Mutational Analysis of the Candidate Internal Fusion Peptide of the Avian Leukosis and Sarcoma Virus Subgroup A Envelope Glycoprotein

The transmembrane subunit (TM) of the avian leukosis and sarcoma virus (ALSV) envelope glycoprotein (Env) contains a stretch of conserved hydrophobic amino acids internal to its amino terminus (residues 21 to 42). By analogy with similar sequences in other viral envelope glycoproteins, this region has been proposed to be a fusion peptide. We investigated the role of this region by changing each of three hydrophobic residues (Ile-21, Val-30, and Ile-39) to glutamatic acid and lysine in the ALSV subgroup A Env. Like wild-type (wt) Env, all six mutant Env proteins were proteolytically processed, oligomerized, and expressed at the cell surface in a form that bound Tva, the ALSV subgroup A receptor. Like wt Env, Ile21Glu, Ile21Lys, Val30Glu, and Val30Lys changed conformation upon binding Tva, as assayed by sensitivity to thermolysin. Ile39Glu and Ile39Lys were cleaved by thermolysin in both the absence and presence of Tva. Although incorporated into virus particles at approximately equal levels, all mutant Envs were compromised in their ability to support infection. The mutants at residues 21 and 30 showed levels of infection 2 to 3 orders of magnitude lower than that of wt Env. The mutants at residue 39 were noninfectious. Furthermore, none of the mutants displayed activity in a cell-cell fusion assay. Our results support the contention that residues 21 to 42 of ALSV subgroup A Env constitute its fusion peptide.     

Rational site-directed mutations of the LLP-1 and LLP-2 lentivirus lytic peptide domains in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41 indicate common functions in cell-cell fusion but distinct roles in virion envelope incorporation

Two highly conserved cationic amphipathic alpha-helical motifs, designated lentivirus lytic peptides 1 and 2 (LLP-1 and LLP-2), have been characterized in the carboxyl terminus of the transmembrane (TM) envelope glycoprotein (Env) of lentiviruses. Although various properties have been attributed to these domains, their structural and functional significance is not clearly understood. To determine the specific contributions of the Env LLP domains to Env expression, processing, and incorporation and to viral replication and syncytium induction, site-directed LLP mutants of a primary dualtropic infectious human immunodeficiency virus type 1 (HIV-1) isolate (ME46) were examined. Substitutions were made for highly conserved arginine residues in either the LLP-1 or LLP-2 domain (MX1 or MX2, respectively) or in both domains (MX4). The HIV-1 mutants with altered LLP domains demonstrated distinct phenotypes. The LLP-1 mutants (MX1 and MX4) were replication defective and showed an average of 85% decrease in infectivity, which was associated with an evident decrease in gp41 incorporation into virions without a significant decrease in Env expression or processing in transfected 293T cells. In contrast, MX2 virus was replication competent and incorporated a full complement of Env into its virions, indicating a differential role for the LLP-1 domain in Env incorporation. Interestingly, the replication-competent MX2 virus was impaired in its ability to induce syncytia in T-cell lines. This defect in cell-cell fusion did not correlate with apparent defects in the levels of cell surface Env expression, oligomerization, or conformation. The lack of syncytium formation, however, correlated with a decrease of about 90% in MX2 Env fusogenicity compared to that of wild-type Env in quantitative luciferase-based cell-cell fusion assays. The LLP-1 mutant MX1 and MX4 Envs also exhibited an average of 80% decrease in fusogenicity. Altogether, these results demonstrate for the first time that the highly conserved LLP domains perform critical but distinct functions in Env incorporation and fusogenicity.     

Mutations in the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein impair the incorporation of Env proteins into mature virions.

