Nef-mediated enhancement of virion infectivity and stimulation of viral replication are fundamental properties of primate lentiviruses

Abstract

Nef is a multifunctional accessory protein of primate lentiviruses. Recently, it has been shown that the ability of Nef to downmodulate CD4, CD28, and class I major histocompatibility complex is highly conserved between most or all primate lentiviruses, whereas Nef-mediated downregulation of T-cell receptor-CD3 was lost in the lineage that gave rise to human immunodeficiency virus type 1 (HIV-1). Whether or not other Nef activities are preserved between different groups of primate lentiviruses remained to be determined. Here, we show that nef genes from a large variety of HIVs and simian immunodeficiency viruses (SIVs) enhance virion infectivity and stimulate viral replication in human cells and/or in ex vivo infected human lymphoid tissue (HLT). Notably, nef alleles from unpassaged SIVcpz and SIVsmm enhanced viral infectivity, replication, and cytopathicity in cell culture and in ex vivo infected HLT as efficiently as those from HIV-1 and HIV-2, their human counterparts. Furthermore, nef genes from several highly divergent SIVs that have not been found in humans were also highly active in human cells and/or tissues. Thus, most primate lentiviral Nefs enhance virion infectivity and stimulate viral replication. Moreover, our data show that SIVcpz and SIVsmm Nefs do not require adaptive changes to perform these functions in human cells or tissues and support the idea that nef alleles from other primate lentiviruses would also be capable of promoting efficient virus spread in humans.

FIG. 1.

Generation of HIV-1 NL4-3 constructs expressing heterologous HIV and SIV nef alleles. Restriction sites used to clone heterologous HIV and SIV nef alleles into the HIV-1 NL4-3 IRES-eGFP (61, 62) and HIV-1 NL4-3nef⁺Δ1Δ2 (46) constructs are indicated. Critical cis-regulatory elements encompassing the TPI region as well as the start of the LTR region and the position of the 266-bp U3 deletion downstream of nef in the NL4-3nef⁺Δ1Δ2 clones (46) are shown at the bottom.

FIG. 2.

The ability of Nef to enhance the infectivity of HIV-1 IRES-eGFP reporter viruses is conserved among different groups of primate lentiviruses. (A) P4-CCR5 indicator cells were infected with HIV-1 NL4-3 IRES-eGFP constructs containing the indicated HIV and SIV nef genes or defective nef alleles. Infections were performed in triplicate with three different virus stocks containing 5 ng of p24 antigen. Shown are average values of the nine measurements ± SDs compared to the infectivity of the virus expressing wild-type NL4-3 Nef (100%). The graph of values for sources of the various Nef alleles is color-coded as follows: black bars HIV-1 and HIV-2; gray bars, the simian counterparts SIVcpz and SIVsmm; blue bars, descendants of the viruses that recombined to become SIVcpz; white bars, all other SIVs. The lower dotted line indicates the infectivity of the nef-defective (nef⁻)control virus and the upper dotted line indicates the HIV-1 construct expressing the control wild-type NL4-3 nef allele. (B) Correlation between Nef-mediated enhancement of viral infectivity in P4-CCR5 and TZM-bl cells. (C) Enhancement of virion infectivity by nef alleles that do (group 2, green) or do not (group 1, red) downmodulate TCR-CD3. Average infectivities (± SEM) of HIV-1 IRES-eGFP constructs expressing nef alleles derived from HIV-1, SIVcpz, SIV-CP, HIV-2, SIVsmm, and all other SIVs (SIV AO). The numbers above the bars in panels A and C indicate the relative infectivity enhancement (n-fold) compared to the nef⁻ control virus. Ptt, P. t. troglodytes; Pts, P. t. schweinfurthii.

FIG. 3.

Various primate lentiviral Nef proteins increase HIV-1 infectivity. (A) P4-CCR5 cells were infected with recombinant HIV-1 constructs expressing the indicated nef alleles. Infections were performed in triplicate (each) with three independent virus stocks. (B) Correlation between Nef-mediated enhancement of NL4-3 TPI and IRES-eGFP constructs. (C) Average infectivities (± SD) of HIV-1 TPI constructs expressing nef alleles derived from the indicated groups of primate lentiviruses (see legend of Fig. 1 for details). Ptt, P. t. troglodytes; Pts, P. t. schweinfurthii; PPT, polypurine tract. AO, all other.

FIG. 4.

