Figure 1.
Sensitivity of HIV-1 to heat is Env-dependent.
HIV-1 PSVs on a pSG3Δenv backbone and bearing Env from strains ADA or JR-CSF, were concentrated (20×) in PBS and incubated for 1 h at 1, 2 or 5 times their original concentration at various temperatures for 1 h prior to determination of infectivity using TZM-bl cells. (A) Absolute infectivity following heat treatment of HIV-1 PSVs at different virus input levels over a >10-fold range (1×105–2×106 RLU/0.1 ml, demarcated by triangles). (B) Data from panel A normalized for input infectivity, indicating T90 value is independent of virus input. HIV-1 PSVs used in panel A were examined for relative levels of p24 (C), and gp120 (D), using p24 or gp120 ELISA, respectively. (E) Reproducibility of T90 determination using independent stocks of HIV-1 PSVs (JR-CSF and ADA). (F) Relative infectivities of HIV-1 PSVs (JR-CSF and ADA) following several different pre-incubation temperatures and times, as determined using the TZM-bl assay. (G) Relative infectivity over time at physiological temperature (37°C) of HIV-1 PSVs (JR-CSF and ADA). Half-life (t1/2) of infectivity was calculated as described in the Materials and Methods and is indicated near the point of half-maximal infectivity for each curve.
Table 1.
Sensitivity of HIV-1 infectivity to heat (T90) is linked to Env.
Figure 2.
Sensitivity of HIV-1 to urea and GuHCl.
(A) Treatment of HIV-1 with denaturant abrogates virion infectivity under conditions in which virion-associated RT remains active. Culture supernatants containing infectious HIV-1LAI-JR-CSF (MC) virions, passaged once in MT-2/CCR5ΔCT cells, were treated using different concentrations of urea and GuHCl. Following extensive washing to remove denaturant, samples were assayed for apparent RT activity (closed symbols) as well as for infectivity on TZM-bl cells (open symbols). Data plotted are normalized to untreated samples. (B and C) HIV-1 (PSVs) produced in 293T cells using backbone plasmid pSG3Δenv, and Env plasmids pSVIII-JR-CSF (solid line) and pSVIII-ADA (dotted line) were incubated with indicated concentrations of (B) urea, or (C) GuHCl. Prior to infectivity determination using TZM-bl cells, virions were pelleted and washed with PBS to remove residual denaturant. Results are an average of duplicate samples, and representative of at least two independent experiments. (D) Sensitivity of HIV-1 to pH. HIV-1 PSVs, prepared and treated as in Fig. 3, except that citric acid (pH 2–6.5) or ethanolamine (pH 7–10) buffers were used. Results are an average of duplicate samples, and representative of three independent experiments.
Figure 3.
Presence of an excess of unprocessed gp160 in PSV but not MC HIV-1 virion preparations.
(A) SDS-PAGE of HIV-1LAI-JR-CSF PSVs (pcDNA) showing excess of uncleaved gp160, and HIV-1LAI-JR-CSF MC (pLAI; sequence matched in Env) showing only the much fainter gp120 band, as well as gp41. Virus loaded was normalized by p24 ELISA. (B) BN-PAGE of samples in panel A, showing mostly oligomeric Env (PSVs and MCs), with HIV-1LAI-JR-CSF PSVs showing greater heterogeneity in staining with the gp120 mAb cocktail, and with MCs showing much less abundant, but mainly trimeric Env. Input virus was normalized as in A.
Figure 4.
Thermally induced dissociation of HIV-1 Env trimers visualized using BN-PAGE.
HIV-1 LAI-chimeric MCs bearing Envs of JR-CSF (top), JR-FL (middle) and ADA (bottom) were treated for 1 h at temperatures ranging from 37°C to 57°C, and then subjected to BN-PAGE and Western blot analysis. Blotted membranes were probed using mAb cocktails to gp120 (IgGs b12, 2G12 and B4e8) or to gp41 (IgGs 2F5, 4E10 and Z13e1). Positions of molecular weight standards are indicated (left), as are positions of monomeric gp120 and native gp120/gp41 trimers (right). The down arrow (↓) on each panel indicates the T90 of the cognate virus, as reported in Table 1.
Figure 5.
Time course incubation of HIV-1 Env trimers at physiological temperature (37°C) visualized using BN-PAGE.