In-frame stop codons were introduced into the coding region of human immunodeficiency virus type 1 (HIV-1) transmembrane protein (gp41). Truncation of 147 amino acids from the carboxyl terminus of gp41 (TM709) significantly decreased the stability and cell surface expression of the viral Env proteins, while truncation of 104 amino acids (TM752) did not. Truncation of 43 or more amino acids from the carboxyl terminus of gp41 generated mutant viruses which were noninfectious in several human CD4+ T lymphoid cell lines and fresh peripheral blood mononuclear cells. Analysis of the noninfectious mutant virions revealed significantly reduced incorporation of the Env proteins compared with the wild-type virions. Comparable amounts of Env proteins were detected on the surfaces of wild-type- and TM752-transfected cells, suggesting that the structures of gp41 required for efficient incorporation of Env proteins were disrupted in mutant TM752. Truncation of the last 12 amino acids (TM844) from the carboxyl terminus of gp41 did not significantly affect the assembly and release of virions or the incorporation of Env proteins into mature virions. However, the TM844 virus had dramatically decreased infectivity compared with the wild-type virus. This suggests that the cytoplasmic domain of gp41 also plays a role in other steps of virus replication.     

Multimerization potential of the cytoplasmic domain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41

We previously demonstrated that an envelope mutant of human immunodeficiency virus type 1 lacking the entire cytoplasmic domain interferes in trans with the production of infectious virus by inclusion of the mutant envelope into the wild-type envelope complex. We also showed that the envelope incorporation into virions is not affected when the wild-type envelope is coexpressed with the mutant envelope. These results suggest that an oligomeric structure of the cytoplasmic domain is functionally required for viral infectivity. To understand whether the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 has the potential to self-assemble as an oligomer, in the present study we fused the coding sequence of the entire cytoplasmic domain at 3' to the Escherichia coli malE gene, which encodes a monomeric maltose-binding protein. The expressed fusion protein was examined by chemical cross-linking, sucrose gradient centrifugation, and gel filtration. The results showed that the cytoplasmic domain of gp41 assembles into a high-ordered structural complex. The intersubunit interaction of the cytoplasmic domain was also confirmed by a mammalian two-hybrid system that detects protein-protein interactions in eucaryotic cells. A cytoplasmic domain fragment expressed in eucaryotic cells was pulled down by glutathione-Sepharose 4B beads via its association with another cytoplasmic domain fragment fused to the C terminus of the glutathione S-transferase moiety. We also found that sequences encompassing the lentiviral lytic peptide-1 and lentiviral lytic peptide-2, which are located within residues 828-856 and 770-795, respectively, play a critical role in cytoplasmic domain self-assembly. Taken together, the results from the present study indicate that the cytoplasmic domain of gp41 by itself is sufficient to assemble into a multimeric structure. This finding supports the hypothesis that a multimeric form of the gp41 cytoplasmic domain plays a crucial role in virus infectivity.     

Wild-type-like viral replication potential of human immunodeficiency virus type 1 envelope mutants lacking palmitoylation signals

Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4+ T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T- and M-tropic mutants also productively replicated in human primary CD4+ T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle.     

Effect of extension of the cytoplasmic domain of human immunodeficiency type 1 virus transmembrane protein gp41 on virus replication

The biological significance of the presence of a long cytoplasmic domain in the envelope (Env) transmembrane protein gp41 of human immunodeficiency virus type 1 (HIV-1) is still not fully understood. Here we examined the effects of cytoplasmic tail elongation on virus replication and characterized the role of the C-terminal cytoplasmic tail in interactions with the Gag protein. Extensions with six and nine His residues but not with fewer than six His residues were found to severely inhibit virus replication through decreased Env electrophoretic mobility and reduced Env incorporation compared to the wild-type virus. These two mutants also exhibited distinct N glycosylation and reduced cell surface expression. An extension of six other residues had no deleterious effect on infectivity, even though some mutants showed reduced Env incorporation into the virus and/or decreased cell surface expression. We further show that these elongated cytoplasmic tails in a format of the glutathione S-transferase fusion protein still interacted effectively with the Gag protein. In addition, the immediate C terminus of the cytoplasmic tail was not directly involved in interactions with Gag, but the region containing the last 13 to 43 residues from the C terminus was critical for Env-Gag interactions. Taken together, our results demonstrate that HIV-1 Env can tolerate extension at its C terminus to a certain degree without loss of virus infectivity and Env-Gag interactions. However, extended elongation in the cytoplasmic tail may impair virus infectivity, Env cell surface expression, and Env incorporation into the virus.     

The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions.

