HIV-1 strains differ significantly in their dependence on Nef for optimal infectivity (43
), and in the case of HIV-1SF2
it has been shown that the relatively high Nef dependency of this particular isolate is determined by its env
). A role for Env in the function of Nef is also suggested by the observation that pseudotyping of HIV-1 particles with the glycoproteins of some unrelated viruses largely suppresses the requirement for Nef (3
). In particular, Nef was no longer required after pseudotyping with glycoproteins that fuse at low pH. Based on these findings, it has been proposed that the effect of Nef on infectivity depends on whether entry occurs at the cell surface or through endocytosis (3
). However, it is noteworthy that a substantial effect of Nef on infectivity has been observed only when retroviral glycoproteins were used to complement HIV-1 particles (21
). Thus, it has not been ruled out that the dependency on Nef is a specific property of retroviral Env proteins. Consistent with this possibility, it was recently reported that Nef stimulates the formation of pseudotypes with the glycoproteins of certain retroviruses but not with those of other viruses (65
In a study by Zhou and Aiken, Nef differentially affected the infectivity resulting from intervirion fusion after mixing donor HIV-1 particles containing defective cores with target virions pseudotyped with the HIV-1 receptors (81
). Intriguingly, Nef enhanced infection only when present in the donor virus. This finding supported the hypothesis that Nef affects the function of HIV-1 Env during virus production and therefore does not act on receptor pseudotypes. However, our results now show that Nef can increase the infectivity of receptor pseudotypes for Env-expressing target cells, and they thus rule out the possibility that the presence of a viral glycoprotein during virus production is required for the function of Nef.
While Nef enhanced the infectivity of CD4/CXCR4 and Tva receptor pseudotypes, it had no effect on pseudotype formation, because the incorporation of viral receptors into Env-deficient HIV-1 particles was unaffected. This observation contrasts with a recently described effect of simian immunodeficiency virus Nef on the incorporation of glycoproteins derived from distantly related retroviruses, which appeared to be due to increased colocalization of Gag and Env proteins in late endosomes (65
). However, it is perhaps worth noting in this regard that we have not observed any effects of Nef on the incorporation of HIV-1 Env protein into HIV-1 particles (57
Interestingly, the effect of Nef on CD4-chemokine receptor pseudotypes was inhibited by dominant-negative dynamin 2, which also inhibits the effect of Nef on the infectivity of authentic HIV-1 virions (57
). Furthermore, as has been shown for HIV-1 virions lacking Nef (19
), the infectivity of nef
-negative receptor pseudotypes could be enhanced to levels close to those obtained with nef
-positive pseudotypes by disrupting the actin cytoskeleton in target cells. These parallels suggest that Nef enhances the infectivity of authentic virions and of receptor pseudotypes by a similar mechanism.
It has been suggested that Nef enhances viral infectivity by increasing the levels of newly synthesized cholesterol in progeny virions (80
). However, we recently observed that dominant-negative dynamin 2 counteracts the effect of Nef on infectivity without decreasing overall virion cholesterol levels (57
). More recently, Brugger et al. have analyzed the lipidome of HIV-1 particles produced in T lymphocytes by a highly sensitive and quantitative mass spectrometry approach, and they failed to detect any appreciable impact of Nef on the overall cholesterol content of HIV-1 virions (17
). Thus, although Nef affects cholesterol homeostasis (53
), the study by Brugger et al. clearly demonstrates that Nef can enhance virion infectivity without affecting overall virion cholesterol levels.
Nef did not enhance the infectivity of HIV-1 particles pseudotyped with EnvA, a retroviral glycoprotein that requires exposure to low pH to elicit full fusion and to mediate viral penetration into the cytosol (9
). While this result appeared consistent with the model that entry through an endosomal compartment circumvents the requirement for Nef, we also observed that Nef did significantly enhance viral infectivity if the orientation of the EnvA-Tva interaction was reversed. Previous work has suggested that Tva pseudotypes are duplicating the events of normal viral entry, because the functional requirements for Tva and EnvA are the same whether they are on the virion or on the target cell surface (7
). Furthermore, our results show that the infectivity of HIV-1(Tva) pseudotypes for cells expressing EnvA is profoundly inhibited by baf A1
Cl, implying that Tva pseudotypes maintain the requirement for exposure to low pH in an acidic intracellular compartment. We thus infer that an entry pathway that involves fusion at the plasma membrane is not an absolute requirement for the enhancement of infectivity by Nef.
Why did the effect of Nef on infectivity differ depending on the direction of the EnvA-Tva interaction? It is possible that this difference is related to the markedly different baseline infectivities of the EnvA and Tva pseudotypes. Consistent with this notion, the magnitude of the effect of Nef on HIV-1 infectivity appears to correlate inversely with the baseline infectivity observed in the absence of Nef (73
). Also, the requirement for Nef for optimal HIV-1 infectivity can be reduced by increasing the amount of Env on the virion or the receptor density on the target cells (73
). Thus, Nef may become more important if the number or affinity of functional Env-receptor interactions is relatively low, which we assume tends to be the case if the normal direction of the interaction is reversed. However, the effect of Nef is not strictly linked to baseline infectivity, because Nef did not enhance the relatively low infectivity of naked HIV-1 virions for cells expressing VSV G.
It has been demonstrated that Nef does not affect the transfer of a β-lactamase-Vpr protein chimera from virions into target cells as a result of Env-mediated fusion (20
). However, this observation does not rule out that Nef facilitates an entry step subsequent to the formation of an initial fusion pore, such as fusion pore expansion, which is necessary to deliver the relatively bulky viral core into the cytosol. Indeed, a study which monitored individual retroviral fusion events by fluorescence microscopy indicates that pore expansion to allow the cytosolic delivery of viral cores occurs only slowly after Env-induced lipid mixing (49
). Also, it is thought that fusion pore expansion is the most energy-demanding fusion stage that requires the participation of the largest number of activated fusion proteins (23
). As has been pointed out previously (20
), a role of Nef in facilitating this apparently rate-limiting step would be consistent with the finding that Nef enhances the cytoplasmic delivery of HIV-1 cores (67
) and could also explain the dependence of the effect of Nef on the Env-receptor interaction. However, by showing that receptor pseudotypes remain responsive to Nef, the present study argues against a simple model in which Nef would act directly on Env.