The HIV-1 accessory protein Vif (virion infectivity factor) is required for the production of infectious virions by CD4(+) lymphocytes. Vif facilitates particle infectivity by blocking the inhibitory activity of APOBEC3G (CEM15), a virion-encapsidated cellular protein that deaminates minus-strand reverse transcript cytosines to uracils. We report that HIV-1 Vif forms a complex with human APOBEC3G that prevents its virion encapsidation. HIV-1 Vif did not efficiently form a complex with mouse APOBEC3G. Vif dramatically reduced the amount of human APOBEC3G encapsidated in HIV-1 virions but did not prevent encapsidation of mouse or AGM APOBEC3G. As a result, these enzymes are potent inhibitors of wild-type HIV-1 replication. The species-specificity of this interaction may play a role in restricting HIV-1 infection to humans. Together these findings suggest that therapeutic intervention that either induced APOBEC3G or blocked its interaction with Vif could be clinically beneficial.

In the human genome the apolipoprotein B mRNAediting enzyme catalytic polypeptide (APOBEC)3 gene has expanded into a tandem array of genes termed APOBEC3A-G. Two members of this family, APOBEC3G and APOBEC3F, have been found to have potent activity against virion infectivity factor deficient (⌬vif) human immunodeficiency virus 1 (HIV-1). These enzymes become encapsidated in ⌬vif HIV-1 virions and in the next round of infection deaminate the newly synthesized reverse transcripts. The lentiviral Vif protein prevents the deamination by inducing the degradation of APOBEC3G and APOBEC3F. We report here that two additional APOBEC3 family members, APOBEC3B and APOBEC3C, have potent antiviral activity against simian immunodeficiency virus (SIV), but not HIV-1. Both enzymes were encapsidated in HIV-1 and SIV virions and were active against ⌬vif SIV mac and SIV agm . SIV Vif neutralized the antiviral activity of APOBEC3C, but not that of APOBEC3B. APOBEC3B induced abundant G 3 A mutations in both wild-type and ⌬vif SIV reverse transcripts. APOBEC3C induced substantially fewer mutations. APOBEC3F was found to be active against SIV and sensitive to SIV mac Vif. These findings raise the possibility that the different APOBEC3 family members function to neutralize specific lentiviruses.

The nef gene, which encodes related cytoplasmic proteins in both human (HIV) and simian (SIV) immunodeficiency viruses is dispensable for viral replication in vitro. In contrast, in vivo experiments have revealed that SIV nef is required for efficient viral replication and development of AIDS in SIV infected rhesus monkeys, thus indicating that nef plays an essential role in the natural infection. We show that expression of the Nef protein from the HIV‐1 NL43 isolate in transgenic mice perturbs development of CD4+ T cells in the thymus and elicits depletion of peripheral CD4+ T cells. Thymic T cells expressing NL43 Nef show altered activation responses. In contrast, Nef protein of the HIV‐1 HxB3 isolate does not have an overt effect on T cells when expressed in transgenic animals. The differential effects of the two HIV‐1 nef alleles in transgenic mice correlate with down‐regulation of CD4 antigen expression on thymic T cells. The differential interactions of the NL43 and HxB3 nef alleles with CD4 were reproduced in a transient assay in human CD4+ CEM T cells. Down‐regulation of CD4 by nef in both human and transgenic murine T cells indicates that the relevant interactions are conserved in these two systems and suggests that the consequences of Nef expression on the host cell function can be analyzed in vivo in the murine system. Our observations from transgenic mice suggest that nef‐elicited perturbations in T cell signalling play an important role in the viral life cycle in vivo, perhaps resulting in elimination of infected CD4+ T cells.

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.Several murine models have been developed for studies of AIDS pathogenesis. Mice transgenic for either the entire or partial human immunodeficiency virus type 1 (HIV-1) genome develop symptoms with similarities to AIDS. In one model, mice expressing HIV-1 Nef developed a wasting syndrome characterized by the loss of CD4 ϩ cells (21, 45). In another model, SCID mice were reconstituted with human peripheral blood lymphocytes or fetal thymus and liver and then inoculated with HIV-1 (37). These have been useful for studies on mechanisms of CD4 ϩ cell depletion and for evaluation of therapeutic strategies.Current murine models lack a central feature of HIV-1-induced pathogenesis: virus replication. Inoculation of mice or rodents such as rats, hamsters, and guinea pigs with high-titer HIV-1 does not result in detectable viremia, nor does the virus replicate in murine cells in culture (37) or infect human-CD4 (hu-CD4) transgenic mice (33). Low levels of virus replication have been detected in experimentally infected rabbits (13,15,20,26,43) and cotton rats (30), but this does not induce pathogenesis. Development of a system in which HIV-1 could replicate in mice would allow the investigation of features...

PCR was used to clone isolates of the human immunodeficiency virus type 1 (HIV-1) nef gene directly from peripheral blood leukocytes of HIIV-1-infected individuals. A transient expression system with human CEM T ceils was used to assess the effect of nefon CD4 antigen expression on the ceHl surface. We show that CD4 down-regulation is a frequent property of primary HIV-1 nefafleles. Mutations in conserved amino acid motifs of Nef disrupted CD4 down-regulation. Our observations strongly suggest that CD4 down-regulation reflects a conserved function of nef, which is selected in vivo in human HIV-1 infection. Methodology described here provides quantitative assays to establish whether alterations in nef correlate with the dynamics of disease progression in human AIDS.