Faculty Bibliography

Murine cells do not support efficient assembly and release of human immunodeficiency virus type 1 (HIV-1) virions. HIV-1-infected mouse cells that express transfected human cyclin T1 synthesize abundant Gag precursor polyprotein, but inefficiently assemble and release virions. This assembly defect may result from a failure of the Gag polyprotein precursor to target to the cell membrane. Plasma membrane targeting of the precursor is mediated by the amino-terminal region of polyprotein. To compensate for the assembly block, we substituted the murine leukemia virus matrix coding sequences into an infectious HIV-1 clone. Transfection of murine fibroblasts expressing cyclin T1 with the chimeric proviruses resulted in viruses that were efficiently assembled and released. Chimeric viruses, in which the cytoplasmic tail of the transmembrane subunit, gp41, was truncated to prevent potential interference between the envelope glycoprotein and the heterologous matrix, could infect human and murine cells. They failed to further replicate in the murine cells, but replicated with delayed kinetics in human MT-4 cells. These findings may be useful for establishing a murine model for HIV-1 replication

Murine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication. Mus musculus lymphoid cell lines EL4 and L1-2 and Mus dunni fibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infected M. dunni tail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release

To enter its target cells, human immunodeficiency virus (HIV) must interact with CD4 and one of a family of chemokine receptors. CCR5 is widely used by the virus in this context, and its ligands can prevent HIV entry. Amino-terminal modified chemokine variants, in particular AOP-RANTES (aminooxypentane-linked regulated on activation normal T cell expressed and secreted), exhibit enhanced HIV entry inhibition. We have previously demonstrated that a non-allelic isoform of macrophage inflammatory protein (MIP)-1alpha, termed MIP-1alphaP, is the most active naturally occurring inhibitor of HIV entry known. Here we report the properties of a variant of MIP-1alphaP with an AOP group on the amino terminus. We show that, like RANTES, the addition of AOP to MIP-1alphaP enhances its interactions with CCR1 and CCR5, allows more effective internalization of CCR5, and increases the ligand's potency as an inhibitor of HIV entry through CCR5. Importantly, AOP-MIP-1alphaP is about 10-fold more active than AOP-RANTES at inhibiting HIV entry, making it the most effective chemokine-based inhibitor of HIV entry through CCR5 described to date. Surprisingly, the enhanced receptor interactions of AOP-MIP-1alphaP do not translate into increased chemotaxis or coupling to calcium ion fluxes, suggesting that this protein should be viewed as a partial, rather than a full, agonist for CCR1 and CCR5

CD40 ligand (CD40L) is a cell surface molecule of CD4(+)T cells that interacts with its receptor CD40 on antigen presenting cells to mediate thymus-dependent humoral immunity and inflammatory reactions. We report here that treating monocyte-derived macrophages (MDM) with a trimeric soluble form of CD40L (CD40LT) induced them to secrete high levels of the beta-chemokines RANTES, MIP-1alpha and MIP-1beta that are ligands for CCR5 and able to inhibit HIV-1 entry. CD40LT inhibited the entry of M-tropic HIV-1 reporter viruses. Furthermore, supernatants obtained from CD40LT-stimulated macrophages protected CEMx174-CCR5 cells from infection by HIV-1(JRFL)reporter virus. The inhibitory activity appeared to be due to beta-chemokines present in the supernatant, since pretreating them with a cocktail of antibodies to RANTES, MIP-1alpha and MIP-1beta neutralized the inhibitory activity of the supernatants. In addition, treating monocytes with CD40LT caused CCR5 and CD4 to be downregulated from the cell surface. In vivo, macrophages activated through CD40 could interfere with HIV replication

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

Chemokines play diverse roles in inflammatory and non-inflammatory situations via activation of heptahelical G-protein-coupled receptors. Also, many chemokine receptors can act as cofactors for cellular entry of human immunodeficiency virus (HIV) in vitro. CCR5, a receptor for chemokines MIP-1alpha (LD78alpha), MIP-1beta, RANTES, and MCP2, is of particular importance in vivo as polymorphisms in this gene affect HIV infection and rate of progression to AIDS. Moreover, the CCR5 ligands can prevent HIV entry through this receptor and likely contribute to the control of HIV infection. Here we show that a non-allelic isoform of human MIP-1alpha (LD78alpha), termed LD78beta or MIP-1alphaP, has enhanced receptor binding affinities to CCR5 (approximately 6-fold) and the promiscuous beta-chemokine receptor, D6 (approximately 15-20-fold). We demonstrate that a proline residue at position 2 of MIP-1alphaP is responsible for this enhanced activity. Moreover, MIP-1alphaP is by far the most potent natural CCR5 agonist described to date, and importantly, displays markedly higher HIV1 suppressive activity than all other human MIP-1alpha isoforms examined. In addition, while RANTES has been described as the most potent inhibitor of CCR5-mediated HIV entry, MIP-1alphaP was as potent as, if not more potent than, RANTES in HIV-1 suppressive assays. This property suggests that MIP-1alphaP may be of importance in controlling viral spread in HIV-infected individuals

