马传染性贫血病毒白细胞弱毒疫苗株及其亲本毒驴强毒株前病毒基因组比较分析

为阐明马传染性贫血白细胞弱毒疫苗株(EIAVDLV)的致弱和免疫保护机理,对EIAVDLV121及其亲本驴强毒株(EIAVDV117)前病毒全基因组序列进行了测定,并结合准种理论,分析了EIAV疫苗致弱过程中基因组进化特点。利用LA-PCR技术对EIAVDV117和EIAVDLV121的前病毒基因组分两段进行扩增,分别获得4个和10个前病毒全基因组序列。EIAVDV117前病毒基因组平均为8236bp,G C含量38.0。EIAVDLV121前病毒基因组平均8249bp,G C含量37.3。两者的前病毒基因组平均差异率为2.8。其中S2、LTR和env基因差异较大,分别为4.1、3.9和3.1。此外,S2、S3和env推导的氨基酸的差异明显,分别为10.4、5.6和4.8(gp90为6.8)。EIAVDLV121各基因的异质性均显著高于EIAVDV117。研究发现体外培养的EIAVDLV121至少有5种类型的LTR混合存在。在gp90推导的氨基酸序列上,EIAVDV117比EIAVDLV121平均多2个N-糖基化位点,总数为19,其中3个为EIAVDV117特有。EIAVDLV121有1个疫苗株特有N-糖基化位点。研究结果为进一步探讨马传染性贫血弱毒疫苗生物学特性提供信息。     

Detailed mapping of the antigenicity of the surface unit glycoprotein of equine infectious anemia virus by using synthetic peptide strategies.

We describe here a detailed analysis of the antigenic determinants of the surface unit glycoprotein (gp90) of equine infectious anemia virus (EIAV), using a comprehensive panel of synthetic peptides in enzyme-linked immunosorbent assays with immune serum from naturally and experimentally infected horses and with a panel of gp90-specific neutralizing and nonneutralizing monoclonal antibodies. The results of these studies identify immunoreactive segments throughout the conserved and variable domains of gp90 but localize immunodominant (100% reactivity) determinants to the amino and carboxyl termini of the glycoprotein molecule. Analysis of peptide reactivities with longitudinal serum samples taken from experimentally infected ponies revealed that antibody responses to conserved B-cell determinants appeared earlier and at higher titers than do antibodies specific for determinants contained in the variable domain of gp90. These observations suggest an evolution of antibody responses in EIAV-infected ponies that may correspond to the establishment of immunological control of virus replication and disease routinely observed in EIAV infections. In addition, the mapping of monoclonal antibody epitopes to peptides of 9 to 12 amino acids demonstrated that all of the neutralizing epitopes are located in the variable domain of gp90. The arrangement of neutralizing epitopes and critical structural considerations suggest that EIAV gp90 contains a principal neutralizing domain similar to the V3 loop of human immunodeficiency virus type 1. These antigenic analyses provide an important foundation for further analyzing the protective immune response generated during persistent EIAV infections and also provide potential peptide substrates for diagnostic assays and for vaccine strategies.     

Efficacy of inactivated whole-virus and subunit vaccines in preventing infection and disease caused by equine infectious anemia virus.

We report here on a series of vaccine trials to evaluate the effectiveness of an inactivated equine infectious anemia virus (EIAV) whole-virus vaccine and of a subunit vaccine enriched in EIAV envelope glycoproteins. The inactivated vaccine protected 14 of 15 immunized ponies from infection after challenge with at least 10(5) 50% tissue culture-infective doses of the homologous prototype strain of EIAV. In contrast, it failed to prevent infection in any of 15 immunized ponies that were challenged with the heterologous PV strain. Levels of PV virus replication and the development of disease, however, were significantly reduced in 12 of the 15 ponies so challenged. The subunit vaccine prevented infection from homologous challenge in four of four ponies tested but failed to prevent infection in all four challenged with the PV strain. Two of the four subunit vaccinates had more severe symptoms of equine infectious anemia than nonimmunized ponies infected in parallel. Both vaccines stimulated EIAV-specific cell-mediated immunity. The in vitro lymphoproliferative response was shown to be mediated by T lymphocytes and appeared to be indistinguishable from that induced by EIAV infection. Significant differences were observed in the in vivo lymphocyte responses following challenge with the two virus strains. While peripheral blood mononuclear cells from the inactivated virus vaccinates were equally stimulated by both the prototype and PV strains, the subunit vaccinates challenged with PV exhibited lower levels of spontaneous proliferation and serine esterase activity. This diminished cellular response to PV was correlated with more severe clinical disease in the same ponies. These studies demonstrate for the first time that both an EIAV inactivated whole-virus vaccine and a viral envelope glycoprotein-based subunit vaccine can provide protection against rigorous challenge levels of homologous virus but are unable to protect against similar challenge levels of a heterologous virus. Moreover, the data demonstrate that protection can be achieved in the absence of detectable levels of virus-specific neutralizing antibody in the vaccine recipients at the time of virus challenge. While vaccine-induced virus-specific cell-mediated immune responses were detected, their role in conferring protection was not obvious. Nevertheless, protection from disease appeared to be correlated with the induction of high levels of serine esterase activity following challenge. A significant observation is that while the whole-virus vaccine was usually capable of preventing or markedly moderating disease in the PV-infected ponies, the subunit vaccine appeared to have a high potential to enhance the disease induced by PV infection.(ABSTRACT TRUNCATED AT 400 WORDS)     

