INTRODUCTION

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The phenomenon of homologous viral interference described for human immunodeficiency virus (HIV) as well as for animal retroviruses commonly correlates with the down-modulation of retrovirus cell receptors (51, 61, 62, 68). To date, very few models of HIV homologous viral interference independent of viral receptor exposure have been described; noninfectious pol-defective HIV virions are able to induce interference in coinfections of Jurkat cells with wild-type HIV (4). Moreover, after HIV infection at high multiplicity, CEM cells become resistant to homologous superinfection during short intervals (24 to 48 h), soon before the CD4 down-regulation takes place (69). Conversely, cells expressing the nondefective, nonproducer F12-HIV type 1 (HIV-1) variant are stably resistant to superinfection by replication-competent HIV, despite an unmodified level of CD4 exposure (16-18).

F12-HIV is an HIV-1 genome cloned from a Hut-78 cell clone infected with the supernatant of the peripheral blood lymphocytes (PBLs) of an HIV-seropositive patient. This variant is unable to code for even aberrant HIV particles as a consequence of a heavily altered structural viral protein pattern (19). Nucleotide sequence analysis failed to highlight genomic deletions, but the presence of many amino acid substitutions scattered along the whole viral genome (mainly in vif and pol genes) was observed (10).

Experimental evidence has suggested that F12-HIV-induced homologous viral interference could be the consequence of the inhibiting action of some F12-HIV viral protein(s). In fact, superinfecting HIV is able to enter into CD4⁺ F12-HIV-expressing cells and to retrotranscribe its genome, thus ruling out any effect of F12-HIV expression on the penetration of superinfecting HIV (17). Moreover, no encapsidation of the F12-HIV genome in virions emerging from either HIV-superinfected, F12-HIV-expressing cells (17) or cells chronically infected with HIV transduced with the F12-HIV genome (7) was observed, thereby excluding a direct role of F12-HIV genomic RNA.

To individuate the F12-HIV protein(s) involved in viral interference, vectors expressing each of the nine F12-HIV genes were separately transfected into highly HIV-susceptible HeLa CD4 cells that were subsequently infected with HIV-1. In this paper, we demonstrate that HeLa CD4 cells expressing F12-HIV Gag, Vif, or Nef protein are protected from infection with T-cell-line-adapted (TCLA) HIV-1 strains. Furthermore, expression of F12-HIV Vif or Nef protein driven by retroviral vectors is able to inhibit both the replication of T-tropic clinical HIV-1 isolates in transduced CEMss cells and the infectivity of virus released from cells chronically infected with HIV-1. Finally, the levels of action at which F12-HIV Vif or Nef protein inhibits HIV replication were determined.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Construction of expressing vectors. A schematic representation of the different molecular constructs used is given in Fig. 1. Intronless F12-HIV genes and rev-responsive elements (RRE) were obtained through PCR amplification from plasmid pUc/F12-HIV (10) or, to obtain wild-type vif or nef genes, from plasmid pNL4-3 (1). tat and rev genes were amplified by reverse transcriptase (RT)-PCR of total RNA extracted from parental Hut-78/F12 cells. DNA and RNA-PCR were performed as described elsewhere (55). The sequences of primers used for amplifications are reported in Table 1. The same couples of primers were used to amplify vif and nef genes from plasmids pUc/F12-HIV and pNL4-3. PCR products were purified and digested with enzymes whose recognition sequences are included on the 5' side of each primer in Table 1. F12-HIV vpr, vpu, env, tat, and NL4-3 and F12-HIV nef genes and RRE sequences were inserted into the polylinker of the immediate-early cytomegalovirus (IE-CMV)-promoted pcDNAI (Invitrogen, San Diego, Calif.)-expressing vector (Fig. 1I). Conversely, constructs expressing F12-HIV gag and pol and either NL4-3 or F12-HIV vif (Fig. 1II) were obtained by inserting each gene in the pcDNAI vector in which RRE sequences were previously inserted in BamHI-EcoRI sites. rev-expressing retrovirus vector (Fig. 1III) was obtained by inserting F12-HIV rev cDNA in a retrovirus vector (L-NGFr) expressing the cDNA of the low-affinity human nerve growth factor receptor (NGFr) (32) truncated in its cytoplasmic domain and driven by the Moloney murine leukemia virus (MLV) long terminal repeat (LTR). The L-NGFr retrovirus vector was obtained by inserting the NGFr cDNA excised from the pUc19 vector with EcoRI and HindII sites in EcoRI and HpaI sites of the polylinker of an LXSN retroviral vector (43) previously deleted (by BamHI-NcoI cuts) from the simian virus 40 (SV40) promoter-G418 resistance cassette. F12-HIV rev cDNA was placed under the control of the SV40 promoter, and the SV40-rev cassette was finally inserted in the XhoI site of the L-NGFr retrovirus vector. Retroviral constructs expressing F12-HIV nef (Fig. 1IV) and vif (Fig. 1V) genes were obtained by inserting, in the filled XhoI site of the L-NGFr retrovirus vector, the IE-CMV promoter-nef cassette (recovered from the pcDNAI-nef-expressing vector after HhaI-NotI digestion and filling) and the IE-CMV promoter-vif-RRE cassette (obtained from the pcDNAI-vif-RRE-expressing vector by BsaI-XbaI fragment digestion and filling), respectively.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Schematic map of pcDNAI-based vectors expressing rev-independent (I) or rev-dependent (II) F12-HIV genes. The structures of rev-, nef-, and vif-expressing retrovirus vectors (III, IV, and V, respectively) are also shown.

