Functional Stability of Unliganded Envelope Glycoprotein Spikes among Isolates of Human Immunodeficiency Virus Type 1 (HIV-1)

The HIV-1 envelope glycoprotein (Env) spike is challenging to study at the molecular level, due in part to its genetic variability, structural heterogeneity and lability. However, the extent of lability in Env function, particularly for primary isolates across clades, has not been explored. Here, we probe stability of function for variant Envs of a range of isolates from chronic and acute infection, and from clades A, B and C, all on a constant virus backbone. Stability is elucidated in terms of the sensitivity of isolate infectivity to destabilizing conditions. A heat-gradient assay was used to determine T90 values, the temperature at which HIV-1 infectivity is decreased by 90% in 1 h, which ranged between ∼40 to 49°C (n = 34). For select Envs (n = 10), the half-lives of infectivity decay at 37°C were also determined and these correlated significantly with the T90 (p = 0.029), though two ‘outliers’ were identified. Specificity in functional Env stability was also evident. For example, Env variant HIV-1ADA was found to be labile to heat, 37°C decay, and guanidinium hydrochloride but not to urea or extremes of pH, when compared to its thermostable counterpart, HIV-1JR-CSF. Blue native PAGE analyses revealed that Env-dependent viral inactivation preceded complete dissociation of Env trimers. The viral membrane and membrane-proximal external region (MPER) of gp41 were also shown to be important for maintaining trimer stability at physiological temperature. Overall, our results indicate that primary HIV-1 Envs can have diverse sensitivities to functional inactivation in vitro, including at physiological temperature, and suggest that parameters of functional Env stability may be helpful in the study and optimization of native Env mimetics and vaccines

N-linked glycosylation of HIV-1 and Hantaan virus glycoproteins. Implications for vaccination and therapy

Enveloped viruses have a great advantage comparedto the non-enveloped viruses due to theirability to acquire not only a host membrane butalso host-specific glycosylation of their surfaceglycoproteins. These carbohydrate structures,mainly of the N-linked glycan class, have a lowimmunogenicity and this feature enables envelopedviruses to keep a low profile towards theimmune system by shielding crucial epitopesfrom antibody recognition. This phenomenonconstitutes hurdles for development of vaccinesand therapies targeting highly glycosylated virusesincluding HIV and Hantaan virus. The aimsof the present work were to identify specific Nlinkedglycans that are especially active in thisvirus-shielding process and develop DNA vaccinesand therapeutic vectors to the two virusesmentioned.An N-linked glycan attached to N306 in the V3loop of the HIV-1 gp120 is involved in protectingthe virus from antibodies. A DNA vaccinewas created, where this particular glycosylationsite was eliminated using site-directed mutagenesis.Mice were immunised intranasally withthe mutant and a wild type vaccine, and theimmune responses were analysed. The protocolincluded an analysis of animals immunised withDNA only and also those given a protein boost.The mutant DNA followed by protein boostinduced a significantly higher IgG response togp120 as measured by ELISA. Both DNA vaccinesinduced similar levels of HIV-neutralisingantibodies in serum. The IgA response was analysedboth for BAL and faeces specimens. SecretedIgA was induced by both DNA vaccinesbut protein boost was required to obtain virusneutralisingantibodies.Attempts were made to construct an adenovirusvector, with receptor specificity retargeted togp120 of the HIV-infected cell surface. To obtaina suitable affinity module for gp120 a combinatorialprotein library of 108 members, based onthe 58 amino acid residue staphylococcal proteinA (Affibody), was screened against gp120.The selected affibody (zg120) and its bivalentform had Kd- values of 100 nM and 10 nM,respectively, towards gp120. The affibody constructswere introduced to the adenovirus 5 fibregene, and the recombinant fibres bound selectivelyto gp120 in a biosensor analysis and togp160, transiently expressed in GMK cells. Thesuccessful selection of a gp120-binding affibodyligand indicates that future affibody-basedstrategies might evolve to complement antibodybasedefforts for HIV-1 therapy.The same strategy as described above for HIV-1was also applied to develop a DNA vaccine toHantaan virus glycoproteins. All six potentialglycosylation sites (N1-N6) of the two Hantaanvirus glycoproteins G1 and G2 were eliminatedand expressed as single mutations in a DNAvaccine. Mice were immunized by Gene Gunwith these constructs. One mutated DNA vaccineconstruct, N6, was found to induce equallyhigh neutralisation titres as did the wild typeDNA vaccine. However, the mutant but not thewild type DNA vaccine was able to induce partialprotection against Hantaan virus challenge.In conclusion, manipulation of glycosylationsites of viral glycoprotein genes is still open as aroad to an improved DNA vaccine capable ofeliciting high titres of broadly neutralising antibodies,but it is clear that there are still obstaclesto be overcome

Time course incubation of HIV-1 Env trimers at physiological temperature (37°C) visualized using BN-PAGE.

