A novel U2 and U11/U12 snRNP protein that associates with the pre-mRNA branch site.

Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa protein (p14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this protein by microsequencing a 14 kDa protein isolated from U2-type spliceosomes. This protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded protein precipitated the 14 kDa protein cross-linked to the branch adenosine, confirming the identity of the p14 cDNA. A combination of immunoblotting, protein microsequencing and immunoprecipitation revealed that p14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2- and U12-type pre-mRNAs, respectively. p14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immuno precipitations indicated that p14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.

thy/liv-SCID-hu mice: a system for investigating the in vivo effects of multidrug therapy on plasma viremia and human immunodeficiency virus replication in lymphoid tissues.

Modified, human immunodeficiency virus (HIV)-inoculated thy/liv-SCID-hu mice were used to evaluate the in vivo efficacy of antiretroviral drugs. Ritonavir treatment alone initially suppressed plasma viremia, but the viremia recurred with the appearance of ritonavir-resistant HIV isolates. Multidrug therapy suppressed plasma HIV RNA to undetectable levels; however, plasma viremia returned after therapy was stopped, showing that the therapy did not completely suppress HIV infection in the thymic implant. When thy/liv-SCID-hu mice were treated with a combination of zidovudine, lamivudine, and ritonavir immediately after inoculation with HIV, cocultures of the thymic implants remained negative for HIV even 1 month after therapy was discontinued, suggesting that acute treatment can prevent the establishment of HIV infection. Thus, these modified thy/liv-SCID-hu mice should prove to be a useful system for evaluating the effectiveness of different antiretroviral therapies on acute and chronic HIV infection.

SCID-hu mice: a model for studying disseminated HIV infection.

Modifications that we introduced into the implantation of human fetal thymus and liver into SCID mice (thy/liv-SCID-hu mice) markedly increased the population of human T cells and monocytes present in the peripheral blood and peripheral lymphoid compartment of these mice. As a result, the modified thy/liv-SCID-hu mice developed disseminated HIV infection after intraimplant or i.p. inoculation. After chronic HIV infection of these mice, depletion of the peripheral human T cells was observed as reported in HIV-infected individuals. In addition, these mice also developed plasma viremia after infection with HIV. The peripheral blood mononuclear cells were responsive to in-vivo cytokine regulation as evidenced by induction of human IFN-gamma gene expression by human IL-12 and inhibition by human IL-10. Acute treatment with human IL-10 but not with human IL-12 inhibited the development of plasma viremia and HIV infection in thy/liv-SCID-hu mice inoculated with HIV-1(59), a clinical isolate. SCID mice transplanted with cultured human fetal bone marrow displayed significant engraftment of the mouse bone marrow with human precursor cells and population of the peripheral blood with human B cells and monocytes. The peripheral blood of these bone marrow-transplanted SCID mice also became populated with human T cells after they were implanted with human thymic tissue due to migration of human precursor cells from the mouse bone marrow to the implanted human thymus. Thus, these modified SCID-hu mice should prove to be a valuable in-vivo model for studying the immunopathogenesis of HIV infection and for examining the in-vivo efficacy of immunomodulatory, drug and gene therapy in modifying HIV infection.

Inhibition of acute in vivo human immunodeficiency virus infection by human interleukin 10 treatment of SCID mice implanted with human fetal thymus and liver.

To improve the usefulness of in vivo mode for the investigation of the pathophysiology of human immunodeficiency virus (HIV) infection, we modified the construction of SCID mice implanted with human fetal thymus and liver (thy/liv-SCID-hu mice) so that the peripheral blood of the mice contained significant numbers of human monocytes and T cells. After inoculation with HIV-1(59), a primary patient isolate capable of infecting monocytes and T cells, the modified thy/liv-SCID-hu mice developed disseminated HIV infection that was associated with plasma viremia. The development of plasma viremia and HIV infection in thy/liv-SCID-hu mice inoculated with HIV-1(59) was inhibited by acute treatment with human interleukin (IL) 10 but not with human IL-12. The human peripheral blood mononuclear cells in these modified thy/liv-SCID-hu mice were responsive to in vivo treatment with exogenous cytokines. Human interferon gamma expression in the circulating human peripheral blood mononuclear cells was induced by treatment with IL-12 and inhibited by treatment with IL-10. Thus, these modified thy/liv-SCID-hu mice should prove to be a valuable in vivo model for examining the role of immunomodulatory therapy in modifying HIV infection. Furthermore, our demonstration of the vivo inhibitory effect of IL-10 on acute HIV infection suggests that further studies may be warranted to evaluate whether there is a role for IL-10 therapy in preventing HIV infection in individuals soon after exposure to HIV such as for children born to HIV-infected mothers.