Cell cultures and transfections. HeLa CD4, Cos-1, GP+E86 (39), and AM12 (42) cells were grown in Dulbecco modified minimal essential medium supplemented with 10% decomplemented fetal calf serum (FCS) and, for HeLa CD4 cells only, 0.5 mg of G418 (70% activity; GIBCO-BRL, Gaithersburg, Md.) per ml. D10 (an Hut-78 cell clone chronically infected by an HIV-1 isolate) (19), C8166, CEMss, and H9/HTLVIII_B cells were maintained in RPMI 1640 medium supplemented with 10% decomplemented FCS. Human PBLs from healthy donors were activated for 48 h with phytohemagglutinin (PHA) and were then cultivated in RPMI 1640 medium supplemented with 20% FCS and 50 U of interleukin-2 (Roche, Nutley, N.J.) per ml.

HIV infections, transfections, and detection. Supernatants from acutely infected CEMss cells were used as the source of TCLA HIV-1 strains (i.e., NL4-3 and HTLVIII_B). Titers (ranging from 10⁶ to 10⁷ 50% tissue culture infective doses [TCID₅₀]/ml) were measured as previously described (18) by scoring the syncytium number on C8166 cells 5 days after the infection of serially diluted virus preparations. HIV-1 clinical isolates were obtained as PBL supernatants from AIDS patients. Titers (calculated as 50% infectious doses by infecting fresh PHA-stimulated PBLs from healthy donors with serially diluted supernatants) ranged from 5 × 10⁴ and 5 × 10⁵ infectious doses/ml. Supernatants of Cos-1 cells transfected with the 6.9 molecular clone (21) were used as the source of a vif-deleted HIV strain whose titers were measured in nanograms of HIV p24 per milliliter by an enzyme-linked immunosorbent assay (ELISA) (Antigen Capture Assay; Abbott, North Chicago, Ill.)