Accumulating evidence suggests that the matrix (MA) protein of retroviruses plays a key role in virus assembly by directing the intracellular transport and membrane association of the Gag polyprotein. In this report, we show that the MA protein of human immunodeficiency virus type 1 is also critical for the incorporation of viral Env proteins into mature virions. Several deletions introduced in the MA domain (p17) of human immunodeficiency virus type 1 Gag polyprotein did not greatly affect the synthesis and processing of the Gag polyprotein or the formation of virions. Analysis of the viral proteins revealed normal levels of Gag and Pol proteins in these mutant virions, but the Env proteins, gp120 and gp41, were hardly detectable in the mutant virions. Our data suggest that an interaction between the viral Env protein and the MA domain of the Gag polyprotein is required for the selective incorporation of Env proteins during virus assembly. Such an interaction appears to be very sensitive to conformational changes in the MA domain, as five small deletions in two separate regions of p17 equally inhibited viral Env protein incorporation. Mutant viruses were not infectious in T cells. When mutant and wild-type DNAs were cotransfected into T cells, the replication of wild-type virus was also hindered. These results suggest that the incorporation of viral Env protein is a critical step for replication of retroviruses and can be a target for the design of antiviral strategies.     

Role of the carboxy terminus of polypeptide D1 in the assembly of a functional water-oxidizing manganese cluster in photosystem II of the cyanobacterium Synechocystis sp. PCC 6803: assembly requires a free carboxyl group at C-terminal position 344.

The D1 polypeptide of the photosystem II (PSII) reaction center is synthesized as a precursor polypeptide which is posttranslationally processed at the carboxy terminus. It has been shown in spinach that such processing removes nine amino acids, leaving Ala344 as the C-terminal residue [Takahashi, M., Shiraishi, T., & Asada, K. (1988) FEBS Lett. 240, 6-8; Takahashi, Y., Nakane, H., Kojima, H., & Satoh, K. (1990) Plant Cell Physiol. 31, 273-280]. We show here that processing on the carboxy side of Ala344 also occurs in the cyanobacterium Synechocystis 6803, resulting in the removal of 16 amino acids. By constructing a deletion strain of Synechocystis 6803 that lacks the three copies of the psbA gene encoding D1, we have developed a system for generating psbA mutants. Using this system, we have constructed mutants of Synechocystis 6803 that are modified in the region of the C-terminus of the D1 polypeptide. Characterization of these mutants has revealed that (1) processing of the D1 polypeptide is blocked when the residue after the cleavage site is changed from serine to proline (mutant Ser345Pro) with the result that the manganese cluster is unable to assemble correctly; (2) the C-terminal extension of 16 amino acid residues can be deleted with little consequence either for insertion of D1 into the thylakoid membrane or for assembly of D1 into a fully active PSII complex; (3) removal of only one more residue (mutant Ala344stop) results in a loss of assembly of the manganese cluster; and (4) the ability of detergent-solubilized PSII core complexes (lacking the manganese cluster) to bind and oxidize exogenous Mn2+ by the secondary donor, Z+, is largely unaffected in the processing mutants (the Ser345Pro mutant of Synechocystis 6803 and the LF-1 mutant of Scenedesmus obliquus) and the truncation mutant Ala344stop. Our results are consistent with a role for processing in regulating the assembly of the photosynthetic manganese cluster and a role for the free carboxy terminus of the mature D1 polypeptide in the ligation of one or more manganese ions of the cluster.     

Requirement of N-terminal amino acid residues of gp41 for human immunodeficiency virus type 1-mediated cell fusion.

