In Vivo Pathogenesis of a Human Immunodeficiency Virus Type 1 Reporter Virus

Our understanding of human immunodeficiency virus type 1 (HIV-1)-induced pathogenesis is hampered by the inability to detect HIV-1 gene expression in infected viable cells. In this report, we describe two HIV-1 reporter constructs that are replication competent and cytopathic in vivo. These constructs contain DNA regions of two different lengths that bear the cDNA for the murine heat-stable antigen in the vpr region of a CXCR4-tropic virus. We used the SCID-hu mouse model and these reporter viruses to perform detailed kinetic studies of HIV-1 infection of human thymocytes in vivo. We document that the CD4⁺/CD8⁺ thymocytes are the first to express virus and that this subset demonstrates the most rapid and extensive HIV-1-induced cell depletion. Following depletion of this subset, subsequent virus expression occurs predominantly in phenotypically CD4⁻ cells, suggesting that CD4 down-regulation occurs in HIV-1-infected thymocytes in vivo. These results demonstrate the utility of these HIV-1 reporter constructs to monitor HIV pathogenesis in vitro and in vivo.     

Regions of Human Immunodeficiency Virus Type 1 nef Required for Function In Vivo

In vivo studies in monkeys and humans have indicated that immunodeficiency viruses with Nef deleted are nonpathogenic in immunocompetent hosts, and this has motivated a search for live attenuated vaccine candidates. However, the mechanisms of action of Nef remain elusive. To define the regions of human immunodeficiency virus type 1 (HIV-1) Nef which mediate in vivo pathogenicity, a series of mutated isogenic viruses were inoculated into human thymic implants in SCID-hu mice. Mutation of several regions, including the myristoylation site at the second glycine and a region encompassing amino acids 41 through 49 of Nef, profoundly affected pathogenicity. Surprisingly, mutations of prolines in either of the two distant PXXP SH3 binding domains did not affect pathogenicity, indicating that these regions are not required for Nef activity in developing T-lineage cells. These data suggest that some functions of Nef described in vitro may not be relevant for in vivo pathogenicity.     

Human Immunodeficiency Virus Inhibits Multilineage Hematopoiesis In Vivo

Human immunodeficiency virus type 1 (HIV-1)-infected individuals often exhibit multiple hematopoietic abnormalities reaching far beyond loss of CD4⁺ lymphocytes. We used the SCID-hu (Thy/Liv) mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues), which provides an in vivo system whereby human pluripotent hematopoietic progenitor cells can be maintained and undergo T-lymphoid differentiation and wherein HIV-1 infection causes severe depletion of CD4-bearing human thymocytes. Herein we show that HIV-1 infection rapidly and severely decreases the ex vivo recovery of human progenitor cells capable of differentiation into both erythroid and myeloid lineages. However, the total CD34⁺ cell population is not depleted. Combination antiretroviral therapy administered well after loss of multilineage progenitor activity reverses this inhibitory effect, establishing a causal role of viral replication. Taken together, our results suggest that pluripotent stem cells are not killed by HIV-1; rather, a later stage important in both myeloid and erythroid differentiation is affected. In addition, a primary virus isolated from a patient exhibiting multiple hematopoietic abnormalities preferentially depleted myeloid and erythroid colony-forming activity rather than CD4-bearing thymocytes in this system. Thus, HIV-1 infection perturbs multiple hematopoietic lineages in vivo, which may explain the many hematopoietic defects found in infected patients.     

Context-Dependent Phenotype of a Human Immunodeficiency Virus Type 1 Nucleocapsid Mutation

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid mutation R10A/K11A abolishes viral replication when present in proviral clone HIV-1(HXB-2), but it was found to have minimal effect on replication of the closely related HIV-1(NL4-3). Functional mapping demonstrated that a nonconservative amino acid change at nucleocapsid residue 24 (threonine in HIV-1(HXB-2), isoleucine in HIV-1(NL4-3)) is the major determinant of the different R10A/K11A phenotypes in these two proviruses. Threonine-isoleucine exchanges appear to modify the R10A/K11A phenotype via effects on virion RNA-packaging efficiency. The improved packaging seen with hydrophobic isoleucine is consistent with solution structures localizing this residue to a hydrophobic pocket that contacts guanosine bases in viral genomic RNA stem-loops critical for packaging.     

Human Immunodeficiency Virus Type 1-Induced Hematopoietic Inhibition Is Independent of Productive Infection of Progenitor Cells In Vivo

Human immunodeficiency virus (HIV)-infected individuals exhibit a variety of hematopoietic dysfunctions. The SCID-hu mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues) can be used to model the loss of human hematopoietic precursor cell function following HIV infection and has a distinct advantage in that data can be obtained in the absence of confounding factors often seen in infected humans. In this study, we establish that HIV type 1 (HIV-1) bearing a reporter gene inserted into the viral vpr gene is highly aggressive in depleting human myeloid and erythroid colony-forming precursor activity in vivo. Human CD34(+) progenitor cells can be efficiently recovered from infected implants yet do not express the viral reporter gene, despite severe functional defects. Our results indicate that HIV-1 infection alone leads to hematopoietic inhibition in vivo; however, this effect is due to indirect mechanisms rather than to direct infection of CD34(+) cells in vivo.     