Molecular analyses. RNA analyses were performed on total RNA extracted with the RNA-FastI kit (Molecular Systems, San Diego, Calif.) following the manufacturer's recommendations. Northern blots were performed as described elsewhere (55) and hybridized with random-primed, ³²P-labelled F12-HIV-specific genes or T-tropic HIV coreceptor CXCR4 cDNA (20). Clones of HeLa CD4 rev-expressing cells transfected with the pcDNAI/RRE expression vector were selected on the basis of transcription of the F12-HIV RRE sequences. On the other hand, the abilities of HeLa CD4 pools to produce the respective F12-HIV or NL4-3 protein were tested by Western blotting or radioimmunoprecipitation assay (RIPA). Western blots were performed by lysing cells in phosphate-buffered saline (PBS)-1% Triton X-100. Samples of 50 µg of protein were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Hybond-ECL; Amersham, Buckinghamshire, United Kingdom) by electroblotting. Filters were blocked overnight at 4°C by 10% nonfat dry milk-0.1% Triton X-100 in PBS. The membranes were then incubated for 1 h at room temperature (r.t.) with the specific antibodies, washed twice with PBS, and incubated for 1 h at r.t. with the appropriate dilution of the horseradish peroxidase-labelled secondary antibody (Amersham). Finally, the filters were incubated in an enhanced chemioluminescence detection reagent (Amersham) for 1 min at r.t. and were exposed for 1 to 5 min to Hyperfilm-ECL (Amersham).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Isolation and characterization of HeLa CD4 cell clones expressing single HIV genes. As previously reported, structural HIV genes (i.e., gag, pol, and env) contain instability sequences that can be counteracted by the binding of the HIV Rev (and, consequently, of cellular factors) to the RRE sequences (6, 13, 22, 30, 50). Data on the stability of vif-specific RNA are more controversial (5, 25), but we observed that both wild-type and F12-HIV vif RNAs were degraded in the absence of Rev-RRE interaction (not shown). Thus, in vectors expressing rev-dependent genes, RRE sequences were added in 3' with respect to each HIV gene (except in vector expressing the env gene that itself contains the RRE sequences). These constructs were transfected in HeLa CD4 cells homogeneously expressing the NGFr-rev retrovirus vector, whereas vectors expressing rev-independent genes (i.e., tat, vpr, vpu, and nef) were transfected in parental HeLa CD4 cells.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Western blot analyses of HeLa CD4 pools transfected with vectors expressing either the F12-HIV gag, pol, tat, vpu, nef, or vif gene or transduced with rev-expressing retrovirus vector. Hygromycin-resistant HeLa CD4 cells (C) and parental Hut78/F12 cells were used as negative and positive controls, respectively. Molecular size markers (in kilodaltons) are given to the left of each panel.

View larger version (39K): [in this window] [in a new window]   FIG. 3.   RIPA of HeLa CD4 pools transfected with env (I)- or vpr (II)-expressing vectors. Hygromycin-resistant HeLa CD4 cells (C) and Hut-78/F12 cells were used as negative and positive controls, respectively. Cell lysates were incubated with normal () or hyperimmune (+) specific rabbit antisera. Molecular size markers (in kilodaltons) are given to the left of each panel.

View larger version (33K): [in this window] [in a new window]   FIG. 4.   CD4 fluorescence-activated cell sorter analyses of HeLa CD4 pools transfected with vector expressing each F12-HIV protein or with the hygromycin B resistance vector only (p220.2). HeLa and parental HeLa CD4 cells were used as negative and positive controls, respectively. Dotted lines identify higher values of fluorescence intensity detected in each pool labelled with unspecific phycoerythrin-conjugated mouse immunoglobulin Gs.

rev-RRE-dependent genes. Lower amounts of the F12-HIV Gag protein were produced in transfected HeLa CD4 cells with respect to the parental Hut-78/F12 cells (Fig. 2). In addition, no Gag cleavage products were detected (not shown), likely as the consequence of the absence of the virus-specific protease encoded in HIV by the pol gene. However, even when the F12-HIV gag gene was expressed in the context of the whole viral genome, no or a very low level of Gag cleavage was always observed (17, 19). In contrast to what was reported for other cells expressing HIV Gag or Gag-Pol polyprotein (34, 49, 58) and similarly to what was observed in parental Hut-78/F12 cells (19), no release of virus-like particles was observed in the F12-HIV gag-expressing HeLa CD4 cells (not shown).

rev-RRE-independent genes. HeLa CD4 cells homogeneously expressing the F12-HIV rev gene were obtained by transducing the cells with the L-NGFr-rev retroviral vector described above.

Effects of F12-HIV proteins on HIV replication: inhibitory action induced by gag, vif, and nef gene expression. The main goal of the present work was to determine which F12-HIV gene(s) is involved in F12-HIV-induced homologous viral interference. Thus, HeLa CD4 cells separately expressing each of the nine F12-HIV genes were infected with TCLA HIV-1 strains (i.e., NL4-3 or, in replicated experiments, HTLVIII_B) at a MOI of from 0.01 to 5. Titers of challenging virus were determined for C8166 cells and were confirmed for HeLa CD4 cells, except at the time of viral outcome, which was delayed in HeLa CD4 cells by about 4 to 6 days with respect to C8166 cells.

