Adaptation of HIV-1 to cells with low expression of the CCR5 co-receptor.

Products Related

Product# 1039 HIV-1 YU2 (M Tropic)Envelope Glycoprotein gp41

Product# 1081 HIV-1 gp120 (ADA)

Product# 1011 HIV-1 gp120 (subtype C)

Introduction

The metastable human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer ((gp120/gp41)3) mediates entry into target cells (Wyatt and Sodroski, 1998). Virus entry is triggered by Env binding sequentially to CD4 and a coreceptor, most often CCR5 but also CXCR4 (Klatzman et al., 1984; Dalgleish et al., 1984; Cocchi et al., 1995; Deng et al., 1996; Feng et al., 1996; Alkhatib et al., 1996; Choe et al., 1996; Doranz et al., 1996; Dragic et al., 1996). CD4 binding drives Env from its unliganded conformation (State 1) through an intermediate (State 2) to the full CD4-bound conformation (State 3) (Munro et al., 2014; Herschhorn et al., 2016). In State 3, Env assumes a pre-hairpin intermediate conformation in which the CCR5-binding site on gp120 and the heptad repeat (HR1) coiled coil on gp41 are formed and exposed (Wu et al., 1996; Trkola et al., 1996, Furuta et al., 1998; Si et al., 2004; He et al., 2003; Koshiba and Chan, 2003). The hydrophobic fusion peptide at the gp41 N-terminus is thought to interact with the target cell membrane during this process (Freed et al., 1990; Kowalski et al., 1987). CCR5 binding (for CCR5-tropic (R5) HIV-1) or CXCR4 binding (for CXCR4-tropic (X4) HIV-1) triggers the pre-hairpin intermediate (State 3) to form the gp41 six-helix bundle (Lu et al., 1995; Chan et al., 1997; Tan et al., 1997; Weissenhorn et al., 1997). The formation of this very stable, post-fusion six-helix bundle is thought to promote the fusion of the viral and target cell membranes (Melikyan et al., 2000).

HIV-1 variants with lower requirements for CD4, including viruses that are completely CD4-independent, have been generated in the laboratory (Kolchinsky et al., 1999; Edwards et al., 2001; Dumonceaux et al., 1998; Zhang et al., 2002). CD4-independent viruses exhibit the ability to sample downstream Env conformations (State 2 and/or State 3) spontaneously, a property dictated by determinants in both gp120 and gp41 subunits (Haim et al., 2011; Kolchinsky et al., 2001a,b; Edwards et al., 2002; LaBranche et al., 1999; Hoffman et al., 1999; Dumonceaux et al., 2001; Musich et al., 2011). Natural HIV-1 variants derived from the central nervous system often exhibit a reduced dependence on CD4 and efficiently infect macrophages and microglia, which express low levels of CD4 (Martin et al., 2001; Thomas et al., 2007; Peters et al., 2004; Gorry et al., 2002; O’Connell et al., 2013).

The vast majority of transmitted/founder HIV-1 and most HIV-1 strains in individuals with established infections are CCR5-tropic (Peters et al., 2004; Lin et al., 2012; Keele et al., 2008; Melby et al., 2006). Clinical observations suggest that blocking Env-CCR5 binding will suppress HIV-1 infection. For example, in 2009, an HIV-1-infected patient with acute myeloid leukemia received a stem cell transplant from a donor homozygous for CCR5Δ32, which encodes an N-terminally deleted CCR5 protein that does not support HIV-1 infection (Liu et al., 1996). Since then, this individual has had an undetectable viral load and a sustained reconstitution of his immune system in the absence of antiretroviral therapy (Hutter et al., 2009; Allers et al., 2011).

