 37) and dramatically enhance immune responses induced by DNA vaccine (27). Since this new Gag expression vector is Rev/RRE-independent and species-independent, it provides a feasible approach to systematically evaluating the strategies that could lead to the maximum induction of cellular immune responses against HIV Gag molecules in animal models. Intramuscular (i.m.) administration of a DNA vaccine represents a simple and effective means of inducing both humoral and cellular immune responses (10). There are three potential pathways responsible for antigen presentation after i.m. injection of DNA. First, muscle cells could take up the DNA, express the encoded protein antigen, and present it to immune cells. Recent data suggest that this pathway is rather unlikely in vivo (40). Second, dendritic cells attracted to the site of injection may take up the DNA, express the encoded protein, and present it to T and B cells. Third, muscle cells may take up the DNA and express the protein antigen, with the antigen then being transmitted to dendritic cells for presentation. If the second possibility is the case, a protein that is synthesized and degraded in the cytoplasm of dendritic cells would be an excellent target for major histocompatibility complex (MHC) class I presentation and induction of CTL responses. Alternatively, if the third scenario were true, a protein synthesized in the muscle cells that could be targeted efficiently to dendritic cells would induce the best CTL response. To distinguish among these different possibilities, we have now constructed and compared three different forms of HIV-1 Gag DNA vaccine vectors for the induction of immune responses. These different forms of Gag include (i) a standard Gag (St-Gag) that assembles into particles, which are efficiently released from cells and become surrounded by host-cell-derived lipid membrane acquired during virus budding; (ii) a cytoplasmic form of Gag (Cy-Gag) that fails to target the plasma membrane and therefore remains in the cytoplasm; and (iii) a secreted form of Gag (Sc-Gag) that is synthesized on the cytoplasmic face of the rough endoplasmic reticulum (ER), transported through the ER and Golgi apparatus, and released as a secreted protein (i.e., not surrounded by a lipid membrane). When these forms of Gag were administered to mice as DNA vaccine, we found that the DNA vector encoding the Sc-Gag generated better primary CTL and T-helper responses than did the DNA vector encoding Cy-Gag. Furthermore, the DNA vector encoding the Sc-Gag also generated a higher level of secondary CTL responses than did the DNA vector encoding Cy-Gag after DNA priming and recombinant vaccinia virus-Gag boost. Vaccinia virus titers were notably reduced in the ovaries of mice immunized with Gag DNA vaccine more than 125 days before infection, as compared to the titer in mice that received only the control DNA vector. These data indicate that CD8+ T-cell memory elicited by DNA vaccination is functionally relevant and provides protective immunity in this system. Again, the DNA vector encoding the Sc-Gag provided better protection against recombinant vaccinia virus-Gag than did the DNA vector encoding Cy-Gag.

