Accomplishing functional insertion of CD4 and CXCR4 into HIV-1 vector envelopes is the initial step towards targeting HIV-1 infected cells with the future long-term goal to inhibit HIV-1 replication. The assembly and functionality of vector particles carrying different chimeric CD4 proteins in their envelopes revealed that protein insertion into vector envelopes was readily achieved by either DNA co-transfections or by using cells that constitutively express CD4 or CD4/gpi. Significant differences in the targeted transduction of Env+
cells were not detected after replacing the transmembrane and cytoplasmic domains of CD4 with corresponding regions of either the HIV-1 envelope protein, the VSV glycoprotein G or the gpi anchor of the cellular DAF protein [42
]. This indicates that the origin of the CD4 anchor was not critical for receptor function during infections, which is consistent with earlier observations that Env binding needed for infection maps near the amino terminus (amino acids 40–60) of CD4 [43
For targeted transduction of cells that constitutively express the T cell tropic HIV-1 Env protein, vector insertion of both, CD4 and the CXCR4 coreceptor is needed. The level of endogenous CXCR4 expression in HeLa cells was sufficient for functional insertion of the coreceptor into vector particles and CXCR4 DNA co-transfection was not required. We conclude that HIV-1 readily inserts many viral and nonviral membrane proteins, and a large number of cellular proteins have previously been identified in HIV-1 envelopes [11
The transduction efficiencies of six different HIV(CD4) pseudotypes generated by co-transfection of HIV-1-Neo with a CD4 expression plasmid were very similar, giving rise to approximately 70 neomycin-resistant colonies per ng p24 (Figure ). HIV-1-Neo DNA transfection of HeLaT4 or S2 cells, which stably express CD4 and CD4/gpi, respectively, produced higher titers of pseudotyped particles yielding transduction efficiencies of approximately 280 colonies per ng p24. This compares favorably with wild-type HIV-1, which, at 1–3 × 103
infectious units per ng p24 [44
], is only three to ten fold more infectious. Thus exchanging the roles of viral and cellular membranes during membrane fusion does not dramatically reduce viral infectivity.
HIV-1-Neo(CD4/CXCR4) particles, independent of the type of CD4 anchor, were able to target and selectively infect cells expressing T cell tropic HIV-1 Env. In homogenous Env+
cell populations, this transduction was efficient. Unexpectedly, CD4+
particles also adhered very efficiently to a variety of human and animal cell types that do not express HIV-1 Env without leading to cell transduction. The competition between Env+
cells for CD4+
particles in mixed cell populations (Figures and ), the time course of CD4+
particle binding to Env+
cells (Figure ) and the inhibition of CD4+
particle adhesion by anti-CD4 antibody and by sCD4 (Figure ) all suggest that the specific Env-dependent and the Env-independent cell binding by these particles may have similar avidity. CD4-Env binding has a dissociation constant (KD
) of 5 nM, which is comparable to that of a good antibody-antigen binding complex [45
]. Our studies focused on the adhesion of entire virus particles to cells. Polyclonal anti-CD4 antibodies, which blocked HIV-1-Neo(CD4/CXCR4) or HIV-1-Neo(CD4/gpi/CXCR4) transduction of Env+
cells (Table ), did not completely prevent Env-independent cell adhesion (Figure , left panel). This suggests either a CD4 domain that may be involved in the Env-independent adhesion was not blocked by the antibody, or alternatively, CD4 may have recruited an additional membrane component into the vector envelope, which causes the new cell adhesion by the particles. In the latter case, the same or a similar membrane component must be present on the various cell types used in the studies.