HIV-inhibiting F12-HIV proteins. HeLa CD4 cells expressing either F12-HIV gag, vif, or nef were resistant (at different degrees) to HIV infection (Fig. 5).

View larger version (23K): [in this window] [in a new window]   FIG. 5.   RT activities (measured in counts per minute per milliliter of supernatant and normalized for 106 cells after background subtraction) at different days post-HIV infection (p.i.) of nef-expressing HeLa CD4 cells (MOIs of 0.2 and 0.5 [I and II, respectively]); gag- or rev-expressing HeLa CD4 cells (MOI of 0.01 [III]); gag-, vif-, or rev-expressing HeLa CD4 cells (MOI of 0.2 [IV]); and vif- or rev-expressing HeLa CD4 cells (MOI of 0.5 [V]). As a control, hygromycin-resistant (Hr) HeLa CD4 cells were utilized. Challenging virus was adsorbed in a small volume (e.g., 0.15 ml in 24-well plates) in semiconfluent cell cultures for 1 h at 37°C, and then cells were refed with fresh complete medium. Data from a representative of three different experiments are reported.

View larger version (13K): [in this window] [in a new window]   FIG. 6.   RT activities in supernatants at different days post-HIV infection (p.i.) (MOI of 0.1) of rev-, NL4-3 vif-, or F12-HIV vif-expressing HeLa CD4 cells. As control, hygromycin-resistant (Hr) HeLa CD4 cells were utilized. Challenge with HIV was performed as described in the legend to Fig. 5. Data from a representative of two different experiments are reported.

F12-HIV proteins enhancing or not affecting HIV. Expression in HeLa CD4 cells of either F12-HIV rev, tat, or vpu led to an increased rate in HIV release at any MOI used (Fig. 7I and II [in which data from the infection with a MOI of 0.2 are reported]). This positive effect was not surprising, considering that (i) no amino acid substitutions with respect to replication-competent HIV strains have been detected in any of these three genes (10); (ii) Rev and Tat proteins play essential roles in the progression of the HIV life cycle; and (iii) data about the increase of HIV release in vpu-expressing cells have already been reported elsewhere (56).

View larger version (16K): [in this window] [in a new window]   FIG. 7.   RT activities in supernatants at different days post-HIV infection (p.i.) (MOI of 0.2) of rev-, env-, or pol-expressing HeLa CD4 cells (I); tat- or vpu-expressing HeLa CD4 cells (II); and vpr-expressing HeLa CD4 cells (III). As a control, hygromycin-resistant (Hr) HeLa CD4 cells were utilized. Challenge with HIV was performed as described in the legend to Fig. 5. Data from a representative of two different experiments are reported.

F12-HIV vif or nef expression also inhibits replication of T-tropic HIV-1 clinical isolates. To establish whether the above-described anti-HIV effects also operate against HIV-1 clinical isolates, HeLa CD4 cells were separately challenged with supernatants obtained from PBLs of 21 patients containing viral isolates characterized as T-tropic HIV strains. Even if a minority of these HIV isolates were able to penetrate parental HeLa CD4 cells (as assessed by infecting HeLa CD4-LTR--gal cells (67a), in no case were levels of HIV replication sufficient to be detected by RT or ELISA achieved.

View larger version (13K): [in this window] [in a new window]   FIG. 8.   RT activities in supernatants of CEMss cells transduced with retrovirus vectors expressing F12-HIV Vif or Nef protein at different days after infection (p.i.) (MOI of 1) with three different T-tropic HIV-1 clinical isolates. As a control, CEMss cells transduced with the empty L-NGFr retrovirus vector were also infected. A total of 106 cells were incubated for 24 h with the appropriate volumes of supernatants containing the challenging virus and were then washed and resuspended in fresh medium. Data from a representative of two different experiments are reported.