An expression vector was designed to test the structural requirements of the gp41 N terminus for human immunodeficiency virus type 1-induced membrane fusion. Mutations in the region coding for the N terminus of gp41 were found to disrupt glycoprotein expression because of deleterious effects on the Rev-responsive element (RRE). Insertion of an additional RRE in the 3'-noncoding sequence of env made possible efficient glycoprotein expression, irrespective of the mutations introduced into the RRE in the natural location. This permitted the insertion of the unique restriction site SpeI within the N-terminal sequences of gp41, allowing convenient and efficient mutation of the gp41 N terminus by using double-stranded synthetic oligonucleotides. Mutants with deletions of 1 to 7 amino acids of the N terminus were constructed. Expression and cleavage of all mutants were confirmed by Western immunoblot analysis with anti-gp41 antibodies. The capability of mutants to induce membrane fusion was monitored following transfection of HeLa-T4+ cell lines with wild-type and mutant expression vectors by electroporation and microinjection. The efficiency of cell-fusing activity decreased drastically with deletion of 3 and 4 amino acids and was completely lost with deletion of 5 amino acids. Cotransfection of the parent and mutant expression vectors resulted in reduced cell-fusing activity. The extent of this dominant interference by mutant glycoprotein paralleled the decrease in cell-fusing activity of the mutants alone. This suggests the existence of a specific N-terminal structure required for fusing activity. However, there does not appear to be a stringent requirement for the precise length of the N terminus. This finding is supported by the length variation of this region among natural human immunodeficiency virus type 1 isolates and is in contrast to the apparent stringency in the length of analogous N-terminal structures of influenza A virus and paramyxovirus fusion glycoproteins.     

Mutations in the Leucine Zipper-Like Heptad Repeat Sequence of Human Immunodeficiency Virus Type 1 gp41 Dominantly Interfere with Wild-Type Virus Infectivity

It has been previously shown that a proline substitution for any of the conserved leucine or isoleucine residues located in the leucine zipper-like heptad repeat sequence of human immunodeficiency virus type 1 (HIV-1) gp41 renders viruses noninfectious and envelope (Env) protein unable to mediate membrane fusion (S. S.-L. Chen, C.-N. Lee, W.-R. Lee, K. McIntosh, and T.-M. Lee, J. Virol. 67:3615–3619, 1993; S. S.-L. Chen, J. Virol. 68:2002–2010, 1994). To understand whether these variants could act as trans-dominant inhibitory mutants, the ability of these mutants to inhibit wild-type (wt) virus infectivity was examined. Comparable amounts of cell- and virion-associated gag gene products as well as virion-associated gp41 were found in transfection with wt or mutant HIV-1 provirus. Viruses obtained from coexpression of wt provirus with mutant 566 or 580 provirus inhibited more potently the production of infectious virus than did viruses generated from cotransfection of wt provirus with other mutant proviruses. Nevertheless, all viruses produced from mixed transfection showed decreased infectivity compared with that of the wt virus when a multinuclear-activation β-galactosidase induction assay was performed. The ability of wt Env to induce cytopathic effects was inhibited by coexpression with mutant Env. Coexpression of mutants inhibited the ability of the wt protein to mediate virus-to-cell transmission, as demonstrated by an env trans-complementation assay with a defective HIV-1 proviral vector. These observations indicated that mutant Env, per se, interferes with wt Env function. Moreover, cotransfection of wt and mutant proviruses produced amounts of cell- and virion-associated gag gene products comparable to those produced by transfection of wt provirus. Similar amounts of gp41 were also found in virions generated from wt-mutant cotransfection as well as from wt transfection alone. These results indicated that the inhibitory effect conferred by mutants on the wt virus infectivity does not involve the late steps of Gag protein assembly and budding, but they suggest that the wt and mutant Env proteins form a dysfunctional hetero-oligomer which is impaired in an early step of the virus replication cycle. Our study demonstrates that mutations in the HIV-1 gp41 leucine zipper-like heptad repeat sequence dominantly inhibit infectious virus production.     

Identification of the LWYIK motif located in the human immunodeficiency virus type 1 transmembrane gp41 protein as a distinct determinant for viral infection

