Human immunodeficiency virus type 1 (HIV-1) does not replicate in murine cells. We investigated the basis of this block by infecting a murine NIH 3T3 reporter cell line that stably expressed human CD4, CCR5, and cyclin T1 and contained a transactivatable HIV-1 long terminal repeat (LTR)-green fluorescent protein (GFP) cassette. Although the virus entered efficiently, formed provirus, and was expressed at a level close to that in a highly permissive human cell line, the murine cells did not support M-tropic HIV-1 replication. To determine why the virus failed to replicate, the efficiency of each postentry step in the virus replication cycle was analyzed using vesicular stomatitis virus G pseudotypes. The murine cells supported reverse transcription and integration at levels comparable to those in the human osteosarcoma-derived cell line GHOST.R5, and human cyclin T1 restored provirus expression, consistent with earlier findings of others. The infected murine cells contained nearly as much virion protein as did the human cells but released less than 1/500 the amount of p24 gag into the culture medium. A small amount of p24 gag was released and was in the form of fully infectious virus. Electron microscopy suggested that aberrantly assembled virion protein had accumulated in cytoplasmic vesicular structures. Virions assembling at the cell membrane were observed but were rare. The entry of M-tropic JR.FL-pseudotyped reporter virus was moderately reduced in the murine cells, suggesting a minor reduction in coreceptor function. A small reduction in the abundance of full-length viral mRNA transcripts was also noted; however, the major block was at virion assembly. This could have been due to a failure of Gag to target to the cell membrane. This block must be overcome before a murine model for HIV-1 replication can be developed.Several murine models have been developed for studies of AIDS pathogenesis. Mice transgenic for either the entire or partial human immunodeficiency virus type 1 (HIV-1) genome develop symptoms with similarities to AIDS. In one model, mice expressing HIV-1 Nef developed a wasting syndrome characterized by the loss of CD4 ϩ cells (21, 45). In another model, SCID mice were reconstituted with human peripheral blood lymphocytes or fetal thymus and liver and then inoculated with HIV-1 (37). These have been useful for studies on mechanisms of CD4 ϩ cell depletion and for evaluation of therapeutic strategies.Current murine models lack a central feature of HIV-1-induced pathogenesis: virus replication. Inoculation of mice or rodents such as rats, hamsters, and guinea pigs with high-titer HIV-1 does not result in detectable viremia, nor does the virus replicate in murine cells in culture (37) or infect human-CD4 (hu-CD4) transgenic mice (33). Low levels of virus replication have been detected in experimentally infected rabbits (13,15,20,26,43) and cotton rats (30), but this does not induce pathogenesis. Development of a system in which HIV-1 could replicate in mice would allow the investigation of features...

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) evolved to stimulate the expression of intronless viral messages. To determine whether this ability to enhance expression could be useful in nonviral and heterologou s viral gene delivery systems, we analyzed the ability of the WPRE to elevate the expression of a cDNA encoding the green fluorescent protein (GFP) in these contexts. We find that the WPRE can stimulate the expression of GFP when the gene is delivered by transfection or transduction with recombinant adeno-associated virus (AAV). Enhancement occurred both during transient expression and when the gene is stably incorporated into the genome of target cells. This enhancement required that the WPRE be located in cis within the GFP message, and was observed in both transformed cell lines and primary human fibroblasts. These results demonstrate that the WPRE will be an effective tool for increasing the long-term expression of transgenes in gene therapy. OVERVIEW SUMMARYThe optimization of transgene expression at the posttranscriptional level will enhance the capacity of vector delivery systems to produce therapeutic results in target tissues. In this study, the woodchuck hepatitis virus posttranscriptional regulatory elem ent (WPRE) is shown to enhance the expression of an eGFP reporter gene in both transient and stable contexts, when delivered either by transfection or by transduction with recombinant AAV. This enhancement of transgene expression is observed both in cell lines and in primary cells. Inclusion of the WPRE in transgenic messages will better the prospects for therapeutic levels of transgene expression using currently existing gene delivery technologies.