There is increasing evidence that CD8+ cytotoxic T lymphocytes (CTL) may play an important role in controlling human immunodeficiency virus type 1 (HIV-1) infection. Containment of primary HIV-1 infection in infected individuals correlates with the emergence of virus-specific CTL responses (3, 12, 22). In chronically infected individuals, a high-frequency CTL response against HIV-1 is also correlated with low viral load and slow disease progression (19, 20). An HIV-1-specific CTL response has also been demonstrated in certain highly exposed seronegative individuals (2, 13, 28). Broad, cross-clade CTL responses recognizing conserved epitopes in HIV-1 Gag have been detected in HIV-1-infected individuals (7, 18). It is therefore reasonable to hypothesize that induction of an effective CTL response against conserved internal virion proteins of HIV-1 such as Gag is essential for the development of a safe and effective HIV-1 vaccine. In order to generate an efficient major histocompatibility complex (MHC) class I-restricted cellular immune response to a vaccine, viral proteins must be synthesized endogenously. Efficient production of CTL responses requires endogenous antigen synthesis, usually achieved by using a live, attenuated virus or recombinant virus vectors. Concerns about using a live, attenuated virus vaccine for HIV-1 include potential pathogenic replication and disease development over a longer period of time as well as potential adverse effects of integrated viral DNA. Using recombinant virus-based vectors, it is difficult to achieve repeated boosting because of the strong immune response generated against the viral proteins of the virus vector. Certain virus vectors, such as vaccinia virus, may also inhibit class I MHC-restricted CTL responses (32). Recently, a new approach (DNA vaccination) has been used to express antigens in vivo for the generation of both humoral and cellular immune responses (6). Several groups have used the DNA vaccination approach against HIV-1 (10, 17, 21, 34). Unfortunately, expression of HIV-1 Gag, Pol, and Env proteins by DNA vectors has been hampered by the presence of multiple inhibitory sequences (INS) in the structural genes encoding Gag, Pol, and Env proteins of HIV-1. This makes expression of the structural HIV-1 proteins dependent on the viral regulatory protein Rev, which is responsible for the nuclear export and efficient expression of unspliced HIV-1 mRNAs (5, 8, 23, 24). Rev binds specifically to an RNA site within HIV-1 mRNA named RRE. In the absence of functional Rev/RRE, mRNAs containing INS are either retained in the nucleus or degraded rapidly; therefore, little protein can be expressed from these mRNAs. Furthermore, even with Rev and RRE, expression of HIV-1 Gag, Pol, or Env is very low in certain murine cell lines (10, 33), limiting our ability to study the DNA vaccine-induced immune response against Gag or Pol using a mouse model. It has been reported that type D retroviruses contain a cis-acting posttranscriptional control element (CTE) that can replace the Rev and RRE regulation of HIV-1 (4, 36). When a CTE is inserted into an HIV-1 Rev−/RRE− clone, replication of the CTE-containing clone can approach that of the wild-type HIV-1 clone in cell lines (4, 36). The CTEs of different type D retroviruses are approximately 200 nucleotides in length and form extensive RNA stem-loop structures. They contain binding sites for cellular factors that contain nuclear export signals (9). CTEs function in a wide spectrum of cell types and species (31) and have been identified in both type D simian retroviruses (4, 36) and murine intracisternal-A particles (31). A current hypothesis about the function of INS is that they are binding sites for cellular factors that contribute to mRNA instability (1). Recently, it has been demonstrated that substitutions of AT-rich regions in HIV-1 gag, mostly in third-site positions, without changing the amino acid sequence of the produced Gag protein can result in efficient expression of Gag in a Rev- and RRE-independent fashion (29, 30). In the present study, we have constructed a series of HIV-1 Gag DNA expression vectors and compared the influence of the constitutive transport element (CTE) or of silent-site mutations within HIV-1 gag on Gag expression in a variety of cells of human, simian, and mouse origin. We found that silent-site mutations in HIV-1 gag allowed efficient Gag expression in a species-independent fashion and induced both humoral and CTL responses after naked DNA vaccination in mice. On the other hand, CTE only increased Gag expression moderately in monkey-derived COS cells and negligibly in HeLa (human) or several mouse cell lines. A DNA vaccine containing wild-type gag coding sequence plus CTE did not induce any detectable humoral or CTL response in mice. In contrast, mutated gag not containing INS induced detectable humoral and CTL responses. We have also achieved stable expression of HIV-1 Gag protein in mouse p815 cells and shown that these cells can be used as CTL target cells. Reagents described in this study should facilitate the development and evaluation of HIV-1 DNA vaccine strategies against conserved viral Gag protein in animal models.

Human cytidine deaminase APOBEC3C (A3C) acts as a potent inhibitor of SIVagm and can be regulated by both HIV-1 and SIVagm Vif. The mechanism by which Vif suppresses A3C is unknown. In the present study, we demonstrate that both HIV-1 and SIVagm Vif can act in a proteasome-dependent manner to overcome A3C. SIVagm Vif requires the Cullin5-ElonginB-ElonginC E3 ubiquitin ligase for the degradation of A3C as well as the suppression of its antiviral activity. Mutation of a residue critical for the species-specific recognition of human or monkey A3G by HIV-1 Vif or SIVagm Vif in A3C had little effect on HIV-1 or SIVagm Vif-mediated degradation of A3C. Although the amino-terminal region of A3G was not important for Vif-mediated degradation, the corresponding region in A3C was critical. A3C mutants that were competent for Vif binding but resistant to Vif-mediated degradation were identified. These data suggest that primate lentiviral Vif molecules have evolved to recognize multiple host APOBEC3 proteins through distinct mechanisms. However, Cul5-E3 ubiquitin ligase appears to be a common pathway hijacked by HIV-1 and SIV Vif to defeat APOBEC3 proteins. Furthermore, Vif and APOBEC3 binding is not sufficient for target protein degradation indicating an important but uncharacterized Vif function.

Cytidine deaminase APOBEC3G (A3G) has broad antiviral activity against diverse retroviruses and/or retrotransposons, and its antiviral functions are believed to rely on its encapsidation into virions in an RNA-dependent fashion. However, the cofactors of A3G virion packaging have not yet been identified. We demonstrate here that A3G selectively interacts with certain polymerase III (Pol III)-derived RNAs, including Y3 and 7SL RNAs. Among A3G-binding Pol III-derived RNAs, 7SL RNA was preferentially packaged into human immunodeficiency virus type 1 (HIV-1) particles. Efficient packaging of 7SL RNA, as well as A3G, was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. A3G mutants that had reduced 7SL RNA binding but maintained wild-type levels of mRNA and tRNA binding were packaged poorly and had impaired antiviral activity. Reducing 7SL RNA packaging by overexpression of SRP19 proteins inhibited 7SL RNA and A3G virion packaging and impaired its antiviral function. Thus, 7SL RNA that is encapsidated into diverse retroviruses is a key cofactor of the antiviral A3G. This selective interaction of A3G with certain Pol III-derived RNAs raises the question of whether A3G and its cofactors may have as-yet-unidentified cellular functions.