Primate lentiviral Nef proteins enhance HIV-1 infectivity independently of the viral coreceptor tropism. (A) P4-CCR5 cells were infected with recombinant R5-tropic HIV-1 constructs expressing the indicated nef alleles. Infections were performed in triplicate (each) with three independent virus stocks. Numbers above the bars indicate the relative infectivity enhancement (n-fold) compared to the nef⁻ control virus. (B) Correlation between the infectivities of X4- and R5-tropic HIV-1 nef recombinants. PPT, polypurine tract; Ptt, P. t. troglodytes; Pts, P. t. schweinfurthii.

FIG. 5.

HIV-1 Group M, N, and O and SIVcpzPtt and SIVcpzPts nef alleles enhance viral replication in human PBMCs with comparable efficiencies. (A) Representative replication kinetics of wild-type NL4-3 and nef recombinants. PBMCs were infected with HIV-1 TPI variants containing the indicated nef genes or a disrupted nef codon immediately after isolation and stimulated 3 days later. Virus production was monitored by RT assay. PSL, photon-stimulated luminescence. (B) Average levels of replication of HIV-1 NL4-3 recombinants expressing HIV-1 group M, N, and O or SIVcpz nef alleles. Results were derived from duplicate infections of two independent virus stocks. Similar results were obtained with cells derived from a different PBMC donor, except that the levels of replication in the absence of Nef were slightly higher and reached about 20% of that of wild-type NL4-3. (C) Average virus production in infected PBMC cultures. PBMCs were inoculated with the indicate NL4-3 nef recombinants, and cumulative production of p24 over 10 days was measured. Presented are means ± SEM as percentages of those measured in cultures infected with wild-type NL4-3. uninf, uninfected.

FIG. 6.

Replication of X4-tropic HIV-1 recombinants expressing nef alleles from HIV-2 and other heterologous primate lentiviruses. (A) Representative replication kinetics of the indicated HIV-1 nef recombinants. Infections were performed, and virus production was detected as described in the legend to Fig. 3. (B) Average levels of replication of HIV-1 NL4-3 recombinants expressing HIV-2, SIVsmm, SIV-CP, and all other (AO) SIV nef alleles. Results represent mean values of duplicate infections performed with two independent virus stocks of cells derived from one PBMC donor. (C) Cumulative virus production in PBMC cultures inoculated with the indicated NL4-3 nef recombinants relative to wild-type virus. Presented are means ± SEM. PSL, photon-stimulated luminescence; uninf, uninfected.

FIG. 7.

Replication of R5-tropic HIV-1 nef recombinants in PBMCs. (A) Representative replication kinetics of the indicated R5-tropic HIV-1 nef recombinants. (B) Cumulative virus production in PBMC cultures inoculated with the indicated X4- or R5-tropic HIV-1 NL4-3 nef recombinants relative to the respective construct expressing the wild-type NL4-3 nef allele. PBMCs from a single blood donor were infected in triplicate (each) with two independent virus stocks. Infections, sampling, and RT assay were all performed in the same experiment. Shown are average values ± SD of cumulative RT activities measured at days 5, 7, 10, 12, and 14 postinfection. (C) Correlation between the average RT production (n = 6) by PBMCs infected with X4- or R5-tropic HIV-1 nef recombinants. Ptt, P. t. troglodytes; uninf, uninfected. PSL, photon-stimulated luminescence.

FIG. 8.

SIVcpz nef alleles enhance HIV-1 replication and CD4⁺ T-cell depletion in HLT ex vivo. For each of the indicated HIV-1 nef variants, 18 tissue blocks were inoculated with virus stocks containing 0.5 ng of p24 antigen and medium was collected every 3 days. (A) Representative replication kinetics of wild-type NL4-3 and the indicated recombinants. (B and C) Average virus production and depletion of CD4⁺ T-cells in HLTs infected ex vivo with the HIV-1 nef recombinants. Tissues from five donors were infected with the indicated nef variants and cumulative p24 production by the tissue blocks over 15 days (B) or CD4⁺ T-cell depletion at the end of culture (C) was determined as described in the methods section. Shown are means ± SEM of these values as percentages compared to culture infected with the wild-type. PSL, photon-stimulated luminescence.

FIG. 9.

Replication and cytopathicity of HIV-1 recombinants expressing nef alleles derived from HIV-2 and different SIVs. (A) Representative replication kinetics of the indicated HIV-1 NL4-3 nef recombinants in ex vivo infected HLT. (B) Average amount of p24 released into the culture medium of the tissue blocks infected with the NL4-3 nef variants over a 15-day period. Values in panels B and C give means ± SEM of tissues from four donors. (C) CD4⁺ T-cell depletion in tissue blocks infected with HIV-1 constructs expressing various HIV and SIV nef alleles. (D) Correlation between p24 virus production and CD4⁺ T-cell depletion in HIV-1-infected HLT ex vivo.