Rev proteins of human and simian immunodeficiency virus enhance RNA encapsidation

The main function attributed to the Rev proteins of immunodeficiency viruses is the shuttling of viral RNAs containing the Rev responsive element (RRE) via the CRM-1 export pathway from the nucleus to the cytoplasm. This restricts expression of structural proteins to the late phase of the lentiviral replication cycle. Using Rev-independent gag-pol expression plasmids of HIV-1 and simian immunodeficiency virus and lentiviral vector constructs, we have observed that HIV-1 and simian immunodeficiency virus Rev enhanced RNA encapsidation 20- to 70-fold, correlating well with the effect of Rev on vector titers. In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev. Binding of Rev to the RRE or to a heterologous RNA element was required for Rev-mediated enhancement of RNA encapsidation. In addition to specific interactions of nucleocapsid with the packaging signal at the 5' end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral RNA.     

Synthesis and processing of human immunodeficiency virus type 1 envelope proteins encoded by a recombinant human adenovirus.

A recombinant adenovirus was constructed by inserting the human immunodeficiency virus type 1 (HIV-1) envelope gene downstream from the early region 3 (E3) promoter of adenovirus type 5 (Ad5), replacing the coding sequences of E3. The recombinant virus replicated as efficiently as the parent virus in all cell lines tested. Human cells infected with the recombinant virus synthesized the HIV-1 envelope precursor gp160, which was efficiently processed to the envelope glycoproteins gp120 and gp41. A human T-lymphoblast line (Molt-4) infected with the recombinant virus expressed HIV-1 envelope glycoproteins on the cell surface, leading to syncytium formation. The envelope gene was expressed from the E3 promoter at early times after infection and at late times from the major late promoter. When cotton rats were infected with the recombinant virus, antibodies against the HIV-1 envelope glycoproteins could be expressed in an immunoreactive form by the recombinant adenovirus, further illustrating the usefulness of adenoviruses as expression vectors.     

Coreceptor-dependent inhibition of the cell fusion activity of simian immunodeficiency virus Env proteins

The cytoplasmic tail (R peptide) sequence is able to regulate the fusion activity of the murine leukemia virus (MuLV) envelope (Env) protein. We have previously shown that this sequence exerts a profound inhibitory effect on the fusion activity of simian immunodeficiency virus (SIV)-MuLV chimeric Env proteins which contain the extracellular and transmembrane domains of the SIV Env protein. Recent studies have shown that SIV can utilize several alternative cellular coreceptors for its fusion and entry into the cell. We have investigated the fusion activity of SIV and SIV-MuLV chimeric Env proteins using cells that express different coreceptors. HeLa cells were transfected with plasmid constructs that carry the SIV or SIV-MuLV chimeric Env protein genes and were overlaid with either CEMx174 cells or Ghost Gpr15 cells, which express the Gpr15 coreceptor for SIV, or Ghost CCR5 cells, which express CCR5, an alternate coreceptor for SIV. The R-peptide sequence in the SIV-MuLV chimeric proteins was found to inhibit the fusion with CEMx174 cells or Ghost Gpr15 cells. However, a significant level of fusion was still observed when HeLa cells expressing the chimeric Env proteins were cocultivated with Ghost CCR5 cells. These results show that the R-peptide sequence exerts differential effects on the fusion activity of SIV Env proteins using target cells that express alternative coreceptors.     

Transcomplementation of simian immunodeficiency virus Rev with human T-cell leukemia virus type I Rex.