F12-HIV vif or nef gene expression decreases the infectivity of HIV-1 released from chronically infected cells. We already observed that the expression of the whole F12-HIV genome was able to block the infectivity of virions released by cells chronically infected with HIV-1 (7). Attempting to test whether the above-described anti-HIV effects of F12-HIV Vif or Nef protein could be reproduced in chronically infected cells, retroviral vectors expressing the relative F12-HIV genes were utilized to transduce a Hut-78 cell clone (D10) chronically infected with HIV-1 (19). As a control, D10 cells were also transduced with the empty L-NGFr retroviral vector. Supernatants from equal numbers of homogeneously expressing NGFr cells were tested in terms of amounts of retroviral particles by RT assaying and in terms of HIV infectivity by viral titration. As shown in Table 3, whereas no significant variations in RT activity were detected, a strong impairment in the infectivity of the released virus could be readily observed (more than 95 and 99% of reduction in vif- and nef-expressing D10 cells, respectively). Thus, the ability of both F12-HIV Vif and Nef proteins to inhibit HIV infectivity in a model of HIV chronic infection was demonstrated.

At which level does F12-HIV Vif or Nef protein interfere with the HIV replicative cycle? (i) F12-HIV Vif. To investigate at which level the anti-HIV effects of F12-HIV Vif protein act, we followed the fate of HIV infecting F12-HIV vif-expressing cells through single-cycle infection experiments.

View larger version (56K): [in this window] [in a new window]   FIG. 9.   (I) PCR analysis of lysates of HeLa CD4 cells infected with supernatants obtained from a single HIV replication cycle in F12-HIV rev- or vif-expressing HeLa CD4 cells and, as controls, in parental HeLa CD4 or in CD4-negative HIV-resistant HeLa cells. Semiconfluent cell cultures were infected with HIV (MOI of 2), and, after 1 h of adsorption, free virus was removed by several washings followed by trypsin treatment (see Materials and Methods). After an additional 16 h of incubation, supernatants were collected and titrated for HIV p24 content. Levels of HIV p24 in supernatants of challenged HeLa cells were consistently below the sensitivity threshold of the ELISA. After DNase treatment, volumes of each supernatant normalized for HIV p24 content (except for the supernatant from infected HeLa cells) were used to infect parental HeLa CD4 cells (200 pg for 5 × 104 cells). These cells were finally harvested at both 6 and 16 h after infection and lysed. Samples corresponding to 2 × 104 cells were amplified at the same time with HIV-LTR (forward and reverse 1) (A)- and -globin (B)-specific primers. The HIV-challenged cell types from which the supernatants were obtained to reinfect HeLa CD4 cells are indicated above each lane. Times (in hours) of cell collection after infection (p.i.) of HeLa CD4 cells are indicated at the bottom. Molecular sizes (in base pairs) of amplification products are shown. (II) PCR analysis of lysates of HeLa CD4 cells infected with DNase-treated supernatants obtained 48 h after cotransfections on rev-expressing HeLa CD4 cells of infectious molecular clone pNL4-3 HIV-1, together with vectors expressing wild-type or F12-HIV Vif protein. Infections and PCR analyses were performed as described above. Samples corresponding to 2 × 104 cells were amplified at the same time with HIV-LTR (A)- and -globin (B)-specific primers. The plasmids transfected in rev-expressing HeLa CD4 cells giving rise to the HIV used to reinfect HeLa CD4 cells are indicated above each lane. C+, amplification products obtained from a lysate of 104 CEMss cells acutely infected with strain NL4-3. Times (in hours) of cell collection after infection (p.i.) of HeLa CD4 cells are indicated at the bottom. Molecular sizes (in base pairs) of amplification products are shown.

View larger version (17K): [in this window] [in a new window]   FIG. 10.   (I) Amounts of HIV p24 in supernatants of PHA-activated human PBLs transduced with F12-HIV Vif-expressing retrovirus vector at different days after infection (p.i.) (MOI of 0.1) with the NL4-3 HIV-1 strain. As a control, PBLs transduced with the empty L-NGFr retrovirus vector were also infected. Challenging virus was adsorbed to 2 × 105 cells in 50 µl for 2 h at 37°C, and then cells were washed and refed with 1 ml of complete medium. (II) RT activities in supernatants of CEMss cells transduced with F12-HIV vif-expressing retrovirus vector at different days after infection (p.i.) with the vif-deleted HIV clone 6.9 (viral input, 2 and 20 ng of HIV p24/106 cells). As a control, CEMss cells transduced with the empty L-NGFr retrovirus vector were infected under the same conditions. Challenge with HIV was performed as already described for CEMss cells. Data from a representative of three different experiments are reported.

