The Sydney Blood Bank Cohort is a group of patients with slowly progressive infection by a human immunodeficiency virus strain containing spontaneous deletions within the nef long terminal repeat region. In 1999, 18 years after the initial infection, one of the members (D36) developed AIDS. In this work, we used an ex vivo human lymphoid cell culture system to analyze two viral isolates obtained from this patient, one prior to the onset of AIDS in 1995 and one after disease progression in 1999. Both D36 isolates were less potent in depleting CD4(+) T cells than a reference dualtropic, nef-bearing viral isolate. However, the 1999 isolate was measurably more cytotoxic to CD4(+) T cells than the 1995 isolate. Interestingly, although both isolates were nearly equally potent in depleting CCR5(+) CD4(+) T cells, the cytotoxic effect of the 1999 isolate toward CCR5(-) CD4(+) T cells was significantly higher. Furthermore, GHOST cell infection assays and blocking experiments with the CXCR4 inhibitor AMD3100 showed that the later D36 1999 isolate could infect both CCR5(+) and CCR5(-) CXCR4(+) cells efficiently, while infection by the 1995 isolate was nearly completely restricted to CCR5(+) cells. Sequence analysis of the V1/V2 and V3 regions of the viral envelope protein gp120 revealed that the more efficient CXCR4 usage of the later isolate might be caused by an additional potential N-glycosylation site in the V1/V2 loop. In conclusion, these data show that an in vivo evolution of the tropism of this nef-deleted strain toward an X4 phenotype was associated with a higher cytopathic potential and progression to AIDS.

Amino Acid Sequence↗

PubMed2002

Human immunodeficiency virus type 1 Nef binds to tumor suppressor p53 and protects cells against p53-mediated apoptosis.

The nef gene product of human immunodeficiency virus type 1 (HIV-1) is important for the induction of AIDS, and key to its function is its ability to manipulate T-cell function by targeting cellular signal transduction proteins. We reported that Nef coprecipitates a multiprotein complex from cells which contains tumor suppressor protein p53. We now show that Nef interacts directly with p53. Binding assays showed that an N-terminal, 57-residue fragment of Nef (Nef 1-57) contains the p53-binding domain. Nef also interacted with p53 during HIV-1 infection in vitro. As p53 plays a critical role in the regulation of apoptosis, we hypothesized that Nef may alter this process. Nef inhibited UV light-induced, p53-dependent apoptosis in MOLT-4 cells, with Nef 1-57 being as effective as its full-length counterpart. The inhibition by Nef of p53 apoptotic function is most likely due its observed ability to decrease p53 protein half-life and, consequently, p53 DNA binding activity and transcriptional activation. These data show that HIV-1 Nef may augment HIV replication by prolonging the viability of infected cells by blocking p53-mediated apoptosis.

Apoptosis↗

PubMed2002

Mechanical stimulation improves tissue-engineered human skeletal muscle.

Human bioartificial muscles (HBAMs) are tissue engineered by suspending muscle cells in collagen/MATRIGEL, casting in a silicone mold containing end attachment sites, and allowing the cells to differentiate for 8 to 16 days. The resulting HBAMs are representative of skeletal muscle in that they contain parallel arrays of postmitotic myofibers; however, they differ in many other morphological characteristics. To engineer improved HBAMs, i.e., more in vivo-like, we developed Mechanical Cell Stimulator (MCS) hardware to apply in vivo-like forces directly to the engineered tissue. A sensitive force transducer attached to the HBAM measured real-time, internally generated, as well as externally applied, forces. The muscle cells generated increasing internal forces during formation which were inhibitable with a cytoskeleton depolymerizer. Repetitive stretch/relaxation for 8 days increased the HBAM elasticity two- to threefold, mean myofiber diameter 12%, and myofiber area percent 40%. This system allows engineering of improved skeletal muscle analogs as well as a nondestructive method to determine passive force and viscoelastic properties of the resulting tissue.

Biocompatible Materials↗

PubMed1997

Detection and subgrouping of respiratory syncytial virus directly from nasopharyngeal aspirates.

OBJECTIVE: To develop a reverse transcription-polymerase chain reaction (RT-PCR)-based assay to identify the subgroup of the infecting respiratory syncytial virus (RSV) strain directly from nasopharyngeal aspirates (NPAs). METHODS: A total of 154 NPAs which were positive for RSV antigen by direct immunofluorescence were subjected to RT-PCR. The primers used were designed to give products for subgroup A and B which were of different sizes and easily visualized on agarose electrophoresis. The PCR products were further analyzed by restriction analysis using enzymes which were unique or rare cutters within the PCR amplimer. RESULTS: It was possible to confirm RSV infection in 70% of the NPA samples studied. Of these, 92.6% belonged to the A group, and only 7.4% to the B group. Within the A group, six subgroups were identified using restriction analysis, while all B-group samples were identical to the prototype B strain, 18537. CONCLUSION: RT-PCR performed on RNA isolated directly from NPAs provides a quick, easy-to-use, reasonably sensitive method to identify and group the infecting RSV strain.

Journal Article↗