Small-molecule CCR5 antagonists have been used to treat HIV-1 infection (Anastassopoulou et al., 2011; Lalezari et al., 2005; Roche et al., 2011; Tilton et al., 2010a). Maraviroc (MVC) is an FDA-approved HIV-1 entry inhibitor that binds in the hydrophobic pocket formed by the CCR5 transmembrane helices and stabilizes a CCR5 conformation that resists efficient gp120 binding (Garcia-Perez et al., 2011; Tan et al., 2013). HIV-1-infected individuals treated with MVC exhibited reduced viral loads, followed by selection for previously undetected CXCR4-using viruses or the evolution of CCR5-tropic viruses capable of using MVC-bound CCR5 as a coreceptor (Lalezari et al., 2005; Tilton et al., 2010a,b; Westby et al., 2007; Jiang et al., 2015; Berro et al., 2011; Westby et al., 2007). These latter MVC-resistant viruses have altered residues in the gp120 V3 stem that enhance binding affinity to the CCR5 N-terminus and to the drug-bound extracellular loops, despite overall decreases in entry and replication capacity (Roche et al., 2011; Tilton et al., 2010a,b; Berro et al., 2012). HIV-1 adapted to vicriviroc, an investigational CCR5 antagonist, displayed altered residues in gp41 that have been proposed to lead to increased triggering of the fusion peptide as well as gp120 changes that enhanced affinity for the CCR5 N-terminus (Lalezari et al., 2005; Berro et al., 2012). These studies indicate that HIV-1 can evolve to use conformationally altered CCR5 for viral entry.

Strategies to interfere with CCR5 binding would benefit from additional knowledge about the structures of the CCR5-bound Env trimer and downstream intermediates, the Env determinants of these conformational transitions, and the molecular dynamics required for viral entry. Here, we generate HIV-1 isolates that infect cells with low levels of CCR5, hypothesizing that the adaptation-associated changes in Env will identify regions that are critical for CCR5 binding or CCR5-triggered membrane fusion. We tested the extent to which HIV-1 can evolve to adapt to limiting amounts of CCR5 by progressively reducing the levels of CCR5 expressed on target cells and monitoring the compensatory changes in the adapted HIV-1 Env. The starting virus for these studies was a chimeric HIV-1 with the envelope glycoproteins from HIV-1JR-FL, a macrophage-tropic brain-derived virus that was already able to infect cells with moderately low levels of CCR5 (O’Brien et al., 1990). After extensive passage, viruses that can infect cells with ~1300 CCR5 molecules per cell were generated. The adapted viruses demonstrated enhanced infectivity compared to the starting virus during cell-cell transmission, but cell-free infectivity was poor. The Env changes required for the adaptation to low CCR5 usage did not result in an increase in CCR5 binding. However, the adapted viruses were more sensitive to CD4 triggering and to neutralization by particular antibodies, indicating that the Env from these viruses is predisposed to make transitions from a State 1 conformation.

Go to:

Results

Adaptation of HIV-1NL4.3(JR-FL) to replicate in cells expressing low levels of CCR5

We adapted an HIV-1 with the HIV-1JR-FL Env to infect cells with low levels of CCR5 by serially passaging the virus in cells in which the level of CCR5 expression was gradually decreased. Cf2Th canine cells constitutively expressing human CD4 and expressing human CCR5 in a Tet-regulated fashion were used as target cells for the adaptation (Fig. 1). Two Cf2Th clones that express high levels of CD4 and either low or high ranges of cell-surface CCR5 upon doxycycline treatment, herein called R5-Low and R5-High cells, were used for HIV-1 adaptation. An initial stock of HIV-1NL4.3(JR-FL) was prepared by transfecting 293T cells with the proviral plasmid and harvesting the cell supernatant three days later. Cf2Th-CD4/CCR5 cells, which constitutively express human CD4 and human CCR5, were incubated with the 293T cell supernatants and cultured. Reverse transcriptase activity was detected in the supernatants by 30 days of culture (data not shown). Cell supernatants with reverse transcriptase activity were used to reinfect Cf2Th-CD4/CCR5 in the presence of 2 μg/mL polybrene to enhance infection. Viruses in the supernatants of these cells were used for adaptation of HIV-1NL4.3(JR-FL) to CD4-expressing cells with low levels of CCR5.