A molecular clone of the simian immunodeficiency virus SIVSMM isolate PBj14, lacking the ATG initiation codon for Rev protein (PBj-1.5), did not produce virus or large unspliced or singly spliced viral RNA upon transfection of HeLa cells. Low but significant levels of virus and large viral RNA production were observed upon transfection of PBj-1.5 into HeLa Rev cells expressing the rev gene of human immunodeficiency virus type 1. Furthermore, abundant virus and large viral RNA production occurred upon transfection of PBj-1.5 into HeLa Rex cells expressing the rex gene of human T-cell leukemia virus type I. Virus produced from HeLa Rex and HeLa Rev transfections was infectious, produced large amounts of virus, and was cytopathic for Rex-producing MT-4 cells. In contrast, no or only low levels of virus production were observed upon infection of H9 cells. These studies show that a defective SIV rev gene can be transcomplemented with human immunodeficiency virus type 1 Rev and with high efficiency by human T-cell leukemia virus type I Rex, and they suggest that rev-defective viruses could serve as a source for production of a live attenuated SIV vaccine.     

Inhibition of human immunodeficiency virus type 1 Rev function by a Rev mutant which interferes with nuclear/nucleolar localization of Rev.

A nonfunctional mutant of human immunodeficiency virus type 1 Rev was created by deleting seven amino acid residues within the nucleolar targeting signal. This mutant Rev remained in the cytoplasm in expressed cells and strongly inhibited the function of Rev by interfering with the nuclear/nucleolar localization of coexpressed Rev. These findings strongly suggest the multimerization of Rev in the cytoplasm before migration to the nucleus/nucleolus, where wild-type Rev functions as a trans-regulator.     

Vaccine protection by a triple deletion mutant of simian immunodeficiency virus.

Twelve rhesus monkeys were vaccinated with SIVmac316 delta nef (lacking nef sequences), and 12 were vaccinated with SIVmac239 delta3 (lacking nef, vpr, and upstream sequences in U3). SIVmac316 and SIVmac239 differ by only eight amino acids in the envelope; these changes render SIVmac316 highly competent for replication in macrophages. Seventeen of the animals developed persistent infections with the vaccine viruses. Seven of the 24 vaccinated animals, however, developed infections that were apparently transient in nature. Six of these seven yielded virus from peripheral blood when tested at weeks 2 and/or 3, three of the seven had transient antibody responses, but none of the seven had persisting antibody responses. The 24 monkeys were challenged in groups of four with 10 rhesus monkey infectious doses of wild-type, pathogenic SIVmac251 at weeks 8, 20, and 79 following receipt of vaccine. None of the seven with apparently transient infections with vaccine virus were protected upon subsequent challenge. Analysis of cell-associated viral loads, CD4+ cell counts, and viral gene sequences present in peripheral blood in the remainder of the monkeys following challenge allowed a number of conclusions. (i) There was a trend toward increased protection with length of time of vaccination. (ii) Solid vaccine protection was achieved by 79 weeks with the highly attenuated SIV239 delta3. (iii) Solid long-term protection was achieved in at least two animals in the absence of complete sterilizing immunity. (iv) Genetic backbone appeared to influence protective capacity; animals vaccinated with SIV239 delta3 were better protected than animals receiving SIV316 delta nef. This better protection correlated with increased levels of the replicating vaccine strain. (v) The titer of virus-neutralizing activity in serum on the day of challenge correlated with protection when measured against a primary stock of SIVmac251 but not when measured against a laboratory-passaged stock. The level of binding antibodies to whole virus by enzyme-linked immunosorbent assay also correlated with protection.     

Truncations of the simian immunodeficiency virus transmembrane protein confer expanded virus host range by removing a block to virus entry into cells.