(ii) F12-HIV nef. Single-cycle infection experiments similar to those described above were set up on F12-HIV nef-expressing HeLa CD4 cells. We assessed that the expression of the F12-HIV Nef protein does not interfere with the early steps of viral infection. In fact, no inhibition of either retrotranscription process (as observed by PCR analysis [not shown]) or intracellular HIV p24 protein production (which, in contrast, at the latest times after infection considered, was enhanced by almost twofold in F12-HIV nef-expressing cells [Fig. 11I]) was observed. Conversely, a strong decrease in HIV p24 levels was detected in supernatants from F12-HIV nef-expressing cells with respect to those from control cells (Fig. 11II). No significant variations in infectivity between the virus released from F12-HIV nef-expressing cells and that from control ones were observed by titrating equal amounts (as measured in picograms of HIV p24) of HIV (not shown).

View larger version (18K): [in this window] [in a new window]   FIG. 11.   Amounts of intracellular (I) and supernatant (II) HIV p24 in F12-HIV nef-expressing cells in a single-cycle infection experiment. F12-HIV nef and, as a control, hygromycin-resistant (Hr) HeLa CD4 cells were infected with HIV (MOI of 2) and then treated as described in the legend to Fig. 9. Values reported were obtained from cultures of 5 × 104 cells in 0.5 ml that were tested at different hours postinfection (p.i.). Results from a representative of three different experiments are reported.

View larger version (30K): [in this window] [in a new window]   FIG. 12.   (I) Amounts of HIV p24 in supernatants of wild-type (NL4-3) or F12-HIV nef-expressing cells after transfection with the infectious molecular clone HIV-1 NL4-3. A total of 5 × 104 cells were seeded in 24-well plates and, after 24 h, transfected in triplicate with 2 µg of the pNL4-3 plasmid. After an overnight incubation, cell cultures were washed and complete medium was added. Amounts of HIV p24 protein (whose levels 24 h posttransfection were, under all conditions, below the sensitivity threshold of the ELISA) were measured at the indicated times posttransfection (p.t.). (II) Amounts of HIV p24 in supernatants of HeLa CD4 cells cotransfected with the infectious molecular clone HIV-1 pNL4-3 together with vectors expressing wild-type or F12-HIV Nef proteins. A total of 2 × 105 cells were seeded in 6-well plates and after 24 h were cotransfected in duplicate with 1 µg of infectious molecular clone pNL4-3 and a 10-fold molar excess of pcDNAI-based nef-expressing vectors. Measurements of HIV p24 protein were performed at the indicated times posttransfection (p.t.). Values from a representative of three different experiments are reported.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

A number of HIV mutagenized viral proteins able to inhibit the replication of wild-type HIV have been described elsewhere (28, 38, 46, 67). An anti-HIV effect induced by the expression of the wild-type HIV Env glycoprotein has also been reported (63, 64); the latter is, however, correlated with the masking of the CD4 receptors, rather than with a direct negative action on the viral life cycle. Furthermore, wild-type nef gene expression can induce the protection of host cells from HIV infection as the consequence of the down-regulation of CD4 HIV receptors (3, 24). Negative trans-dominant effects on HIV viral replication induced by mutagenized forms of Gag HIV proteins have also been reported (67). These results were obtained by infecting with HIV HeLa CD4 cells stably expressing mutagenized gag genes inserted in an env/nef HIV genome. Conversely, the data here presented describe the inhibitory effects on HIV replication induced by a full-length, naturally occurring F12-HIV Gag protein. The F12-HIV gag gene presents 10 amino acidic substitutions that have no counterparts in any other gag gene from HIV-1 genomes sequenced so far. In particular, two substitutions reside in p17 (five on p24 and three on p15 regions) (Fig. 13), and they do not overlap those reported in the already described HIV-inhibiting gag mutants (67).