 

An external file that holds a picture, illustration, etc.
Object name is nihms877325f1.jpg
An external file that holds a picture, illustration, etc.
Object name is nihms877325f2.jpg

Fig. 2

Adaptation of HIV-1NL4.3(JR-FL) to low levels of CCR5. (A) Replication kinetics of HIV-1NL4.3(JR-FL) at different rounds of adaptation in the indicated cell type and doxycycline concentrations. Cf2Th and Cf2Th-CD4/CCR5 cells were infected with HIV-1NL4.3(JR-FL). A 32P-reverse-transcriptase assay was performed on medium removed at each passage. Results are displayed as cpm of 32P -labeled nucleotides reverse transcribed. Viruses at peak reverse transcriptase activity were used to infect R5-High or R5-Low cells in the next round of adaptation. The dashed line represents reverse transcriptase activity in supernatants from CD4-negative, CCR5-negative Cf2Th cells incubated with virus in Round 12. Numbers in parentheses refer to the concentration of doxycycline in ng/ml. (B) Viruses were derived at various passages in R5-Low cells. Wild-type HIV-1JR-FL or viruses from passage 3 (J3), passage 9 (J9) and passage 11 (J11) were used to infect Cf2Th, Cf2Th-CD4/CCR5, and R5-Low cells. Viral pools were normalized for the amount of p24 Gag protein prior to infection. The reverse transcriptase activity in the culture supernatants is shown.

When additional rounds of virus passage were conducted at decreasing levels of target cell CCR5 expression, we observed decreasing reverse transcriptase activity in the cell supernatants (Fig. 2A). Viral replication was not detected beyond round 12, in which we detected reverse transcriptase activity in the supernatants of R5-Low cells in 12.5 ng/mL doxycycline. Cell-free viruses in the supernatant of the round 12 cultures did not detectably replicate in R5-Low cells maintained in the same levels of doxycycline (data not shown). QuantibritePE was used to estimate the number of CCR5 molecules/cell by quantifying the epitopes for the 2D7 anti-CCR5 antibody per cell (BD Biosciences). Replication of HIV-1NL4.3(JR-FL) was not detected in the CD4-expressing cells with less than ~1300 CCR5 molecules per cell (Fig. 1).

To confirm that the viruses generated by this adaptation procedure could replicate in cells expressing low levels of CCR5, we infected R5-Low cells at 50 and 100 ng/mL doxycycline, as well as Cf2Th-CD4/CCR5 cells, with cell supernatants from round 3 (J3), round 9 (J9) and round 11 (J11) that were normalized for the level of the p24 Gag protein. Fig. 2B compares the replication capacity of the J3 and J9 viruses with that of the parental HIV-1NL4.3(JR-FL) virus for each cell type and doxycycline concentration. In Cf2Th-CD4/CCR5 cells, J3 and J9 viruses infected 2-fold and 4-fold more efficiently than the parental HIV-1NL4.3(JR-FL), respectively. Both J3 and J9 viruses replicated better than HIV-1NL4.3(JR-FL) in R5-Low(100) and R5-Low(50) cells. The production of the J3 virus in R5-Low(100) cells was comparable to that of the parental HIV-1NL4.3(JR-FL) in Cf2Th-CD4/CCR5 cells; by contrast, HIV-1NL4.3(JR-FL) replicated only marginally in the R5-Low(100) cells. Both J3 and J9 viruses replicated in R5-Low(50) cells, whereas no HIV-1NL4.3(JR-FL) reverse transcriptase activity was detected in these cells. These observations indicate that the J3 and J9 viruses have adapted to replicate better than the parental HIV-1NL4.3(JR-FL) virus on cells expressing low levels of CCR5.

The J11 viral pool did not detectably replicate in any cell type, suggesting that some adaptive changes occurring after round 9 were deleterious to cell-free infection in this context.

Adaptive changes in the HIV-1JR-FL Env

We isolated the genomic DNA of the cells collected at the time point of peak reverse transcriptase activity in the supernatant at each round of adaptation, PCR amplified the integrated HIV-1 provirus, and sequenced the env gene. The wild-type HIV-1JR-FL env sequence was maintained throughout multiple rounds of replication in Cf2Th-CD4/CCR5 cells (data not shown). In the viruses adapted to replicate in R5-Low cells, multiple changes were observed. Changes that were retained through multiple rounds of adaptation are shown in Fig. 3A. Three changes, S115N, R564H, and E662K, were found in all three adapted viruses, J3, J9, and J12. Both J9 and J12 had, in addition, an S164N change, and J9 had an E831D change.