Envelope determinants of equine infectious anemia virus vaccine protection and the effects of sequence variation on immune recognition

A highly effective attenuated equine infectious anemia virus (EIAV) vaccine (EIAV(D9)) capable of protecting 100% of horses from disease induced by a homologous Env challenge strain (EIAV(PV)) was recently tested in ponies to determine the level of protection against divergent Env challenge strains (J. K. Craigo, B. S. Zhang, S. Barnes, T. L. Tagmyer, S. J. Cook, C. J. Issel, and R. C. Montelaro, Proc. Natl. Acad. Sci. USA 104:15105-15110, 2007). An inverse correlation between challenge strain Env variation and vaccine protection from disease was observed. Given the striking differences in protective immunity, we hypothesized that analysis of the humoral and cellular immune responses to the Env protein could reveal potential determinants of vaccine protection. Neutralization activity against the homologous Env or challenge strain-specific Env in immune sera from the vaccinated ponies did not correlate with protection from disease. Cellular analysis with Env peptide pools did not reveal an association with vaccine protection from disease. However, when individual vaccine-specific Env peptides were utilized, eight cytotoxic-T-lymphocyte (CTL) peptides were found to associate closely with vaccine protection. One of these peptides also yielded the only lymphoproliferative response associated with protective immunity. The identified peptides spanned both variable and conserved regions of gp90. Amino acid divergence within the principal neutralization domain and the identified peptides profoundly affected immune recognition, as illustrated by the inability to detect cross-reactive neutralizing antibodies and the observation that certain peptide-specific CTL responses were altered. In addition to identifying potential Env determinants of EIAV vaccine efficacy and demonstrating the profound effects of defined Env variation on immune recognition, these data also illustrate the sensitivity offered by individual peptides compared to peptide pools in measuring cellular immune responses in lentiviral vaccine trials.     

Sequence analysis of Gardner-Arnstein feline leukaemia virus envelope gene reveals common structural properties of mammalian retroviral envelope genes

We have sequenced the envelope (env) gene and most of the adjacent 3' long terminal repeat (LTR) of Gardner-Arnstein feline leukaemia virus subtype B. The LTR of this virus contains, at corresponding positions, all signal sequence elements known from other retroviral LTRs. The deduced amino acid sequence of the longest open reading frame was compared with env polypeptide sequences of several murine leukaemia viruses. This allowed us to predict the positions of both p12/15env and gp70 polypeptides as well as a hydrophobic leader polypeptide. The env polypeptides of the different viruses show long stretches of homology and similar hydrophilicity profiles in the p12env region and in the carboxy-terminal half of gp70 (constant region). The most extensive variations are confined to certain parts of the amino-terminal half of gp70 (differential region). In this region, however, feline leukaemia virus and murine mink cell focus forming viruses are still closely related. A correspondingly spaced pattern of identical, short amino acid sequences appears in three different parts of the env polyprotein, suggesting its evolution from a primordial env-related precursor by tandem duplications.     