The highly conserved LWYIK motif located immediately proximal to the membrane-spanning domain of the gp41 transmembrane protein of human immunodeficiency virus type 1 has been proposed as being important for the surface envelope (Env) glycoprotein's association with lipid rafts and gp41-mediated membrane fusion. Here we employed substitution and deletion mutagenesis to understand the role of this motif in the virus life cycle. None of the mutants examined affected the synthesis, precursor processing, CD4 binding, oligomerization, or cell surface expression of the Env, nor did they alter Env incorporation into the virus. All of the mutants, particularly the ΔYI, ΔIK, and ΔLWYIK mutants, in which the indicated residues were deleted, exhibited greatly reduced one-cycle viral replication and the Env trans-complementation ability. All of these deletion mutant proteins were still localized in the lipid rafts. With the exception of the Trp-to-Ala (WA) mutant, which exhibited reduced viral infectivity albeit with normal membrane fusion, all mutants displayed loss of some or almost all of the membrane fusion ability. Although these deletion mutants partially inhibited in trans wild-type (WT) Env-mediated fusion, they were more effective in dominantly interfering with WT Env-mediated viral entry when coexpressed with the WT Env, implying a role of this motif in postfusion events as well. Both T20 and L43L peptides derived from the two gp41 extracellular C- and N-terminal α-helical heptad repeats, respectively, inhibited WT and ΔLWYIK Env-mediated viral entry with comparable efficacies. Biotin-tagged T20 effectively captured both the fusion-active, prehairpin intermediates of WT and mutant gp41 upon CD4 activation. Env without the deletion of the LWYIK motif still effectively mediated lipid mixing but inhibited content mixing. Our study demonstrates that the immediate membrane-proximal LWYIK motif acts as a unique and distinct determinant located in the gp41 C-terminal ectodomain by promoting enlargement of fusion pores and postfusion activities.     

Unique Functional Properties of Conserved Arginine Residues in the Lentivirus Lytic Peptide Domains of the C-terminal Tail of HIV-1 gp41

A previous study from our laboratory reported a preferential conservation of arginine relative to lysine in the C-terminal tail (CTT) of HIV-1 envelope (Env). Despite substantial overall sequence variation in the CTT, specific arginines are highly conserved in the lentivirus lytic peptide (LLP) motifs and are scarcely substituted by lysines, in contrast to gp120 and the ectodomain of gp41. However, to date, no explanation has been provided to explain the selective incorporation and conservation of arginines over lysines in these motifs. Herein, we address the functions in virus replication of the most conserved arginines by performing conservative mutations of arginine to lysine in the LLP1 and LLP2 motifs. The presence of lysine in place of arginine in the LLP1 motif resulted in significant impairment of Env expression and consequently virus replication kinetics, Env fusogenicity, and incorporation. By contrast, lysine exchanges in LLP2 only affected the level of Env incorporation and fusogenicity. Our findings demonstrate that the conservative lysine substitutions significantly affect Env functional properties indicating a unique functional role for the highly conserved arginines in the LLP motifs. These results provide for the first time a functional explanation to the preferred incorporation of arginine, relative to lysine, in the CTT of HIV-1 Env. We propose that these arginines may provide unique functions for Env interaction with viral or cellular cofactors that then influence overall Env functional properties.     

Modifications of the human immunodeficiency virus envelope glycoprotein enhance immunogenicity for genetic immunization

In this study, we have investigated the effect of specific mutations in human immunodeficiency virus type 1 (HIV-1) envelope (Env) on antibody production in an effort to improve humoral immune responses to this glycoprotein by DNA vaccination. Mice were injected with plasmid expression vectors encoding HIV Env with modifications in regions that might affect this response. Elimination of conserved glycosylation sites did not substantially enhance humoral or cytotoxic-T-lymphocyte (CTL) immunity. In contrast, a modified gp140 with different COOH-terminal mutations intended to mimic a fusion intermediate and stabilize trimer formation enhanced humoral immunity without reducing the efficacy of the CTL response. This mutant, with deletions in the cleavage site, fusogenic domain, and spacing of heptad repeats 1 and 2, retained native antigenic conformational determinants as defined by binding to known monoclonal antibodies or CD4, oligomer formation, and virus neutralization in vitro. Importantly, this modified Env, gp140 Delta CFI, stimulated the antibody response to native gp160 while it retained its ability to induce a CTL response, a desirable feature for an AIDS vaccine.     