View larger version (34K): [in this window] [in a new window]   FIG. 13.   Amino acid sequences of F12-HIV Gag, Vif, and Nef proteins. The amino acid substitutions typical (i.e., not detectable in the sequences of any T-tropic HIV-1 isolates [HXB2, BRU, SF2, NL4-3, PV22, and MN] used to construct the reference consensus sequence) of the F12-HIV genes are indicated.

Considering that higher levels of the p55 Gag protein were detected in cells expressing the entire F12-HIV genome with respect to those coexpressing rev and gag genes only, it is likely that the data here presented underestimate the inhibitory effect of F12-HIV Gag protein expressed in the context of the whole viral genome.

The function of wild-type Vif protein was originally correlated with the infectivity of HIV particles. Recently, it has been shown that Vif associates with viral core structures (37) and that defects in Gag processing have been observed in PBLs infected with vif-defective HIV (57). In general, cells that do not sustain the replication of vif-defective HIV strains are defined as vif-nonpermissive cells (e.g., PBLs and H9), whereas cell lines in which Vif protein is dispensable for HIV replication are defined as vif-permissive cells (e.g., C8166, HeLa CD4, and CEMss). This was explained by admitting the presence of a vif cellular homolog in vif-permissive cells or, alternatively, of a vif-responsive HIV inhibitor in vif-nonpermissive ones (23, 54). vif is the most mutated F12-HIV gene, bearing 31 point mutations leading to 14 amino acid substitutions characteristic of the F12-HIV genome (Fig. 13). In this paper, we originally show an inhibitory effect induced by a Vif mutant protein in vif-permissive cells as well as in vif-nonpermissive ones. Thus, the effectiveness of F12-HIV Vif protein in inhibiting HIV replication seems independent of the ability of challenged cells to complement the wild-type vif function(s). Furthermore, inhibition of the replication of vif-deleted HIV in vif-permissive cells constitutively expressing the F12-HIV Vif protein may suggest that this mutant protein is able to negatively influence the vif-complementing activity. Taken together, the data presented here disclose the possibility that F12-HIV Vif protein may counteract both wild-type protein and, possibly, the vif cellular homolog function(s).

Studies of the level of action of the F12-HIV Vif-induced anti-HIV effect indicate that (i) in the first infection cycle, the presence of the F12-HIV Vif protein does not influence HIV replication and (ii) the replication efficiency of HIV emerging from F12-HIV vif-expressing cells after the first replication cycle is markedly reduced, as demonstrated by the consistent inhibition in the early retrotranscription step observed by PCR analysis of infected vif-permissive cells.

The following hypotheses may be considered. (i) The observed reduced amounts of retrotranscription products may have derived from an impaired ability in the cell entry of HIV obtained from F12-HIV vif-expressing cells. This could be the consequence of an altered viral assembly in producer cells, as was already observed through infection with HIV coding for mutagenized Vif proteins (8, 31, 54, 57). (ii) Alternatively (or in addition), F12-HIV Vif protein is encapsidated in (at least) part of the emerging HIV and interferes with the early replication step in subsequent viral cycles. This could be conceivable by admitting a role for Vif protein in the early retrotranscription process, as has been suggested by the evidence that vif HIV strains are inhibited in the early replication steps when they infect cells unable to support the vif deficiency (vif-nonpermissive cells) (59, 70).

The inhibition of retrotranscription was observed in parental HeLa CD4 cells at early times after infection but was not still effective at later times. This was probably due to the replication of HIV escaping (in view of the high MOI used) the F12-HIV Vif-induced inhibition effect during the first replication cycle or, alternatively, to the effect of intracellular vif complementing factor(s) overcoming the possible negative trans-dominant effect of F12-HIV Vif protein. Similar mechanisms could be based on the reduced but not abolished infectivity of retrovirions released by cells chronically infected with HIV-1 that were transduced with F12-HIV vif-expressing retrovirus vector.

We previously published that when the vif gene of the syncytium-inducing, rapid-high NL4-3 strain was replaced by the F12-HIV vif one, the phenotype of the resulting chimeric provirus transfected in vif-permissive cells (i.e., HeLa CD4 and SupT1) was dramatically mutated in a slow-low, non-syncytium-inducing HIV (11). This observation, together with the results reported here, demonstrates that expression of the F12-HIV vif gene is able to negatively control HIV replication either in cis or in trans configurations.