We have investigated how truncation of the cytoplasmic domain of the transmembrane (TM) glycoprotein of simian immunodeficiency virus (SIV) modulates the host range of this virus. Termination codons were introduced into the env gene of SIVmac239 which resulted in the truncation of the transmembrane protein from a wild-type 354 amino acids (TM354) to 207 (TM207) and 193 (TM193) amino acids. Expression of the wild-type and mutant env genes from a simian virus 40-based vector resulted in normal biosynthesis and processing of the glycoproteins to gp130 and gp41 or the truncated TM proteins (gp28 and gp27). When expressed on the surface of COS-1 cells, all three glycoproteins mediated fusion of both CEMX174 and HUT78 cells. Virions containing the wild-type and mutant glycoproteins were capable of efficient replication in macaque peripheral blood lymphocytes and CEMX174 cells; in contrast, only virions that contained TM207 were capable of rapid infection of HUT78 cells. Both truncated glycoproteins were capable of efficiently mediating infection of both CEMX174 and HUT78 cells by an env-deficient human immunodeficiency virus. The wild-type SIV glycoprotein, however, was unable to mediate human immunodeficiency virus infection of HUT78 cells when assayed with this system. An analysis of the protein composition of SIV released from infected CEMX174 cells showed that the mutant virions contained significantly higher levels of glycoprotein compared with the wild type. These results demonstrate that truncation of the SIV cytoplasmic domain removes a block at the level of glycoprotein-mediated virus entry into HUT78 cells and points to a role for glycoprotein density in determining virus tropism.     

In vivo binding of wild-type and mutant human immunodeficiency virus type 1 Rev proteins: implications for function.

The Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1) is required for protein expression from the HIV-1 RNAs which contain a binding site for the Rev protein, termed the Rev-responsive element (RRE). This transactivator acts both at the level of splicing/transport of nuclear RNAs and at the level of translation of cytoplasmic RNAs. We used a monoclonal antibody specific for the HIV-1 Rev protein to immunoprecipitate cellular extracts from HIV-1-infected and -transfected cells. High levels of specific binding of wild-type Rev to the RRE-containing RNAs were found in cytoplasmic, but not nuclear, extracts from these cells. A Rev mutant which lacked both nuclear and cytoplasmic Rev function but retained RNA binding in vivo was generated. This binding was detectable with both nuclear and cytoplasmic extracts. These results verify the existence of direct binding of Rev to HIV-1 RNAs in vivo and conclusively prove that binding of Rev is not sufficient for nuclear or cytoplasmic Rev function. The results also support a direct role for Rev in the nuclear export and translation of HIV-1 RNAs.     

The cytopathicity of a simian immunodeficiency virus Mne variant is determined by mutations in Gag and Env.

Previous studies suggested that the rapidly replicating, highly cytopathic, syncytium-inducing (rapid-high/SI) phenotype of simian immunodeficiency virus Mne variants that evolved in macaques inoculated with a slowly replicating, minimally cytopathic, non-syncytium-inducing (slow-low/NSI) molecular clone was not solely the result of changes in the envelope surface protein (Env SU). To define the viral determinants responsible for the change in phenotype, we molecularly cloned a rapid-high/SI variant (designated SIVMne170) derived from the peripheral blood mononuclear cells (PBMCs) of a pig-tailed macaque that was inoculated with a slow-low/NSI molecular clone, SIVMneCL8. SIVMne170 was SI and replicated with faster kinetics and was more cytopathic than the parent SIVMneCL8 in CEMx174 cells. Additionally, SIVMne170 was more cytopathic for the CD4+ T-cell population than SIVMneCL8 in macaque PBMCs. An analysis of chimeric viruses constructed between the variant SIVMne170 and the parent virus SIVMneCL8 demonstrated that there are determinants encoded within both the 5' and 3' halves of SIVMne170 that independently contribute to its rapid-high/SI phenotype. As we previously observed with other SIVMne variants, the Env SU of SIVMne170 was important for syncytium induction but was not a key determinant of cytopathicity. By contrast, the intracellular domain of the envelope transmembrane protein (Env TM) contributed to both the SI and cytopathic properties of SIVMne170. We also found that the minimal determinant within the 5' half of SIVMne170 that conferred its rapid replication kinetics and cytopathicity mapped to the capsid- and nucleocapsid-encoding regions of gag. Together, these data demonstrate that mutations selected in Gag and Env TM intracytoplasmic tail influence the replication and cytopathicity of SIVMne variants that evolve in the host.     

Pseudotyping with human T-cell leukemia virus type I broadens the human immunodeficiency virus host range.