Equine infectious anemia virus genomic evolution in progressor and nonprogressor ponies

A primary mechanism of lentivirus persistence is the ability of these viruses to evolve in response to biological and immunological selective pressures with a remarkable array of genetic and antigenic variations that constitute a perpetual natural experiment in genetic engineering. A widely accepted paradigm of lentivirus evolution is that the rate of genetic variation is correlated directly with the levels of virus replication: the greater the viral replication, the more opportunities that exist for genetic modifications and selection of viral variants. To test this hypothesis directly, we examined the patterns of equine infectious anemia virus (EIAV) envelope variation during a 2.5-year period in experimentally infected ponies that differed markedly in clinical progression and in steady-state levels of viral replication as indicated by plasma virus genomic RNA assays. The results of these comprehensive studies revealed for the first time similar extents of envelope gp90 variation in persistently infected ponies regardless of the number of disease cycles (one to six) and viremia during chronic disease. The extent of envelope variation was also independent of the apparent steady-state levels of virus replication during long-term asymptomatic infection, varying from undetectable to 10(5) genomic RNA copies per ml of plasma. In addition, the data confirmed the evolution of distinct virus populations (genomic quasispecies) associated with sequential febrile episodes during acute and chronic EIA and demonstrated for the first time ongoing envelope variation during long-term asymptomatic infections. Finally, comparison of the rates of evolution of the previously defined EIAV gp90 variable domains demonstrated distinct differences in the rates of nucleotide and amino acid sequence variation, presumably reflecting differences in the ability of different envelope domains to respond to immune or other biological selection pressures. Thus, these data suggest that EIAV variation can be associated predominantly with ongoing low levels of virus replication and selection in target tissues, even in the absence of substantial levels of plasma viremia, and that envelope variation continues during all stages of persistent infection as the virus successfully avoids clearance by host defense mechanisms.     

Proviral genomic sequence analysis of Chinese donkey leukocyte attenuated equine infectious anemia virus vaccine and its parental virus strain Liaoning

Proviral DNA was extracted from donkey leukocyte infected with Chinese donkey leukocyte attenuated equine infectious anemia virus (DLA-EIAV), and peripheral blood lymphocytes (PBL) from a horse infected with the virulent EIAV strain Liaoning (EIAV L). The entire proviral DNA from both viruses was cloned and sequenced. The lengths of complete genomic sequences of DLA-EIAV and EIAV L provirus were 8266 bp and 8235 bp, respectively. Sequence comparison indicated that DLA-EIAV shares 97.0% and 97.5% in sequence homology with EIAV L and donkey-adapted EIAV (DA-EIAV), respectively. Lots of variations occurred in long terminal repeat (LTR, consisting of U3, R, U5), ORF S2, and envregions between DLA-EIAV and EIAV L. The nucleotide sequence differences of the two viruses in U3, R, U5, ORF S2, and env are 13.2%, 7.5%, 5.1%, 3.9%, and 2.7%, respectively, and predicted amino acid sequence differences in env and S2 coding regions are 4.4% and 8.8%, respectively. Six conserved regions are characterized in Gp90. There     

Equine infectious anemia virus envelope evolution in vivo during persistent infection progressively increases resistance to in vitro serum antibody neutralization as a dominant phenotype

Equine infectious anemia virus (EIAV) infection of horses is characterized by well-defined waves of viremia associated with the sequential evolution of distinct viral populations displaying extensive envelope gp90 variation; however, a correlation of in vivo envelope evolution with in vitro serum neutralization phenotype remains undefined. Therefore, the goal of the present study was to utilize a previously defined panel of natural variant EIAV envelope isolates from sequential febrile episodes to characterize the effects of envelope variation during persistent infection on viral neutralization phenotypes and to define the determinants of EIAV envelope neutralization specificity. To assess the neutralization phenotypes of the sequential EIAV envelope variants, we determined the sensitivity of five variant envelopes to neutralization by a longitudinal panel of immune serum from the source infected pony. The results indicated that the evolution of the EIAV envelope sequences observed during sequential febrile episodes produced an increasingly neutralization-resistant phenotype. To further define the envelope determinants of EIAV neutralization specificity, we examined the neutralization properties of a panel of chimeric envelope constructs derived from reciprocal envelope domain exchanges between selected neutralization-sensitive and neutralization-resistant envelope variants. These results indicated that the EIAV gp90 V3 and V4 domains individually conferred serum neutralization resistance while other envelope segments in addition to V3 and V4 were evidently required for conferring total serum neutralization sensitivity. These data clearly demonstrate for the first time the influence of sequential gp90 variation during persistent infection in increasing envelope neutralization resistance, identify the gp90 V3 and V4 domains as the principal determinants of antibody neutralization resistance, and indicate distinct complex cooperative envelope domain interactions in defining sensitivity to serum antibody neutralization.     