It has been reported that Nef protein expression (i) down-regulates the CD4 HIV receptors (3), (ii) increases both proviral DNA synthesis (2) and HIV infectivity (45, 60), and (iii) is involved in SIV-induced AIDS pathogenesis (35). The F12-HIV nef gene shows 19 point mutations resulting in 13 amino acid substitutions (10), 3 of which have no counterparts in any nef gene sequenced so far (Fig. 13). The phenotype induced by the F12-HIV Nef protein seems quite different from that described for wild-type Nef. In fact, not only is F12-HIV Nef unable to down-regulate CD4 receptors, but in HeLa CD4 cells it induces a strong inhibition rather than an increase in HIV infectivity. To our knowledge, this is the first report concerning a negative effect of the Nef protein on the HIV life cycle acting through a mechanism independent of CD4 down-regulation. The results from single-cycle infection experiments demonstrated that the F12-HIV Nef protein exerts its anti-HIV activity in a step subsequent to viral protein synthesis. It has been proposed that wild-type Nef increases HIV viral infectivity acting at the level of viral core formation (2, 44). Our data about the F12-HIV Nef level of action seem compatible with this model, by admitting that the mutated Nef protein is able to act in the appropriate HIV replication step, but likely in a wrong manner. The already reported evidence that Nef could be dispensable for HIV replication in immortalized cell lines (45, 60) may enforce the hypothesis that F12-HIV Nef protein exerts its HIV inhibitory effect through a direct or indirect interaction with viral proteins other than wild-type Nef. This was also suggested by the evidence that expression of the nef mutant inhibits replication of HTLVIII_B, an HIV-1 strain defective in nef expression. The recently described ability of wild-type Nef protein to induce phosphorylation of HIV matrix Gag protein through binding of a cellular kinase (65) could be considered a demonstration of the possibility that the Nef protein influences the structure of other HIV proteins.

HIV-1 emerging from chronically infected cells expressing F12-HIV Nef protein was impaired in its infectivity in spite of essentially unvaried amounts of released retrovirions. These data may apparently contrast with the evidence that in acute HIV infection, an inhibition at the level of virus assembling and/or release could be observed in F12-HIV nef-expressing cells, without any evident impairment in the infectivity of released virus. It is possible that different models of virus-cell interactions (i.e., acute versus chronic infections) allowed detection of different steps during which F12-HIV nef expression could inhibit HIV replication. In particular, in cells chronically infected with HIV, the F12-HIV Nef protein may induce an anti-HIV effect through its encapsidation in released virions (as demonstrated for wild-type Nef proteins) (44, 48), thus exerting its anti-HIV effect on target cells.

The block of HIV replication induced by F12-HIV Vif or Nef protein persists at MOIs of 0.2 and 0.5 TCID₅₀/cell, respectively. This is a substantial improvement with respect to the higher MOI at which viral interference was observed in HeLa CD4 clones expressing the full-length F12-HIV genome (0.017 TCID₅₀/cell) (17), which indeed also expresses HIV-enhancing proteins such as Tat, Rev, and Vpu.

In conclusion, the results regarding the negative effects of either Gag, Vif, or Nef protein in HIV replication seem interesting since they (i) definitively demonstrate the involvement of these proteins in F12-HIV-induced homologous viral interference, (ii) could represent a model by which to study the already largely unknown functions that Vif and Nef proteins play in the HIV life cycle, and (iii) allow us to consider F12-HIV gag, vif, and nef genes as new candidates for experiments in anti-HIV gene therapy. This perspective is strongly encouraged by the evidence that the anti-HIV effect of F12-HIV Vif or Nef protein is not restricted to laboratory-adapted HIV-1 strains but is fully operative also when cells are challenged with T-tropic HIV-1 clinical isolates. Moreover, the ability of retroviral constructs expressing F12-HIV Vif or Nef protein to inhibit the viral infectivity of HIV released from already infected cells is a very important feature regarding the attempt to perform gene therapy experiments in cells also from HIV-infected AIDS patients.