 

An external file that holds a picture, illustration, etc.
Object name is nihms877325f3.jpg
An external file that holds a picture, illustration, etc.
Object name is nihms877325f4.jpg

Fig. 4

Composition and infectivity of single-round recombinant viruses with Envs from wild-type HIV-1JR-FL or from adapted viruses. (A) Immunoblots of viral proteins in recombinant viruses purified by ultracentrifugation through a 20% sucrose cushion. The recombinant viruses contain the wild-type HIV-1JR-FL Env or the indicated Envs from the adapted viruses; the ΔEnv control viruses lack Env. (B) Infectivity of recombinant, luciferase-expressing viruses containing the wild-type HIV-1JR-FL Env or the indicated variant Envs. Infectivity is indicated by the relative luciferase units (RLU) in the lysates of infected cells, after normalization of the input virus by reverse transcriptase activity (RT). The averages of the means from two experiments with duplicate samples are shown.

To evaluate whether the adapted virus Envs retain the ability to support entry, we incubated Cf2Th-CD4/CCR5 and R5-Low cells with equivalent reverse transcriptase units of recombinant single-round virus (Fig. 4B). In this assay, the expression of luciferase in the target cells is dependent on Env-mediated virus entry, as well as subsequent reverse transcription and integration of the viral vector (Helseth et al., 1990). We observed that viruses with the adapted Envs exhibited approximately a 5-fold reduced infectivity compared to viruses with the parental HIV-1JR-FL Env. Viruses with single and double-residue changes introduced into the wild-type HIV-1JR-FL Env did not demonstrate as great an attenuation in infectivity as the viruses with the adapted Envs. The low levels of infectivity of single-round viruses with adaptation-associated changes contrasts with the enhanced infection of Cf2Th-CD4/CCR5 or R5-Low cells by replication-competent viruses with the same changes. This result suggests that although the parental HIV-1JR-FL Env mediates better infection in a cell-free, single-round context (Fig. 4B), the adapted Envs display an enhanced replication capacity in a context in which cell-free and cell-cell infection occurs.

Effect of adaptation-associated changes in HIV-1JR-FL Env on coreceptor use and virus entry requirements

The altered infectivity of the adapted Envs for CD4-positive, CCR5-positive target cells could result from changes in coreceptor tropism, e.g. with a switch to CXCR4 or to coreceptors present in Cf2Th cells. We tested this by incubating single-cycle recombinant viruses with the parental Cf2Th cell line, and with Cf2Th cells stably expressing human CCR5 alone, CD4 and CXCR4, or CD4 and CCR5. Detectable levels of infection were observed only in Cf2Th-CD4/CCR5 cells (Fig. 5A). These results indicate that the viruses with the adapted Envs remain dependent on the CCR5 coreceptor for efficient infection of target cells.

An external file that holds a picture, illustration, etc.
Object name is nihms877325f5.jpg
An external file that holds a picture, illustration, etc.
Object name is nihms877325f6.jpg
An external file that holds a picture, illustration, etc.
Object name is nihms877325f7.jpg

Open in a separate window

Fig. 7

Syncytium-forming ability and cell-to-cell transmission of HIV-1JR-FL Env variants. (A) Cos-1 cells expressing the wild-type HIV-1JR-FL or the adapted virus Envs were incubated with Cf2Th-CD4/CCR5 cells in an alpha complementation assay, as described in the Materials and Methods. The relative light units (RLU) of luciferase activity reflect the degree of cell-cell fusion (syncytium formation) mediated by the Env. The ∆Env is a negative control. (B) Sensitivity of the alpha complementation assay to maraviroc, a CCR5 inhibitor. The % fusion for each Env is shown relative to the value seen in the absence of inhibitor.(C) 293T cells transiently expressing single-round recombinant HIV-1 with the indicated wild-type or variant HIV-1JR-FL Envs were cocultivated with Cf2Th-CD4/CCR5 cells and passaged for approximately 6 days. The luciferase activity (RLU) in the culture was then measured. Means and standard deviations derive from 2 or more coculture samples, normalized to the RLU of viruses with the wild-type HIV-1JR-FL Env. The ∆Env is a negative control virus lacking a functional Env. Student t-test as compared to wild-type HIV-1JR-FL Env: * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.