Several epidemiologic and clinical studies suggest that patients coinfected with human immunodeficiency virus (HIV), the primary etiologic agent in AIDS, and other viruses, such as cytomegalovirus or human T-cell leukemia virus (HTLV), have a more severe clinical course than those infected with HIV alone. Cells infected with two viruses can, in some cases, give rise to phenotypically mixed virions with altered or broadened cell tropism and could therefore account for some of these findings. Such pseudotypes could alter the course of disease by infecting more tissues than are normally infected by HIV. We show here that HIV type 1 (HIV-1) efficiently incorporates the HTLV type I (HTLV-I) envelope glycoprotein and that both HIV-1 and HTLV-II accept other widely divergent envelope glycoproteins to form infectious pseudotype viruses whose cellular tropisms and relative abilities to be transmitted by cell-free virions or by cell contact are determined by the heterologous envelope. We also show that the mechanism by which virions incorporate heterologous envelope glycoproteins is independent of the presence of the homologous glycoprotein or heterologous gag proteins. These results may have important implications for the mechanism of HIV pathogenesis.     

Inhibition of human immunodeficiency virus type 1 replication is enhanced by a combination of transdominant Tat and Rev proteins.

Mutation of either of two critical human immunodeficiency virus type 1 (HIV-1) regulatory proteins, Tat and Rev, results in marked defects in viral replication. Thus, inhibition of the function of one or both of these proteins can significantly inhibit viral growth. In the present study, we constructed a novel transdominant Tat mutant protein and compared its efficiency in inhibiting HIV-1 replication with that of transdominant mutant Rev M10 when these proteins were stably expressed either alone or in combination in T-lymphocyte cell lines. The transdominant Tat mutant protein alone resulted in a modest inhibition of HIV replication, but it was able to enhance the ability of the M10 Rev mutant protein to inhibit HIV-1 replication. These results suggest a possible synergistic effect of these transdominant mutant proteins in inhibiting HIV-1 replication.     

Shared antigenic epitopes of the major core proteins of human and simian immunodeficiency virus isolates.

Antigenic epitopes on the major core (gag) protein of isolates of simian and human immunodeficiency virus (SIV and HIV) were compared using a panel of eleven mouse monoclonal antibodies (Mabs) that recognized nine distinct gag epitopes. Viral isolates used for comparison were HIV-1IIIb, HIV-2ROD, and SIV isolates from macaque (SIVmac), sooty mangabey (SIVsm-UCD), African green monkey (SIVagm), and stump-tailed macaque (SIVstm-UCD). The relatedness of the various HIV and SIV isolates, as determined by Mabs to core protein epitopes, paralleled that ascertained by genetic sequencing.     

Differential regulation of the antibody responses to Gag and Env proteins of human immunodeficiency virus type 1.

We have studied the antibody responses to Env and Gag antigens of human immunodeficiency virus type 1 (HIV-1) in several cohorts of HIV-1-infected individuals: long-term nonprogressors, progressors to disease, acute seroconvertors, and recipients of HIV-1 protease inhibitors. We conclude that the antibody responses to Env and Gag antigens are differentially regulated and that changes in the plasma viral load in the measurable range (500 to 10(8) RNA copies per ml) do not directly affect the antibody responses to these HIV-1 proteins. We provide quantitative estimates of HIV-1-specific immunoglobulin G concentrations in plasma, which can be in excess of 1 mg/ml for both anti-gp120 and anti-p24 once the immune response to HIV-1 has stabilized after seroconversion. We discuss the apparent paradox that the absence of anti-Gag antibodies (which have, at best, limited antiviral activity) is indicative of disease progression, while the retention of anti-Env antibodies (which do have antiviral activity) is of limited (or no) prognostic value. We show that the disappearance of anti-Gag antibodies during disease progression is highly unlikely to be due to immune complexing; instead, we believe that it reflects the loss of T-cell help that is more necessary for the anti-Gag than the anti-Env response.     

Posttranscriptional regulation by the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins through a heterologous RNA binding site.

