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Related post: infectious clones. MAL could replicate on H9 and SupTl cells, but no viral growth was detected on CEM and U937 cells. In contrast, ELI could infect CEM and U937 cells but not H9 nor SupTl cells. The mechanisms underlying these differences in cell tropism is being investigated by exchanging homologous segments of the ELI and MAL cloned proviruses. (Peden) HIV-1 Envelope Processing . The intracellular transport and processing of the HIV-1 envelope proteins have been studied using wild-type and envelope mutant proviral clones. Pulse-chase experiments in conjunction with several biochemical assays have been conducted on lysates prepared from acutely infected cells or following transfection of wild type or mutant proviral clones. The cumulative results from these analyses indicate that intracellular transport and processing of the gpl60 precursor polyprotein proceeds via the rough endoplasmic reticulum (ER) and Golgi complex. The formation of oligomeric gpl60 structures within the ER may be required for transport; however, appropriate folding after oligomerization is also a critical step. Mutations within different regions of the envelope can alter the intracellular distribution and fate of the gpl60 as well as the envelope cleavage products, gpl20 and gp41. Intracellular Cartia Xt 180 Mg sorting of the HIV-1 envelope glycoproteins appears to very complex and may ultimately determine which proteins become incorporated into viral particles. (Willey and Martin) 12-4 HIV-1 envelope structure and function . Oligonucleotide-directed site-specific mutagenesis has been used to introduce amino acid changes into two discrete regions Cartia Xt 300 Mg of the HIV-1 envelope gene. These regions have been previously reported to be critical for fusing viral and cellular membranes. In addition to affecting the fusion reaction, all of the mutations impair or ablate the replicative capability of HIV-1. Quantitative analyses of envelope processing revealed that some mutations dramatically reduce the intracellular production of the mature envelope components gpl20 and gp41 while others affect the stability of these proteins after processing. These results indicate that individual regions within the viral envelope have multiple roles (viz. envelope structure, function, and processing). (Willey and Martin) Monotropic HTV-1 isolates replicate efficiently in CSF-1 treated, primary monocyte-derived macrophage cultures . We have developed a primary monocyte-derived macrophage tissue culture system in which the growth of the monotropic AD-87 isolate of HIV-1 replicates with an efficiency comparable to other HIV isolates in T-cells. Pre-treatment of the cells in suspension with recombinant human CSF-1 for forty-eight hours, followed by plating at a density of at least 10 6 /well in 24-well plates, gives rise, 9 days later, to a culture competent to replicate the AD-87 isolate with high efficiency. Using this system, in combination with an "inside-out" PCR method developed in our laboratory, we have demonstrated that the AD-87 proviral DNA becomes integrated into the genome of infected monocyte-derived macrophages. (Englund and Martin) Molecular cloning and biological characterization of a monotropic isolate of HIV-1 . Infectious molecular clones of the AD-87 isolate were derived from unintegrated proviral DNA in a primary lymphocyte infection. All of these clones direct the synthesis of HTVs which replicate in primary lymphocytes. One of the clones also generates virus which efficiently infects primary, monocyte-derived macrophages. These results indicate that: 1) The AD-87 isolate is a mixed population of HIV-1, containing both T-cell restricted and broader host-cell range variants, and 2) the broad host-cell range phenotype is a property of an individual molecular clone. (Englund and Martin) UV irradiation increases HIV LTR driven expression of cholaramphenicol acetyl transferase (CAT) in HIV-LTR/CAT transgenic mice . Cartia Xt 180 UV irradiation is known to induce HIV-directed gene expression in tissue culture systems. To determine whether UV light induces HIV gene expression in vivo, transgenic mice harboring HTV-LTR driven CAT gene were exposed to UV radiation. Increased CAT expression (up to 30-fold) was observed in ear samples from irradiated animals. The kinetics of in vivo UV induced CAT activity reached a maximum on day 3 and diminished to baseline levels by day 7. These data suggest that UV light can also increase HIV LTR driven expression in vivo and may prove useful in activation of latent proviruses in animal models. (Frucht, Vicenzi and Martin) Biochemical characterization of the HIV nef protein . HIV nef protein has been reported to share certain biochemical and structural properties with oncogenes of the ras family. To determine whether this is a general property of nef from various HIV isolates, ras and nef 12-5 clones, expressed in the same bacterial and mammalian vectors, were compared biochemically and biologically. Unlike ras, nef proteins lacked GTP binding activity but exhibited autophosphorylation with either GTP or ATP as the phosphate donor. In addition, unlike ras, HIV nef did not exhibit oncogenic potential in focus-forming assays with NTH 3T3 cells nor caused meiotic maturation of Xenopus oocytes. It therefore appears that the biological function of nef does not follow the pattern of G proteins. However, the nef associated phosphorylation, either by protein kinase C or through its autokinase Cartia Xt 240 Mg activity, could be functionally significant in vivo. (Nebreda, Bryan, Segade, Wingfield, Venkatesan, Cartia Xt Discontinued Santos) OTHER RETROVIRUSES HTLV-I Tax and Rex proteins have multiple functional activities . Over 50 point mutations were inserted in the tax gene of HTLV-I and have been used to define functional domains within the tax protein. Experiments correlating structural changes Cartia Xt 120 Mg with tax function are presently in progress. The rex protein has been found to induce increased transcriptional initiations from a variety of eucaryotic promoters. This activity differs from the previously described post-transcriptional function for rex which modulated the nuclear to cytoplasmic transport of viral mRNA. (Semmes, Nelson Cartia Xt 240 and Jeang) Mouse Leukemia Virus Cell Surface Receptors . The mouse leukemia viruses comprise four different subgroups defined by interference patterns and host range. A mouse cDNA that confers susceptibility to the ecotropic host range group was used to map this gene to chromosome 5 confirming that it is likely to be Rec-1, the ecotropic cell surface receptor. This gene was also positioned at the distal end of chromosome 5 by analysis of an interspecies backcross demonstrating Buy Cartia Xt that Rec-1 is not part of the cluster of retrovirus- associated genes near the centromere, and also demonstrating that Rec-1 does not map at or near any known mouse mutation that might suggest its normal role. A second gene, Tea, cloned by C. MacLeod by virtue of its differential expression in T cells, was found to be highly homologous to Rec-1. This homology suggests that Tea and Rec-1 define a new family of membrane spanning proteins and suggests that Tea may represent one of the other
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