Rapid emergence of novel antigenic and genetic variants of equine infectious anemia virus during persistent infection.

Previous results from our laboratory have demonstrated that equine infectious anemia virus displays structural variations in its surface glycoproteins and RNA genome during passage and chronic infections in experimentally infected Shetland ponies (Montelaro et al., J. Biol. Chem. 259:10539-10544, 1984; Payne et al., J. Gen. Virol. 65:1395-1399, 1984). The present study was undertaken to obtain an antigenic and biochemical characterization of equine infectious anemia virus isolates recovered from an experimentally infected pony during sequential disease episodes, each separated by intervals of only 4 to 8 weeks. The virus isolates could be distinguished antigenically by neutralization assays with serum from the infected pony and by Western blot analysis with a monoclonal antibody against the major surface glycoprotein gp90, thus demonstrating that novel antigenic variants of equine infectious anemia virus predominate during each clinical episode. The respective virion glycoproteins displayed different electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels, indicating structural variation. Tryptic peptide and glycopeptide maps of the viral proteins of each virus isolate revealed biochemical alterations involving amino acid sequence and glycosylation patterns in the virion surface glycoproteins gp90 and gp45. In contrast, no structural variation was observed in the internal viral proteins pp15, p26, and p9 from any of the four virus isolates. Oligonucleotide mapping experiments revealed similar but unique RNase T1-resistant oligonucleotide fingerprints of the RNA genomes of each of the virus isolates. Localization of altered oligonucleotides for one virus isolate placed two of three unique oligonucleotides within the predicted env gene region of the genome, perhaps correlating with the structural variation observed in the envelope glycoproteins. Thus these results support the concept that equine infectious anemia virus is indeed capable of relatively rapid genomic variations during replication, some of which result in altered glycoprotein structures and antigenic variants which are responsible for the unique periodic disease nature observed in persistently infected animals. The findings of envelope specific differences in isolates of visna virus and of human T-cell lymphotropic virus III (acquired immune deficiency syndrome-related virus) suggest that this variation may be a common characteristic of the subfamily Lentivirinae.     

A live attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses

Previous evaluations of inactivated whole-virus and envelope subunit vaccines to equine infectious anemia virus (EIAV) have revealed a broad spectrum of efficacy ranging from highly type-specific protection to severe enhancement of viral replication and disease in experimentally immunized equids. Among experimental animal lentivirus vaccines, immunizations with live attenuated viral strains have proven most effective, but the vaccine efficacy has been shown to be highly dependent on the nature and severity of the vaccine virus attenuation. We describe here for the first time the characterization of an experimental attenuated proviral vaccine, EIAV(UK)deltaS2, based on inactivation of the S2 accessory gene to down regulate in vivo replication without affecting in vitro growth properties. The results of these studies demonstrated that immunization with EIAV(UK)deltaS2 elicited mature virus-specific immune responses by 6 months and that this vaccine immunity provided protection from disease and detectable infection by intravenous challenge with a reference virulent biological clone, EIAV(PV). This level of protection was observed in each of the six experimental horses challenged with the reference virulent EIAV(PV) by using a low-dose multiple-exposure protocol (three administrations of 10 median horse infectious doses [HID(50)], intravenous) designed to mimic field exposures and in all three experimentally immunized ponies challenged intravenously with a single inoculation of 3,000 HID(50). In contrast, na?ve equids subjected to the low- or high-dose challenge develop a detectable infection of challenge virus and acute disease within several weeks. Thus, these data demonstrate that the EIAV S2 gene provides an optimal site for modification to achieve the necessary balance between attenuation to suppress virulence and replication potential to sufficiently drive host immune responses to produce vaccine immunity to viral exposure.     

