D Braaten

Cyclophilin A regulates HIV-1 infectivity, as demonstrated by gene targeting in human T cells.

The human immunodeficiency virus type 1 (HIV‐1) Gag polyprotein binds most members of the cyclophilin family of peptidyl‐prolyl isomerases. Of 15 known human cyclophilins, cyclophilin A (CypA) has been the focus of investigation because it was detected in HIV‐1 virions. To determine whether CypA promotes HIV‐1 replication, we deleted the gene encoding CypA (PPIA) in human CD4+ T cells by homologous recombination. HIV‐1 replication in PPIA−/− cells was decreased and not inhibited further by cyclosporin or gag mutations that disrupt Gags interaction with cyclophilins, indicating that no other cyclophilin family members promote HIV‐1 replication. The defective replication phenotype was specific for wild‐type HIV‐1 since HIV‐2/SIV isolates, as well as HIV‐1 bearing a gag mutation that confers cyclosporin resistance, replicated the same in PPIA+/+ and PPIA−/− cells. Stable re‐expression of CypA in PPIA−/− cells restored HIV‐1 replication to an extent that correlated with steady‐state levels of CypA. Finally, virions from PPIA−/− cells possessed no obvious biochemical abnormalities but were less infectious than virions from wild‐type cells. These data formally demonstrate that CypA regulates the infectivity of HIV‐1 virions.

Envelope-Dependent, Cyclophilin-Independent Effects of Glycosaminoglycans on Human Immunodeficiency Virus Type 1 Attachment and Infection

ABSTRACT Cell surface glycosaminoglycans (GAGs), in particular heparan sulfate (HS), have been proposed to mediate the attachment of human immunodeficiency virus type 1 (HIV-1) to target cells prior to virus entry, and both the viral gp120 envelope protein and virion-associated cyclophilin A (CypA) have been shown to directly interact with HS and its analogues. To determine the role of GAGs in HIV attachment and infection, we generated HIV-susceptible derivatives of CHO cell lines that either express high levels of GAGs (CHO-K1) or lack GAGs (pgsA745). Using a panel of HIV-1 envelopes, we found that cell surface GAG-mediated effects on virion attachment and infection vary in an envelope strain-dependent but coreceptor-independent manner. In fact, cell surface GAG-mediated enhancement of infection is confined to isolates that contain a highly positively charged V3-loop sequence, while infection by most strains is apparently inhibited by the presence of GAGs. Moreover, the enhancing and inhibitory effects of polycations and polyanions on HIV-1 infection are largely dependent on the presence of cell surface GAGs. These observations are consistent with a model in which GAGs influence in vitro HIV-1 infection primarily by modifying the charge characteristics of the target cell surface. Finally, the effects of GAGs on HIV-1 infection are observed to an equivalent extent whether CypA is present in or absent from virions. Overall, these data exclude a major role for GAGs in mediating the attachment of many HIV-1 strains to target cells via interactions with virion-associated gp120 or CypA.

An Optimized CD8+ T-Cell Response Controls Productive and Latent Gammaherpesvirus Infection

ABSTRACT Strategies to prime CD8+ T cells against Murine gammaherpesvirus 68 (γHV68; MHV68) latency have, to date, resulted in only limited effects. While early forms of latency (<21 days) were significantly reduced, effects were not seen at later times, indicating loss of control by the primed CD8+ T cells. In the present study, we evaluated CD8+ T cells in an optimized system, consisting of OTI T-cell-receptor (TCR) transgenic mice, which generate clonal CD8+ T cells specific for Kb-SIINFEKL of OVA, and a recombinant γHV68 that expresses OVA (γHV68.OVA). Our aim was to test whether this optimized system would result in more effective control not only of acute infection but also of later forms of latent infection than was seen with previous strategies. First, we show that OTI CD8+ T cells effectively controlled acute replication of γHV68.OVA in liver, lung, and spleen at 8 and 16 days after infection of OTI/RAG mice, which lack expression of B and CD4+ T cells. However, we found that, despite eliminating detectable acute replication, the OTI CD8+ T cells did not prevent the establishment of latency in the OTI/RAG mice. We next evaluated the effectiveness of OTI T cells in OTI/B6 animals, which express B cells—a major site of latency in wild-type mice—and CD4+ T cells. In OTI/B6 mice OTI CD8+ T cells not only reduced the frequency of cells that reactivate from latency and the frequency of cells bearing the viral genome at 16 days after infection (similar to what has been reported before) but also were effective at reducing latency at 42 days after infection. Together, these data show that CD8+ T cells are sufficient, in the absence of B cells and CD4+ T cells, for effective control of acute replication. The data also demonstrate for the first time that a strong CD8+ T-cell response can limit long-term latent infection.