Over the past 10 years, stimuli-responsive polymeric biomaterials have emerged as effective systems for the delivery of therapeutics. Persistent with ongoing efforts to minimize adverse effects, stimuli-responsive biomaterials are designed to release in response to either chemical, physical, or biological triggers. The stimuli-responsiveness of smart biomaterials may improve spatiotemporal specificity of release. The material design may be used to tailor smart polymers to release a drug when particular stimuli are present. Smart biomaterials may use internal or external stimuli as triggering mechanisms. Internal stimuli-responsive smart biomaterials include those that respond to specific enzymes or changes in microenvironment pH; external stimuli can consist of electromagnetic, light, or acoustic energy; with some smart biomaterials responding to multiple stimuli. This review looks at current and evolving stimuli-responsive polymeric biomaterials in their proposed applications.

Accurate and efficient genotyping of HIV-1 strains in vaccine trial populations in East Africa, ascertainment of dual infections, and elucidation of the genesis of recombinant forms in individuals can be facilitated by the application of MHA.

Viruses often contain cis-acting RNA elements, which facilitate the posttranscriptional processing and export of their messages. These elements fall into two classes distinguished by the presence of either viral or cellular RNA binding proteins. To date, studies have indicated that the viral proteins utilize the CRM1-dependent export pathway, while the cellular factors generally function in a CRM1-independent manner. The cis-acting element found in the woodchuck hepatitis virus (WHV) (the WHV posttranscriptional regulatory element [WPRE]) has the ability to posttranscriptionally stimulate transgene expression and requires no viral proteins to function. Conventional wisdom suggests that the WPRE would function in a CRM1-independent manner. However, our studies on this element reveal that its efficient function is sensitive to the overexpression of the C terminus of CAN/Nup214 and treatment with the antimicrobial agent leptomycin B. Furthermore, the overexpression of CRM1 stimulates WPRE activity. These results suggest a direct role for CRM1 in the export function of the WPRE. This observation suggests that the WPRE is directing messages into a CRM1-dependent mRNA export pathway in somatic mammalian cells.The generation of mature cytoplasmic mRNAs requires numerous processing steps, namely, transcription, capping, splicing, polyadenylation, and transport to the cytoplasm. This process is tightly regulated, since aberrant transcripts are degraded in the nucleus and only properly processed mRNAs are exported to cytoplasm (43). Thus, nuclear export ensures that only completely processed mRNAs can be translated into protein.Much of our present understanding of nuclear export has come from the study of how viruses exploit host cell RNA processing and export pathways. The first viral export system studies were those of complex retroviruses, exemplified by human immunodeficiency virus type 1 (HIV-1). HIV-1 replication requires unspliced and partially spliced RNAs to be exported from the nucleus by the virally encoded Rev protein (9,13,44). Rev contains an RNA binding domain, which specifically binds to the Rev response element (RRE), located within the second intron of HIV-1 pre-mRNA, and a nuclear export signal (NES) that interacts with CRM1, a member of the importin ␤ family of transport receptors (6,15,17,44,58).The interaction between Rev and CRM1 is dependent upon CRM1 association with the GTP-bound form of the GTPase Ran protein (RanGTP). Once assembled, the RRE/Rev-CRM1-RanGTP ribonucleoprotein complex interacts with NPs, which trigger its nuclear export. CRM1 has been proposed to mediate this interaction by directly contacting selected nucleoporins (NPs), including CAN/Nup214. Binding of CRM1 to CAN has been mapped to the NP domain located within the extreme carboxy terminus of CAN (16). Overexpression of the isolated NP domain of CAN, termed ⌬CAN, is able to inhibit Rev-mediated export by competing with the NPs for binding to CRM1 (2). Rev-mediated export is also inhibited by the antibiotic leptomycin B (LMB), w...