HIV-1 Nef enhances both membrane expression and virion incorporation of Env products. A model for the Nef-dependent increase of HIV-1 infectivity

The expression of human immunodeficiency virus Nef increases the viral infectivity through mechanisms still not fully elucidated. Here we report that wild-type (wt) human immunodeficiency virus, type 1 (HIV-1), particles were neutralized by higher concentrations of either anti-Env glycoprotein (gp) 41 antibodies or recombinant soluble human CD4 compared with Deltanef HIV-1. This appeared to be the result of a Nef-induced increase of virion incorporation of both gp41 (transmembrane (TM)) and surface gp120 Env products likely originating from enhanced steady-state levels of cell membrane-associated Env products. This, in turn, seemed to be the consequence of a reduced retention of the Env precursor. Most interesting, we found that both the Nef-directed increase of Env membrane expression and the Nef-induced enhancement of HIV-1 infectivity relied on the presence of the intracytoplasmic domain of TM, supporting the hypothesis of a functional correlation between these effects. Mutagenesis studies allowed us to establish that the two leucine residues at the TM C terminus, which are part of a sorting motif involved in the control of Env membrane expression, and the 181-210-residue Nef C-terminal region were critically involved in the Nef/Env functional interaction. In conclusion, we propose that Nef increases the infectivity of HIV-1 at least in part by enhancing the amounts of Env products incorporated into virus particles.     

Amino acid alterations within a highly conserved region of the Rous sarcoma virus src gene product pp60src inactivate tyrosine protein kinase activity.

Bisulfite mutagenesis techniques have been used to introduce single-point mutations within a region of the Rous sarcoma virus src gene defined by a BglI restriction endonuclease cleavage site. The mutants of Rous sarcoma virus that are produced by these techniques encode src proteins which contain single amino acid changes within a highly conserved amino acid sequence encompassing residues 430 to 433. DNA from the mutants CHpm26 ( Ala430 to Val), CHpm9 ( Pro431 to Ser), CHpm6 ( Glu432 to Lys), and CHpm65 ( Ala433 to Thr) each failed to transform chicken cells upon transfection, whereas DNA from CHpm59 (a third base alteration in the codon for Glu432 ) readily transformed chicken cells. Analysis of immune complexes containing the altered src proteins indicates that these proteins have decreased tyrosine protein kinase activity in vitro. In vivo labeling of cells infected with the mutant virus revealed diminished levels of the tyrosine-phosphorylated 34,000-molecular-weight protein. These data indicate that mutations within the sequence Ala430 - Pro431 - Glu432 - Ala433 lead to alterations in pp60src-specific tyrosine protein kinase activity and a concomitant loss of transforming potential of the mutant virus.     

Genetic evidence for an interaction between human immunodeficiency virus type 1 matrix and alpha-helix 2 of the gp41 cytoplasmic tail

The incorporation of envelope (Env) glycoproteins into virions is an essential step in the retroviral replication cycle. Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), encode Env glycoproteins with unusually long cytoplasmic tails, the functions of which have not been fully elucidated. In this study, we examine the effects on virus replication of a number of mutations in a helical motif (alpha-helix 2) located near the center of the HIV-1 gp41 cytoplasmic tail. We find that, in T-cell lines, small deletions in this domain disrupt the incorporation of Env glycoproteins into virions and markedly impair virus infectivity. Through the analysis of viral revertants, we demonstrate that a single amino acid change (34VI) in the matrix domain of Gag reverses the Env incorporation and infectivity defect imposed by a small deletion near the C terminus of alpha-helix 2. These results provide genetic evidence, in the context of infected T cells, for an interaction between HIV-1 matrix and the gp41 cytoplasmic tail and identify domains of both proteins involved in this putative interaction.     

Nuclear import of Epstein-Barr virus nuclear antigen 1 mediated by NPI-1 (Importin alpha5) is up- and down-regulated by phosphorylation of the nuclear localization signal for which Lys379 and Arg380 are essential