CD4+ lymphocytes exhibit variable permissiveness to the replication of HIV-1. A cohort of sexually-exposed-yet-uninfected individuals were previously shown to have CD4+ lymphocytes refractory for M-tropic viral replication. In particular, two individuals from this population whose CD4+ lymphocytes exhibited complete resistance to M-tropic viral replication were later shown to be homozygous for a 32bp (delta32) deletion in the gene encoding for CCR5. In screening diverse populations, HIV-1 infected individuals heterozygous for the delta32 allele were statistically favored in their disease course to harbor lower viral loads and exhibit slower rates of CD4+ cell loss when compared to control CCR5 wild-type individuals. Further comparative analysis between individuals in the exposed but uninfected cohort who demonstrated intermediate levels of in vitro viral replication and CD4+ lymphocytes isolated from uninfected delta32 heterozygous individuals indicate that reduced levels of in vitro M-tropic replication are a CCR5-related phenomenon: CD4+ lymphocytes from these individuals were more sensitive to the HIV-1 blocking effects of recombinant chemokines, displayed lower CCR5 cell surface expression levels and a proportionate increase in the production of RANTES when compared to CD4+ lymphocytes from control individuals. These results suggest that the CCR5 phenotype is important in determining the replicative capacity of M-tropic HIV-1 in vitro. The implications of these results with relation to HIV-1 transmission and disease progression are discussed

A polymorphism in the gene encoding CCR2 is associated with a delay in progression to AIDS in human immunodeficiency virus (HIV)-infected individuals. The polymorphism, CCR2-64I, changes valine 64 of CCR2 to isoleucine. However, it is not clear whether the effect on AIDS progression results from the amino acid change or whether the polymorphism marks a genetically linked, yet unidentified mutation that mediates the effect. Because the gene encoding CCR5, the major coreceptor for HIV type 1 primary isolates, lies 15 kb 3' to CCR2, linked mutations in the CCR5 promoter or other regulatory sequences could explain the association of CCR2-64I with slowed AIDS pathogenesis. Here, we show that CCR2-64I is efficiently expressed on the cell surface but does not have dominant negative activity on CCR5 coreceptor function. A panel of peripheral blood mononuclear cells (PBMC) from uninfected donors representing the various CCR5/CCR2 genotypes was assembled. Activated primary CD4(+) T cells of CCR2 64I/64I donors expressed cell surface CCR5 at levels comparable to those of CCR2 +/+ donors. A slight reduction in CCR5 expression was noted, although this was not statistically significant. CCR5 and CCR2 mRNA levels were nearly identical for each of the donor PBMC, regardless of genotype. Cell surface CCR5 and CCR2 levels were more variable than mRNA transcript levels, suggesting that an alternative mechanism may influence CCR5 cell surface levels. CCR2-64I is linked to the CCR5 promoter polymorphisms 208G, 303A, 627C, and 676A; however, in transfected promoter reporter constructs, these did not affect transcriptional activity. Taken together, these findings suggest that CCR2-64I does not act by influencing CCR5 transcription or mRNA levels

Infection of T cells with HIV-1 induces loss of CD4 and HLA class I from the cell surface. In the present article we have investigated whether changes in expression of other cell surface molecules could be related to HIV infection. To detect HIV-infected cells at the single-cell level, peripheral blood lymphocytes were infected in vitro with HIV-HSA, a reporter virus encoding the murine heat-stable antigen. Expression of HSA on activated primary lymphocytes was an efficient indicator of productive infection. Expression of the majority of the cell surface proteins studied was unaffected by HIV infection (HLA class I, II, CD11a, CD18, CD25, CD27, CD28, CD29, CD30, CD31, CD38, CD44, CD45R0, CD49d, CD57, CD94, CD95, and CXCR4). However, phenotypic changes specific to the productively infected cells were detected. Expression of the CD4 molecule was progressively lost and this was closely associated with loss of CD62L expression, a molecule involved in T cell homing into the lymph nodes. By contrast, T cells productively infected with this T-tropic reporter virus were enriched for CD54, and for CCR5, the main coreceptor for M-tropic viruses. Given the roles of CD62L, CD54, and CCR5 in lymphocyte trafficking, these results suggest that cells productively infected with HIV might have altered homing patterns in vivo