The efficiency of this Env-independent cell adhesion by CD4+
particles was unexpectedly high. At a ratio of 1 Env+
to 8 Env-
cells, the transduction of Env+
cells was inhibited by over ninety percent (Figure ). HIV-1 is a cytolytic retrovirus and the number of cells expressing HIV-1 Env in vivo
are few [46
]. Although many silently infected cells are present in lymph nodes, only a small fraction (1 in 300) generally expresses HIV-1 Env [49
]. Since Env-independent cell adhesion occurred with all CD4+
particles and all cell types tested (Figure and ), we predict that targeted HIV(CD4) particles will be unable to infect Env+
cells at high efficiency in vivo
. This severely limits, if not totally abolishes, the feasibility of such an antiviral approach. Transduction of Env+
cells by HIV(CD4/CXCR4) particles, similar to the particles used in the present study, has previously been reported [11
], however the efficacy of cell targeting was not evaluated in mixed cell populations. Without this challenge, the potential limitations for cell targeting in vivo
were not recognized. Earlier assumptions that HIV(CD4/CXCR4) particles could potentially target HIV-1 infected cells in patients relied on the high specificity and presumed strength of the CD4-Env interaction.
The first clinical trial using a HIV-1-based vector is currently in progress [50
]. A vector was introduced at high efficiency into patients T4 lymphocytes ex vivo
. The purpose of this trial is to evaluate the safety of the HIV-1-based vector and to render the cells resistant to endogenous HIV-1 strains, which previously did not respond to antiviral drug cocktails. For gene therapy, effective transgene expression in vivo
generally requires, besides very high in vivo
grade vector titers, optimal access to the target cell population. The ex vivo
transduction of enriched human T-lymphocytes by a HIV-1-based vector can be expected to be much more efficient because this route of transgene delivery avoids vector infusion into complex cell populations or target tissues.
Vector distribution in vivo
is likely to be affected by proteins that are inserted into the vector envelope during viral budding from the producer cell. The unexpected diversion of targeted HIV(CD4/CXCR4) particles has important general implications for the use of enveloped viral vectors in vivo
. It demonstrates that the vector producer cell can greatly impact on the transduction efficiency of the vector depending on the nature of the target cell within a complex cellular environment in vivo
. Insight from detailed studies by several labs [19
] can be expected to help with the choice of vector producer cells for specific cell targeting applications in the future.
Our study compared the cell adhesion and the infectivity of vectors produced in the absence of Vpu, Nef and Env expression, which allowed to focus on the immediate effects of CD4 and CXCR4 insertion into vector envelopes. During wild-type HIV-1 infection, CD4 and the coreceptors CXCR4 and CCR5 are excluded from the HIV-1 envelope through interactions with HIV-1 Nef protein [40
]. The insertion of endogenous CXCR4 from HeLa and Hek293 cells was sufficient for the generation of infectious HIV-1-Neo(CD4/CXCR4) particles. Without Vpu, Env and Nef expression from both HDPack1 and HIV-1-Neo, insertion of CD4 and CXCR4 appeared undisturbed and the resulting vector particles efficiently infected homogeneous Env+
target cell populations. In fact, their infectivity was surprisingly effective and not dramatically reduced as one might anticipate in comparison to particles carrying the fusogenic VSV glycoprotein.
CD4 down-modulation benefits the release and the infectivity of the released particles. Whether CD4-dependent membrane adhesion already occurs intracellularly in the absence of HIV Env is currently unknown. As shown here, functional vector particles were efficiently released. Additional expression of Env interferes with CD4 cell surface expression and vice versa. Particles containing both Env and small amounts of CD4 could potentially be released from the cell. Upon release, these particles may either adhere directly to the cell from which they originated, or they may bind to and be arrested by adjacent non-host cells. In fact, a chimeric CD4 protein, which lacks the C-terminus and is not down-modulated by proteolytic degradation [55
] may effectively inhibit HIV-1 replication as previously suggested [7
Vpu plays an important role in the down-modulation of CD4. Vpu forms cationic-selective channels [57
]. Amiloride derivatives have been shown to be able to block Vpu channel activity and to reduce HIV-like particle release and viral replication in human macrophages [58
]. From our studies presented here, we infer that the reduced amount of particles released form T4 cells and macrophages in the absence of functional Vpu (or Nef) might carry increased amounts of CD4 in their envelope and thereby potentially decrease the HIV-1 infectivity through cell adhesion to a majority of non-host cell types. More detailed studies on the precise mechanism of the cell adhesion and the resulting particle diversion are necessary to better understand the potential involvement of CD4. If confirmed, inhibiting Vpu and/or Nef activity may potentially help augment the effectiveness of the current highly active antiretroviral therapy.