Sequence of the Long Terminal Repeat and Adjacent Segments of the Endogenous Avian Virus Rous-Associated Virus 0

Rous-associated virus 0 (RAV-0), an endogenous chicken virus, does not cause disease when inoculated into susceptible domestic chickens. An infectious unintegrated circular RAV-0 DNA was molecularly cloned, and the sequence of the long terminal repeat (LTR) and adjacent segments was determined. The sequence of the LTR was found to be very similar to that of replication-defective endogenous virus EV-1. Like the EV-1 LTR, the RAV-0 LTR is smaller (278 base pairs instead of 330) than the LTRs of the oncogenic members of the avian sarcoma virus-avian leukosis virus group. There is, however, significant homology. The most striking differences are in the U(3) region of the LTR, and in this region there are a series of small segments present in the oncogenic viruses which are absent in RAV-0. These differences in the U(3) region of the LTR could account for the differences in the oncogenic potential of RAV-0 and the avian leukosis viruses. I also compared the regions adjacent to the RAV-0 LTR with the available avian sarcoma virus sequences. A segment of approximately 200 bases to the right of the LTR (toward gag) is almost identical in RAV-0 and the Prague C strain of Rous sarcoma virus. The segment of RAV-0 which lies between the end of the env gene and U(3) is approximately 190 bases in length. Essentially this entire segment is present between env and src in the Schmidt-Ruppin A strain of Rous sarcoma virus. Most of this segment is also present between env and src in Prague C; however, in Prague C there is an apparent deletion of 40 bases in the region adjacent to env. In Schmidt-Ruppin A, but not in Prague C, about half of this segment is also present between src and the LTR. This arrangement has implications for the mechanism by which src was acquired. The region which encoded the gp37 portion of env appears to be very similar in RAV-0 and the Rous sarcoma viruses. However, differences at the very end of env imply that the carboxy termini of RAV-0, Schmidt-Ruppin A, and Prague C gp37s are significantly different. The implications of these observations are considered.     

Course and extent of variation of equine infectious anemia virus during parallel persistent infections.

Comparisons of peptide and oligonucleotide maps of glycoproteins and RNA from nine isolates of equine infectious anemia virus (EIAV) that were generated during parallel infections of two Shetland ponies revealed that each isolate was structurally unique. Each EIAV isolate contained a unique subset of variant peptides, oligonucleotides, or both, indicating that structural variation in EIAV is a random and noncumulative process and that a large spectrum of possible EIAV variants can be generated in infected animals.     

Molecular characterization of a neuropathogenic and nonerythroleukemogenic variant of Friend murine leukemia virus PVC-211.

PVC-211 murine leukemia virus (MuLV) is a replication-competent, ecotropic type C retrovirus that was isolated after passage of the Friend virus complex through F344 rats. Unlike viruses in the Friend virus complex, it does not cause erythroleukemia but causes a rapidly progressive hind limb paralysis when injected into newborn rats and mice. We have isolated an infectious DNA clone (clone 3d) of this virus which causes neurological disease in animals as efficiently as parental PVC-211 MuLV. The restriction map of clone 3d is very similar to that of the nonneuropathogenic, erythroleukemogenic Friend murine leukemia virus (F-MuLV), suggesting that PVC-211 MuLV is a variant of F-MuLV and that no major structural alteration was involved in its derivation. Studies with chimeric viruses between PVC-211 MuLV clone 3d and wild-type F-MuLV clone 57 indicate that at least one determinant for neuropathogenicity resides in the 2.1-kb XbaI-ClaI fragment containing the gp70 coding region of PVC-211 MuLV. Compared with nonneuropathogenic ecotropic MuLVs, the env gene of PVC-211 MuLV encodes four unique amino acids in the gp70 protein. Nucleotide sequence analysis also revealed a deletion in the U3 region of the long terminal repeat (LTR) of PVC-211 MuLV clone 3d compared with F-MuLV clone 57. In contrast to the env gene of PVC-211 MuLV, particular sequences within the U3 region of the viral LTR do not appear to be required for neuropathogenicity. However, the changes in the LTR of PVC-211 MuLV may be responsible for the failure of this virus to cause erythroleukemia, because chimeric viruses containing the U3 region of F-MuLV clone 57 were erythroleukemogenic whereas those with the U3 of PVC-211 MuLV clone 3d were not.     

Sequences from myeloblastosis-associated virus MAV-2(O) and UR2AV involved in the formation of plaques and the induction of osteopetrosis, anemia, and ataxia.