The human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins induce cytoplasmic expression of incompletely spliced viral mRNAs by binding to these mRNAs in the nucleus. Each protein binds a specific cis-acting element in its target RNAs. Both proteins also associated with nucleoli, but the significance of this association is uncertain because mutations that inactivate nucleolar localization signals in Rev or Rex also prevent RNA binding. Here we demonstrate that Rev and Rex can function when tethered to a heterologous RNA binding site by a bacteriophage protein. Under these conditions, cytoplasmic accumulation of unspliced RNA occurs without the viral response elements, mutations in the RNA binding domain of Rev do not inhibit function, and nucleolar localization can be shown to be unnecessary for the biological response.     

Cross-neutralization of human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus isolates.

In contrast to infrequent and low-titer cross-neutralization of human immunodeficiency virus type 1 (HIV-1) isolates by HIV-2- and simian immunodeficiency virus (SIV)-positive sera, extensive cross-neutralization of HIV-2NIH-Z, SIVMAC251, and SIVAGM208K occurs with high titer, suggesting conservation of epitopes and mechanism(s) of neutralization. The V3 regions of HIV-2 and SIV isolates, minimally related to the HIV-1 homolog, share significant sequence homology and are immunogenic in monkeys as well as in humans. Whereas the crown of the V3 loop is cross-reactive among HIV-1 isolates and elicits neutralizing antibodies of broad specificity, the SIV and especially HIV-2 crown peptides were not well recognized by cross-neutralizing antisera. V3 loop peptides of HIV-2 isolates did not elicit neutralizing antibodies in mice, guinea pigs, or a goat and together with SIV V3 peptides did not inhibit serum neutralization of HIV-2 and SIV. Thus, the V3 loops of HIV-2 and SIV do not appear to constitute simple linear neutralizing epitopes. In view of the immunogenicity of V3 peptides, the failure of conserved crown peptides to react with natural sera implies a significant role of loop conformation in antibody recognition. Our studies suggest that in addition to their grouping by envelope genetic relatedness, HIV-2 and SIV are neutralized similarly to each other but differently from HIV-1. The use of linear peptides of HIV-2 and SIV as immunogens may require greater attention to microconformation, and alternate subunit approaches may be needed in exploiting these viruses as vaccine models. Such approaches may also be applicable to the HIV-1 system in which conformational epitopes, in addition to the V3 loop, participate in virus neutralization.     

Vaccine protection against simian immunodeficiency virus by recombinant strains of herpes simplex virus

An effective vaccine for AIDS may require development of novel vectors capable of eliciting long-lasting immune responses. Here we report the development and use of replication-competent and replication-defective strains of recombinant herpes simplex virus (HSV) that express envelope and Nef antigens of simian immunodeficiency virus (SIV). The HSV recombinants induced antienvelope antibody responses that persisted at relatively stable levels for months after the last administration. Two of seven rhesus monkeys vaccinated with recombinant HSV were solidly protected, and another showed a sustained reduction in viral load following rectal challenge with pathogenic SIVmac239 at 22 weeks following the last vaccine administration. HSV vectors thus show great promise for being able to elicit persistent immune responses and to provide durable protection against AIDS.     

Virus-specific cytotoxic T-lymphocyte responses select for amino-acid variation in simian immunodeficiency virus Env and Nef.

Cytotoxic T-lymphocyte (CTL) responses to human immunodeficiency virus arise early after infection, but ultimately fail to prevent progression to AIDS. Human immunodeficiency virus may evade the CTL response by accumulating amino-acid replacements within CTL epitopes. We studied 10 CTL epitopes during the course of simian immunodeficiency virus disease progression in three related macaques. All 10 of these CTL epitopes accumulated amino-acid replacements and showed evidence of positive selection by the time the macaques died. Many of the amino-acid replacements in these epitopes reduced or eliminated major histocompatibility complex class I binding and/or CTL recognition. These findings strongly support the CTL 'escape' hypothesis.