Highly Purified Human Immunodeficiency Virus Type 1 Reveals a Virtual Absence of Vif in Virions

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary blood-derived lymphocytes, macrophages, and certain human T-cell lines. It has been shown that Vif is associated with HIV-1 virions purified by sucrose density-equilibrium gradient analysis. However, the specificity of Vif incorporation into virions has not been determined. Moreover, recent studies have demonstrated that standard HIV-1 particle preparations created with sucrose density-equilibrium gradients are contaminated with cell-derived microvesicles. Here we demonstrate, as previously reported, that Vif cosediments with HIV-1 particles in sucrose density-equilibrium gradient analysis. However, we also found that, when Vif was expressed in the absence of all other HIV-1-encoded gene products and then isolated by sucrose density-equilibrium gradient centrifugation from extracellular supernatants, its sedimentation pattern was largely unaltered, suggesting that Vif can be secreted from cells. Using a newly developed OptiPrep velocity gradient method, we were able to physically separate most of the extracellular Vif from the HIV-1 virions without disrupting the infectivity of the virus. By titrating serial dilutions of purified Vif and Gag against the viral peak fraction in the OptiPrep gradient, we demonstrate that <1.0 Vif molecule per virion was present. This study shows that Vif is not significantly present in HIV-1 virions, a finding which is consistent with the idea that Vif functions predominantly in the virus-producing cells during virus assembly. The OptiPrep velocity gradient technique described here could be an easy and rapid way to purify HIV and other enveloped viruses from microvesicles and/or cell debris.     

Comparative Fitness of Multi-Dideoxynucleoside-Resistant Human Immunodeficiency Virus Type 1 (HIV-1) in an In Vitro Competitive HIV-1 Replication Assay

We examined whether human immunodeficiency virus type 1 (HIV-1) fitness was altered upon the acquisition of a set or subset of five mutations (A62V, V75I, F77L, F116Y, and Q151M) in the pol gene, which confers resistance to multiple dideoxynucleosides (MDR), as well as the zidovudine resistance-associated mutation T215Y, using a competitive HIV-1 replication assay in a setting of an HXB2D genetic background. Target H9 cells were exposed to a 50:50 mixture of paired infectious molecular clones, and HIV-1 in the culture supernatant was transmitted to new cultures every 7 to 10 days. The polymerase-encoding region of the virus was sequenced at various time points, and the relative proportion of the two viral populations was determined. In the absence of drugs, the comparative order for replicative fitness was HIV-162/75/77/116/151 > HIV-177/116/151 > HIV-1151 > wild-type HIV-1 (HIV-1wt) > HIV-175/77/116/151 > HIV-1151/215 > HIV-1215. In the presence of zidovudine or didanosine, the order was HIV-162/75/77/116/151 > HIV-177/116/151 > HIV-175/77/116/151 > HIV-1151 > HIV-1215. HIV-1215S(TCC), a putative intermediate infectious clone for HIV-1215, replicated comparably to HIV-1wt, while two putative intermediates for HIV-1151 [HIV-1151L(CTG) and HIV-1151K(AAG)] replicated much less efficiently than HIV-1wt and HIV-1151, suggesting that for HIV-1151 to develop, two base substitutions are likely to occur concurrently or within a short interval. These data may illustrate the molecular basis by which HIV-1151 emerges much less frequently than HIV-1215. The present data also demonstrate that several MDR HIV-1 variants are more fit than HIV-1wt in the absence of drugs and that resistance-associated mutations and drug pressure are critical variates for HIV-1 fitness.     

Loss of Viral Fitness Associated with Multiple Gag and Gag-Pol Processing Defects in Human Immunodeficiency Virus Type 1 Variants Selected for Resistance to Protease Inhibitors In Vivo

We examined the viral replicative capacity and protease-mediated processing of Gag and Gag-Pol precursors of human immunodeficiency virus (HIV) variants selected for resistance to protease inhibitors. We compared recombinant viruses carrying plasma HIV RNA protease sequences obtained from five patients before protease inhibitor therapy and after virus escape from the treatment. Paired pretherapy-postresistance reconstructed viruses were evaluated for HIV infectivity in a quantitative single-cycle titration assay and in a lymphoid cell propagation assay. We found that all reconstructed resistant viruses had a reproducible decrease in their replicative capacity relative to their parental pretherapy counterparts. The extent of this loss of infectivity was pronounced for some viruses and more limited for others, irrespective of the inhibitor used and of the level of resistance. In resistant viruses, the efficiency of Gag and Gag-Pol precursor cleavage by the protease was impaired to different extents, as shown by the accumulation of several cleavage intermediates in purified particle preparations. We conclude that protease inhibitor-resistant HIV variants selected during therapy have an impaired replicative capacity related to multiple defects in the processing of Gag and Gag-Pol polyprotein precursors by the protease.     

Human Immunodeficiency Virus Type 1 Intergroup (M/O) Recombination in Cameroon

Here we describe, for the first time, recombinants between two highly divergent major groups of human immunodeficiency virus type 1 (HIV-1), M and O, within a Cameroonian woman infected with three different HIV-1 strains, a group O virus, a subtype D virus, and a recently reported IBNG (A/G)-like recombinant virus. Using nested extra-long PCR amplification, we sequenced from the pol region to the env region including accessory genes of the viral genome obtained from the patient's uncultured peripheral blood mononuclear cells and examined the phylogenetic position of each gene. Compared with sequential blood samples obtained in 1995 and 1996, there were multiple segmental exchanges between three HIV-1 strains (O, D, and IBNG) and all the recombinants appeared to be derived from a common M/O ancestor. Importantly, recombination between groups M and O occurred, even though the homology between these two groups is 69, 76, 68, and 55% in the gag, pol, vif-vpr, and env regions, respectively. Recombination between strains with such distant lineages may contribute substantially to generating new HIV-1 variants.