Recombinant viruses were made between myeloblastosis-associated virus MAV-2(O) and UR2AV to examine the relationship between regions of the MAV-2(O) genome and disease induction. The env-long terminal repeat (LTR) portion of MAV-2(O), when substituted into UR2AV, was sufficient to induce osteopetrosis identical to that caused by the parent MAV-2(O). When this region was reduced to the gp37 and LTR of MAV-2(O), osteopetrosis more severe than that caused by the parent virus was induced. Recombinant viruses that contained all or part of the MAV-2(O) env gene in the absence of the MAV-2(O) LTR induced a severe, chronic anemia and late-onset osteopetrosis, leading to the conclusion that the MAV-2(O) LTR, in addition to env, was required for rapid induction of osteopetrosis. A viral recombinant, pEU, which contained the gp85 segment of UR2AV substituted into MAV-2(O), induced an ataxia/cerebellar dysfunction not seen during infection with the other chimeric or parent viruses. In vitro studies of the parent and recombinant viruses demonstrated that the ability to form plaques on chicken embryo fibroblasts correlated with the presence of the MAV-2(O) gp37 and LTR except for construct pEU. When the viruses were inoculated into 10-day-old chickens, chimeras containing the env-LTR of gp37-LTR region of MAV-2(O) induced severe regenerative anemia similar to that induced by MAV-2(O). pEU was the exception, suggesting that the unique configuration of this chimera is responsible for its unusual pathogenic properties.     

The R-U5-5'' leader sequence of neurovirulent wild mouse retrovirus contains an element controlling the incubation period of neurodegenerative disease.

The wild mouse ecotropic retrovirus CasBrE causes a spongiform neurodegenerative disease after neonatal inoculation, with an incubation period ranging from 2 to 12 months. We previously showed that introduction of long terminal repeat (LTR) and gag-pol sequences from a strain of Friend murine leukemia virus (FB29) resulted in a dramatic acceleration of the onset of the disease. The chimeric virus FrCasE, which consisted of the FB29 genome containing 3' pol and env sequences from the wild mouse virus, induced a highly predictable, lethal neurodegenerative disease with an incubation period of only 16 days. Here we report that the sequences which are primary determinants of the length of the incubation period are located in the 5' end of the viral genome between a KpnI site in the R region of the LTR and a PstI site immediately 5' of the start codon for pr65gag (R-U5-5' leader). This region contains the tRNA primer binding site, splice donor site for the subgenomic env mRNA, and the packaging sequence. Computer-assisted sequence analysis failed to find evidence of a consensus sequence for a DNA enhancer in this region. In addition, sequences within a region of the genome between a ClaI site at the 3' end of env to the KpnI site in the R region of the LTR (inclusive of U3) also influenced the incubation period of the disease, but the effect was distinctly weaker than that of the R-U5-5' leader sequence. This U3 effect, however, appeared to be independent of the number of direct repeats, since deletion of one of two duplicated 42-base repeats containing consensus sequences of nuclear-factor binding domains had no effect on the incubation period of the disease. On the basis of Southern blot analysis of total viral DNA in the tissues, the effect of these sequences on the incubation period appeared to be related to the level of virus replication in the central nervous system. All of the chimeric viruses analyzed, irrespective of neurovirulence, replicated to comparable levels in the spleen and induced comparable levels of viremia.     

Role of the envelope genetic context in the development of enfuvirtide resistance in human immunodeficiency virus type 1-infected patients

Acquired human immunodeficiency virus type 1(HIV-1) resistance to the fusion inhibitor enfuvirtide (ENF) is primarily associated with mutations within the highly conserved first heptad repeat (HR1) region of gp41. Viral env sequences, however, are remarkably variable, and the envelope genetic background could have an important impact on optimal expression of HR1 mutations. We have examined the genetic evolution of env sequences, ENF susceptibility, and Env replicative capacity in patients failing ENF treatment. Sequential plasma-derived virus populations, obtained from six patients initiating ENF treatment as part of a salvage therapy, were studied using a recombinant phenotypic assay evaluating the entire gp120 and the gp41 ectodomains. Regardless of major differences in the baseline ENF susceptibilities, viral populations with similar phenotypic ENF resistance (50% inhibitory concentration, >3,000 ng/ml) were selected under treatment in four of six patients. As expected, in all patients ENF-resistant viruses harbored one or more HR1 mutations (positions 36, 38, and 43). Interestingly, in five patients the emergence of resistance mutations was not associated with reduced Env replicative capacity. Phylogenetic analysis of env sequences in sequential samples from two patients showed that the HR1 mutations had emerged in the context of env quasi-species that were different from those prevalent at baseline. Thus, the envelope genetic context appears to play a critical role in the selection of HR1 mutations and the expression of ENF resistance, thereby conditioning the evolution of HIV-1 under fusion inhibitor selective pressure.