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is essential for replication of episomal EBV DNAs and maintenance of latency. Multifunctional EBNA-1 is phosphorylated, but the significance of EBNA-1 phosphorylation is not known. Here, we examined the effects on nuclear translocation of Ser phosphorylation of the EBNA-1 nuclear localization signal (NLS) sequence, 379Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser386. We found that Lys379Ala and Arg380Ala substitutions greatly reduced nuclear transport and steady-state levels of green fluorescent protein (GFP)-EBNA1, whereas Pro381Ala, Arg382Ala, Pro384Ala, and Glu378Ala substitutions did not. Microinjection of modified EBNA-1 NLS peptide-inserted proteins and NLS peptides cross-linked to bovine serum albumin (BSA) showed that Ala substitution for three NLS Ser residues reduced the efficiency of nuclear import. Similar microinjection analyses demonstrated that phosphorylation of Ser385 accelerated the rate of nuclear import, but phosphorylation of Ser383 and Ser386 reduced it. However, transfection analyses of GFP-EBNA1 mutants with the Ser-to-Ala substitution causing reduced nuclear import efficiency did not result in a decrease in the nuclear accumulation level of EBNA-1. The results suggest dynamic nuclear transport control of phosphorylated EBNA-1 proteins, although the nuclear localization level of EBNA-1 that binds to cellular chromosomes and chromatin seems unchanged. The karyopherin alpha NPI-1 (importin alpha5), a nuclear import adaptor, bound more strongly to Ser385-phosphorylated NLS than to any other phosphorylated or nonphosphorylated forms. Rch1 (importin alpha1) bound only weakly and Qip1 (importin alpha3) did not bind to the Ser385-phosphorylated NLS. These findings suggest that the amino-terminal 379Lys-Arg380 is essential for the EBNA-1 NLS and that Ser385 phosphorylation up-regulates nuclear transport efficiency of EBNA-1 by increasing its binding affinity to NPI-1, while phosphorylation of Ser386 and Ser383 down-regulates it.     

Antibody neutralization escape mediated by point mutations in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41

The persistence of human immunodeficiency virus type 1 (HIV-1) infection in the presence of robust host immunity has been associated in part with variation in viral envelope proteins leading to antigenic variation and escape from neutralizing antibodies. Previous studies of natural neutralization escape mutants have predominantly focused on gp120 and gp41 ectodomain sequence variations that alter antibody binding via changes in conformation or glycosylation pattern of the Env, likely due to the immune pressure exerted on the exposed ectodomain component of the glycoprotein. Here, we show for the first time a novel mechanism by which point mutations in the intracytoplasmic tail of the transmembrane component (gp41) of envelope can render the virus resistant to neutralization by monoclonal antibodies and broadly neutralizing polyclonal serum antibodies. Point mutations in a highly conserved structural motif within the intracytoplasmic tail resulted in decreased binding of neutralizing antibodies to the Env ectodomain, evidently due to allosteric changes both in the gp41 ectodomain and in gp120. While receptor binding and infectivity of the mutant virus remained unaltered, the changes in Env antigenicity were associated with an increase in neutralization resistance of the mutant virus. These studies demonstrate the structurally integrated nature of gp120 and gp41 and underscore a previously unrecognized potentially critical role for even minor sequence variation of the intracytoplasmic tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.     

A conserved structural determinant located at the interdomain region of mammalian inositol-requiring enzyme 1alpha

Inositol-requiring enzyme 1α (IRE1α), an endoplasmic reticulum-resident sensor for mammalian unfolded protein response, is a bifunctional enzyme containing kinase and RNase domains critical for trans-autophosphorylation and Xbp1 mRNA splicing, respectively, in response to endoplasmic reticulum stress. However, the amino acid residues important for its function and activation remain largely unexplored. Here, through analysis of IRE1α mutants associated with human somatic cancers, we have identified a highly conserved proline residue at position 830 (Pro(830)) that is critical for its structural integrity and hence, the activation of both kinase and RNase domains. Structural analysis revealed that Pro(830) may form a highly conserved structural linker with adjacent tryptophan and tyrosine residues at positions 833 and 945 (Trp(833) and Tyr(945)), thereby bridging the kinase and RNase domains. Indeed, mutation of Pro(830) to leucine (P830L) completely abolished the kinase and RNase activities, significantly decreased protein stability, and prevented oligomerization of IRE1α upon ER stress; similar observations were made for mutations of Trp(833) to alanine (W833A) and to a lesser extent for Y945A. Our finding may facilitate the identification of small molecules to compromise IRE1α function specifically.