<p>(<b>A</b>) HIV-1_LAI-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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021339#pone-0021339-g004" target="_blank"><b>Fig. 4</b></a>. 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>B</b>) The same virus sample as in Panel A was subjected to indicated temperatures for 1 h and analyzed on BN-PAGE, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021339#pone-0021339-g004" target="_blank"><b>Fig. 4</b></a>, except that the electrophoresis run time was shorter causing less separation between different bands.</p

Sensitivity of HIV-1 to urea and GuHCl.

<p>(<b>A</b>) Treatment of HIV-1 with denaturant abrogates virion infectivity under conditions in which virion-associated RT remains active. Culture supernatants containing infectious HIV-1_LAI-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>B</b> and <b>C</b>) 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>B</b>) urea, or (<b>C</b>) 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. (<b>D</b>) Sensitivity of HIV-1 to pH. HIV-1 PSVs, prepared and treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021339#pone-0021339-g003" target="_blank"><b>Fig. 3</b></a>, 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.</p

Time course dissociation of HIV-1 Env trimers in the presence of membrane altering reagents visualized using BN-PAGE.

<p>HIV-1_LAI-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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021339#pone-0021339-g004" target="_blank"><b>Fig. 4</b></a>.</p

Intersubunit disulfide bond (“SOS”) increases apparent thermostability of trimeric Env but does not increase thermostability of infectivity (T₉₀) of HIV-1_LAI-JR-FL.

<p>(<b>A</b>) BN-PAGE analysis of the thermostability of wildtype HIV-1_LAI-JR-FL and SOS-HIV-1_LAI-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>B</b>) Thermostability comparison in the infectivity assay of wildtype HIV-1_LAI-JR-FL and SOS-HIV-1_LAI-JR-FL, using the same heat gradient as in panel A.</p

Presence of an excess of unprocessed gp160 in PSV but not MC HIV-1 virion preparations.

<p>(<b>A</b>) SDS-PAGE of HIV-1_LAI-JR-CSF PSVs (pcDNA) showing excess of uncleaved gp160, and HIV-1_LAI-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>B</b>) BN-PAGE of samples in panel A, showing mostly oligomeric Env (PSVs and MCs), with HIV-1_LAI-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 <b>A</b>.</p

Thermally induced dissociation of HIV-1 Env trimers visualized using BN-PAGE.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021339#pone-0021339-t001" target="_blank"><b>Table 1</b></a>.</p

Change in thermostability of HIV-1_JR2 (ΔT₉₀ values) due to Ala mutations in the MPER of gp41.

<p>T₉₀ values were determined for each Ala mutant and for parental HIV-1_JR2 and the ΔT₉₀ values were calculated (ΔT₉₀ = T₉₀ of parental−T₉₀ of mutant).</p

Sensitivity of HIV-1 infectivity to heat (T₉₀) is linked to Env.

1<p>T₉₀, the temperature at which HIV-1 infectivity decreases by 90% in 1 h, and the standard error of the mean (SEM) of at least three independent experiments.</p>2<p>HIV-1 PSVs, produced by transfection of 293T cells using <i>env</i> expression plasmids pSVIIIexe7 (JR-CSF, JR-FL, ADA), pCAGGS (SF162, SIVmac239), pcDNA3.1 (LAI), or pVSV-G (VSV-G) in combination with the backbone vectors pSG3Δenv (clade B) or Q23Δenv (clade A).</p>3<p>HIV-1 MCs are LAI-chimeras (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021339#s2" target="_blank">Materials and Methods</a>) and fully replication competent. Virions were produced by transfection of 293T cells, or through passage in MT-2/CCR5ΔCT cells, as indicated.</p>4<p><i>nd</i>, not determined, or material unavailable.</p