Summary The cytidine deaminase apolipoprotein B mRNA editing catalytic subunit-3 (APOBEC3) proteins have been identified as potent inhibitors of diverse retroviruses, retrotransposons and hepatitis B virus (HBV). The mechanism of APOBEC3 proteins in the control of HBV infection, however, is less clear. Here we report that APOBEC3B (A3B) displays dual inhibitory effects on both HBsAg and HBeAg expression as well as HBV core-associated DNA synthesis. Heterogeneous nuclear ribonucleoprotein K (hnRNP K), a positive regulator of HBV expression, has been identified as a major interaction partner of A3B protein. A3B protein inhibited the binding of hnRNP K to the enhancer II of HBV (Enh II), and S gene transcription of HBV. Moreover, A3B directly suppressed HBV S gene promoter activity. Individual variation in A3B expression was observed in both normal primary hepatocytes and liver tissues. Interestingly, A3B was able to inhibit CMV and SV40 promoter-mediated gene expression. In conclusion, A3B suppresses HBV replication in hepatocytes by inhibiting hnRNP K-mediated transcription and expression of HBV genes as well as HBV core DNA synthesis. In addition, A3B protein may be a broad antiviral host factor. Thus, regulated A3B expression may contribute to non-cytolytic HBV clearance in vivo.

APOBEC3G (A3G) is a single-stranded DNA cytidine deaminase that targets retroviral minus-strand DNA and has potent antiviral activity against diverse retroviruses. However, the mechanisms of A3G antiviral functions are incompletely understood. Here we demonstrate that A3G, A3F, and, to a lesser extent, the noncatalytic A3GC291S block human immunodeficiency virus type 1 (HIV-1) replication by interfering with proviral DNA formation. In HIV-1 virions, A3G interacted with HIV-1 integrase and nucleocapsid, key viral factors for reverse transcription and integration. Unlike A3G, the weak antiviral A3C cytidine deaminase did not interact with either of these factors and did not affect viral reverse transcription or proviral DNA formation. Thus, multiple steps of the HIV-1 replication cycle, most noticeably the formation of proviral DNA, are inhibited by both cytidine deamination-dependent and -independent mechanisms.

APOBEC3G (A3G) and related cytidine deaminases, such as APOBEC3F (A3F), are potent inhibitors of retroviruses. Formation of infectious human immunodeficiency virus type 1 (HIV-1) requires suppression of multiple cytidine deaminases by Vif. Whether HIV-1 Vif recognizes various APOBEC3 proteins through a common mechanism is unclear. The domains in Vif that mediate APOBEC3 recognitions are also poorly defined. The N-terminal region of HIV-1 Vif is unusually rich in Trp residues, which are highly conserved. In the present study, we examined the role of these Trp residues in the suppression of APOBEC3 proteins by HIV-1 Vif. We found that most of the highly conserved Trp residues were required for efficient suppression of both A3G and A3F, but some of these residues were selectively required for the suppression of A3F but not A3G. Mutant Vif molecules in which Ala was substituted for Trp79 and, to a lesser extent, for Trp11 remained competent for A3G interaction and its suppression; however, they were defective for A3F interaction and therefore could not efficiently suppress the antiviral activity of A3F. Interestingly, while the HIV-1 Vif-mediated degradation of A3G was not affected by the different C-terminal tag peptides, that of A3F was significantly influenced by its C-terminal tags. These data indicate that the mechanisms by which HIV-1 Vif recognizes its target molecules, A3G and A3F, are not identical. The fact that several highly conserved residues in Vif are required for the suppression of A3F but not that of A3G suggests a critical role for A3F in the restriction of HIV-1 in vivo.

Efficient expression of the human immunodeficiency virus type 1 (HIV-1) structural gene products Gag, Pol, and Env involves the regulation by viral Rev and Rev-responsive elements (RRE). Removal of multiple inhibitory sequences (INS) in the coding regions of these structural genes or modification of the codon usage patterns of HIV-1 genes to those used by highly expressed human genes has been found to significantly increase HIV-1 structural protein expression in the absence of Rev and RRE. In this study, we show that efficient and stable expression of the HIV-1 structural gene products Gag and Env could be achieved by transfection with a noncytopathic Sindbis virus expression vector by using HIV-1 sequences from primary isolates without any sequence modification. Stable expression of these Gag and Env proteins was observed for more than 12 months. The fact that the Sindbis virus expression vector replicates its RNA only in the cytoplasm of the transfected cells and the fact that the lack of expression of HIV-1 Gag by the DNA vector containing unmodified HIV-1 gag sequences was associated with a lack of detectable cytoplasmic gag RNA suggest that a major blockage in the expression of HIV-1 structural proteins in the absence of Rev/RRE is caused by inefficient accumulation of mRNA in the cytoplasm. Efficient long-term expression of structural proteins of diverse HIV-1 strains by the noncytopathic Sindbis virus expression system may be a useful tool for functional study of HIV-1 gene products and vaccine research.

A fluorescent sensor, dansyl-l-aspartic acid (1), coupled with BSA was used to specifically detect Hg2+ in a neutral aqueous solution as well as in live cells; the fluorescence emission spectrum underwent an obvious blue shift with an enhancement in fluorescence intensity, and these effects were evident as color changes in fluorescence imaging pictures.