The above results indicate that, in contrast to the presumed advantage of the adaptation-associated Env changes to HIV-1NL4.3(JR-FL) replication in Cf2Th cells expressing CD4 and low levels of CCR5, these changes do not improve cell-free infection or cell-cell fusion mediated by Env. We therefore utilized an assay in which cell-free and cell-to-cell transmission of HIV-1 occur (Helseth et al, 1990), potentially mimicking the conditions under which the viruses were adapted to the lower level of CCR5 expression. In this assay, transfected 293T cells expressing single-round luciferase-expressing viruses were cocultivated with Cf2Th-CD4/CCR5 cells. Thus, the Cf2Th-CD4/CCR5 target cells could be infected by single-cycle recombinant virus via cell-free infection or by cell-to-cell transfer. Because luciferase expression is initially high in the transfected 293T cells, the assay is conducted for at least 6 days, by which time luciferase expression in the transfected 293T cells diminishes; this decrease in luciferase is monitored by using an Env-deleted (ΔEnv) virus that cannot initiate new infections. If the cotransfected Env can support a new round of either cell-free or cell-to-cell transmission from the transfected 293T cells to the Cf2Th-CD4/CCR5 cells, the expression of luciferase from the integrated HIV-1 vector is sustained for long periods of time. Thus, we measured luciferase activity in the cocultures when the luciferase activity associated with the ΔEnv control returned to background levels.

In this assay, the adapted Envs supported cell-to-cell infection better than the parental JR-FL Env (Fig. 7C). The S115N, S164N and E662K mutants, as well as some of the combined mutants, also were very efficient in this assay. The activity of the single and double mutant Envs did not correlate with the level of enhancement seen for these Envs in the virus replication assay. Thus, although this single-round cell-to-cell infection assay does not fully reproduce all of the features of the virus replication assay, the results suggest that the adaptation-associated Env changes can enhance HIV-1 infection when cell-to-cell transmission is allowed.

Effects of adaptation-associated changes in Env on HIV-1 sensitivity to antibody neutralization

HIV-1 variants with decreased dependence on CD4 typically demonstrate an increased sensitivity to antibodies targeting the CD4-binding and coreceptor-binding sites, such as F105 and 17b, respectively (Haim et al., 2011; Hoffman et al., 1999; Kolchinsky et al., 2001a,b). Therefore, we measured the neutralization sensitivity of our adapted Envs by infecting Cf2Th-CD4/CCR5 cells with single-round luciferase-expressing viruses that had been pre-incubated with a panel of antibodies.

Viruses with the J3, J9 and J12 Envs were slightly more sensitive to inhibition by sCD4 and small-molecule CD4-mimetic compounds than the wild-type HIV-1JR-FL (Table 2). The viruses with the adapted Envs exhibited significant increases in sensitivity to neutralization by the F105, 17b, 830A, 19b, and 1.4E antibodies, which did not inhibit the viruses with the wild-type HIV-1JR-FL Env. These anti-gp120 antibodies against the CD4-binding site (F105), CD4-induced gp120 epitopes (17b), V2 region (830A), and V3 region (19b and 1.4E) recognize an intermediate Env conformation (State 2) that is on the HIV-1 entry pathway (Munro et al., 2014; Herschhorn et al., 2016). The adapted Envs apparently sample conformations similar to State 2 more readily than the parental HIV-1JR-FL Env. We observed no significant difference in the inhibition of viruses containing the parental HIV-1JR-FL and adapted Envs by the broadly neutralizing antibodies VRC01, PGT121, and 35O22.

Table 2

Inhibition of viruses with wild-type and mutant HIV-1JR-FL Envs. Recombinant, luciferase-expressing viruses with wild-type or mutant HIV-1JR-FL Envs were incubated with sCD4, small- molecule entry inhibitors or antibodies for one hour at room temperature. The virus-inhibitor mixtures were then incubated with Cf2Th-CD4/CCR5 cells for 2 days prior to cell lysis and measurement of luciferase activity. Viral infection was plotted as a percentage of the relative luciferase activity observed in the absence of inhibitor. The concentration of inhibitors required to inhibit virus infection by 50% (IC50) is reported. The IC50 values for all of the antibodies are in μg/mL. The values shown are means and standard deviations from one or more sets of duplicate samples in typical experiment. N/A – not applicable.