(A) HIV-1LAI-JR2 MC, produced in 293T cells, was incubated for various time intervals up to 96 h at 37°C and aliquots removed for BN-PAGE and Western blot analysis, as in Fig. 4. Down arrows indicate the time interval in which infectivity of the cognate virus decreases by ≥90%. The band smearing in the 72 h lane is an experimental artifact of sample loading. (B) The same virus sample as in Panel A was subjected to indicated temperatures for 1 h and analyzed on BN-PAGE, as in Fig. 4, except that the electrophoresis run time was shorter causing less separation between different bands.
Figure 6.
Time course dissociation of HIV-1 Env trimers in the presence of membrane altering reagents visualized using BN-PAGE.
HIV-1LAI-JR-FL MC, produced in 293T cells, was incubated for 0 h, 4 h, 8 h, 24 h, or 96 h at 37°C in the presence of no compound (left), 70 mM β-cyclodextrin (cholesterol scavenger; center), or 1% DDM (mild detergent; right). Following incubation for the indicated time periods, samples were analyzed by BN-PAGE and Western blot as in Fig. 4.
Figure 7.
Effect of heat pre-treatment on mAb-virion capture efficiency.
HIV-1LAI-JR-FL (MC) virions, produced in 293T cells, were incubated at the indicated temperatures for 1 h and anti-gp120 mAbs 2G12 (outer face), b12 (CD4 binding site), anti-gp41 mAbs 4E10 (MPER), and 7B2 (immunodominant loop), and the irrelevant mAb DEN3 were used to capture the heat treated virions in an in-solution virus capture assay. Amount of virion (p24) equivalents captured were determined using an in-house p24 ELISA and reported relative to background levels captured using DEN3.
Figure 8.
Thermostabilities (T90 values) of acute phase standard panel viruses (PSVs) of clades A, B and C, and lack of a relationship with susceptibility to soluble CD4.
(A) Bar graph indicating the various T90 values with that of the thermostable HIV-1JR-CSF, and thermolabile HIV-1ADA indicated with dashed lines. Env panel members that are thermostable (T90≥48°C), intermediate in thermostability (43°C<T90<48°C), and thermolabile (T90≤43°C) are indicated by filled, hatched and open bars, respectively. (B) Bell curve (normal distribution) in T90 values of HIV-1 Envs used in this study, including Envs from acute phase panels of clades A, B and C (Tables 1 and 2), n = 34. Mean, 44.2°C; Std. Dev., 2.4°C; Range, 40.0–49.0°C. The curve-fit (nonlinear regression lorentzian) was made using Prism software (Graphpad, CA). (C) Lack of correlation between T90 and reported IC50 values of soluble CD4 against HIV-1 standard Env panels (clades B and C). Soluble CD4 IC50 data are taken from Wu et al [79], excluding resistant isolates (IC50>50 µg/ml) for which exact values are undetermined.
Table 2.
Thermostability (T90 values) and sensitivity to soluble (4-domain) CD4 of HIV-1 PSVs bearing Envs from acute phase virion panels (clades A, B and C).
Figure 9.
Change in thermostability of HIV-1JR2 (ΔT90 values) due to Ala mutations in the MPER of gp41.
T90 values were determined for each Ala mutant and for parental HIV-1JR2 and the ΔT90 values were calculated (ΔT90 = T90 of parental−T90 of mutant).
Table 3.
Effect of the presence of soluble CD4 on the thermostability (T90) of HIV-1.
Figure 10.
Relationship between thermostability (T90) and infectivity half-life at 37°C (t1/2) for HIV-1 (PSVs) from clades A, B and C.
Pearson, r = 0.686; P value (2-tailed) = 0.0286 (significant). Identity of Env is shown beside each point. For emphasis, shaded regions designate areas of thermolabile and thermostable Envs. The arrow to the left of the origin indicates the point at which t1/2 = 18 min, which, at 37°C, also corresponds to a T90 = 37°C.
Table 4.
Thermostability (T90) and half-life of infectivity decay at 37°C (t1/2) of select HIV-1 (PSVs) from clades A, B and C.
Figure 11.
Intersubunit disulfide bond (“SOS”) increases apparent thermostability of trimeric Env but does not increase thermostability of infectivity (T90) of HIV-1LAI-JR-FL.
(A) BN-PAGE analysis of the thermostability of wildtype HIV-1LAI-JR-FL and SOS-HIV-1LAI-JR-FL, incubated as whole virions for 1 hr at indicated temperatures, then prepared for BN-PAGE and subsequent blotting using mAb cocktails to gp120 and gp41. (B) Thermostability comparison in the infectivity assay of wildtype HIV-1LAI-JR-FL and SOS-HIV-1LAI-JR-FL, using the same heat gradient as in panel A.