IC50

Inhibitor

Binding Site

WT
JR-FL

J3

J9

J12

ΔV1V2

S115N

R564H

E662K

S115N
S164N

S115N
R564H

S115N
E662K

R564H
E662K

sCD4 (in μg/mL)

CD4

3.7±0.6

1.3±0.1

1.0±0.1

1.6±0.2

>100

0.9±0.2

BMS-806 (in nM)

β20–β21 compound

3.0±.7

3.9±0.6

7.5±2.2

4.1±0.6

>100

9.2±2.8

6.0±2.6

3.5±0.7

28.5±5.0

5.4±1.3

21.1±7.1

2.3±0.2

DMJ-I-228 (in uM)

CD4BS compound

43±7.0

12±2.8

13±4.1

2.0±0.5

9.1±1.0

JP-III-48 (in uM)

CD4BS compound

33.9±10.5

7.7±1.8

9.5±2.0

7.0±1.6

1.3±0.2

VRC01

CD4BS

0.6±0.1

0.6±0.2

0.7±0.1

0.7±0.2

15.9±7.9

0.9±0.2

0.4±0.1

0.6±0.1

0.7±0.2

0.6±0.1

0.9±0.2

0.8±0.2

F105

CD4BS

>100

8.3±1.7

4.6±0.8

8.7±2.5

7.7±4.3

35.1±14.5

>100

>100

22.8±5.6

>100

5.5±1.6

>50

17b

CD4i

>100

16.4±4.3

16.1±3.7

28.0±8.5

6.8±8.3

>100

>100

>100

>50

>50

>100

>100

830A

V2

>50

6.1±2.8

2.4±1.2

0.6±0.3

N/A

902090

V2

>100

34±14

>50

>100

>100

19b

V3

>50

13.7±2.4

5.4±1.0

5.1±+1.4

0.03±0.04

21.1±5.4

>100

>100

>50

40.6±15.6

5.6±1.1

>50

1.4E

V3

>50

4.0±1.2

4.9±1.0

2.7±0.6

8.3±10.6

PGT121

V3 Glycan

2.6±0.4

1.3±0.1

3.2±0.7

1.8±0.3

25.1±49.8

35O22

gp120-gp41

3.2±1.7

7.7±3.9

6.9±3.6

3.1±1.6

0.05±0.02

Open in a separate window

To identify the specific Env changes responsible for this phenotype, we assayed the sensitivity of single and double Env mutants to VRC01, F105, 17b, and 19b. The mutants R564H, E662K, and R564H/E662K exhibited a neutralization profile similar to that of the parental HIV-1JR-FL virus (Fig. 8A, Table 2). The S115N mutant was more sensitive to F105 and 19b neutralization than the wild-type HIV-1JR-FL virus, although not as sensitive as the viruses with the adapted Envs. The virus with the S115N and E662K changes was as sensitive as the viruses with the J3, J9, and J12 Envs to neutralization by F105 and 19b. These results indicate that the S115N change makes a major contribution to the sampling of State 2-like conformations, in which the CD4-binding site and V3 region of gp120 is more exposed (Herschhorn et al., 2016). Other changes, like E662K in gp41, contribute to the degree of this phenotype. Apparently, multiple Env changes in the adapted viruses result in greater sampling of a State 2-like conformation with increased exposure of two epitopes (V3 and CD4-induced) near the CCR5-binding region.



An external file that holds a picture, illustration, etc.
Object name is nihms877325f8.jpg

Open in a separate window

Fig. 8

Sensitivity of viruses with wild-type and variant HIV-1JR-FL Envs to neutralization by antibodies and activation by CD4-mimetic molecules. (A) Recombinant, luciferase-expressing viruses with the wild-type HIV-1JR-FL Env or variant Envs with adaptation-associated changes were incubated with antibody at room temperature. The virus-antibody mixtures were then incubated with Cf2Th-CD4/CCR5 cells for 2 days prior to cell lysis and measurement of luciferase activity. Viral infection is shown as a percentage of the luciferase activity observed in the absence of antibody. The means and standard deviations from duplicate samples in a typical experiment are shown. (B) Recombinant viruses with the wild-type HIV-1JR-FL Env or the adapted virus Envs were incubated with CD4-negative Cf2Th-CCR5 cells in the presence of the indicated concentrations of sCD4 or the CD4-mimetic compound DMJ-I-228. Luciferase activity was measured in the cells two days later and is reported as the percentage of the maximum luciferase activity observed for each virus variant.

The neutralization assays with sCD4 and small-molecule CD4-mimetic compounds suggested that the adapted Envs might be slightly more susceptible to the induction of conformational changes by these ligands. To test this hypothesis, the activation of infection of CD4-negative Cf2Th-CCR5 cells by sCD4 and two CD4-mimetic compounds was evaluated for viruses with the adapted and parental HIV-1JR-FL Envs (Herschhorn et al., 2016; Madani et al., 2016). The infection of Cf2Th-CCR5 cells by the viruses with J3, J9 and J12 Envs was activated at 2-3-fold lower concentrations of sCD4, JP-III-48 and DMJ-I-228 than the wild-type HIV-1JR-FL infection (Fig. 8B and data not shown). These results are consistent with a slight increase in the ability of the adapted viruses to be triggered by CD4.

Effect of the N302Y change on the HIV-1JR-FL low-R5 replication phenotype

The adaptation of HIV-1 to infect cells expressing low levels of CCR5 has been recently reported (Garg et al., 2016). In this system, SupT1 T lymphocytes (L23 cells) stably expressing low levels of human CCR5 were infected with HIV-1YU2, followed by passage for 70 days. The resulting virus contained an N302Y change in the gp120 V3 region that enhanced virus replication in the L23 cells and conferred 5-fold resistance to Maraviroc.

We introduced the N302Y change into the wild-type and J9 mutant Envs in the HIV-1NL4.3(JR-FL) provirus to determine whether the V3 change would enhance replication in R5-Low cells. The N302Y change did not enhance the replication of HIV-1NL4.3(JR-FL) (Fig. 9). We observed no significant improvement in the replication of the J9/N302Y virus in R5-Low cells, compared with that of the J9 virus. The replication of the J9/N302Y virus in Cf2Th-CD4/CCR5 cells was attenuated relative to that of the J9 virus. These results indicate that the N302Y change is not sufficient to confer the ability to replicate efficiently on cells with low CCR5 to HIV-1NL4.3(JR-FL).



An external file that holds a picture, illustration, etc.
Object name is nihms877325f9.jpg

Open in a separate window

Fig. 9

Replication kinetics of viruses with the wild-type HIV-1JR-FL Env or mutant Envs. Cells were transfected with NL4.3 proviruses with the wild-type HIV-1JR-FL Env or the indicated variant Env, and passaged for 15 days. A 32P RT assay was performed on medium removed at each passage. Each point represents an average of duplicate samples from a representative replication kinetics assay. The dashed line represents the average reverse transcriptase activity in supernatants of CD4-negative, CCR5-negative Cf2Th cells incubated with each virus.

Introduction of adaptation-associated changes into HIV-1AD8

The R564H change in the HIV-1JR-FL Env contributed to the replication of the virus in cells with low levels of CCR5. HIV-1AD8 and 92.7 percent of all HIV-1 strains have a histidine residue at position 564. Since the wild-type HIV-1AD8 Env already has a histidine at position 564, we hypothesized that the addition of the other single-residue changes that arose in the adapted HIV-1JR-FL Env would allow HIV-1AD8 to infect cells expressing low CCR5 levels. The addition of these single-residue changes to the Env of the HIV-1NL4.3(AD8) provirus resulted in a virus that did not detectably replicate in Cf2Th-CD4/CCR5 or R5-Low cells (data not shown). Introduction of S115N in single-cycle recombinant HIV-1AD8 with the AD8 Env resulted in increased infectivity in Cf2Th-CD4/CCR5 cells and an increased sensitivity to the 17b and 19b antibodies (Fig. 10). Apparently, some but not all of the phenotypes resulting from the adaptation-associated changes in the HIV-1JR-FL Env can be reproduced in the context of the HIV-1AD8 Env. The adaptation-associated changes may contribute to the ability of a subset of naturally occurring HIV-1 variants to infect cells with different levels of CCR5.



An external file that holds a picture, illustration, etc.
Object name is nihms877325f10.jpg
    Cd4Gp120Gp41Hiv-1

    Leave a comment

    All comments are moderated before being published