Identification of a cis-acting element in human immunodeficiency virus type 2 (HIV-2) that is responsive to the HIV-1 rev and human T-cell leukemia virus types I and II rex proteins.

A simian virus 40 late replacement vector encoding human immunodeficiency virus type 1 (HIV-1) gp120 (pGP120) was used to define a region within the HIV-2 genome that could work as a rev-responsive element (RRE). Our previous work showed that gp120 expression in this system required a functional RRE in cis and required the rev protein in trans (M.-L. Hammarskj?ld, J. Heimer, B. Hammarskj?ld, I. Sangwan, L. Albert, and D. Rekosh, J. Virol. 63:1959-1966, 1989). Using pGP120, we first mapped an RRE to a 1,042-base-pair (bp) Sau3a fragment in the env region of HIV-2. Both HIV-1 rev (rev1) and HIV-2 rev (rev2) could work in conjunction with this fragment. Further mapping showed that a 272-bp subfragment within the 1,042-bp region was sufficient as an RRE. Surprisingly, the smaller fragment worked only with the rev1 protein and not with its homologous rev2 protein. In addition, the rev2 protein failed to function together with the RRE from HIV-1. We also utilized this system to examine the ability of the rex genes of human T-cell leukemia virus types I and II to functionally substitute for rev. These experiments showed that complementation by both the rexI and rexII proteins required the presence of an RRE. The rex proteins worked well in conjunction with either the HIV-1 or the HIV-2 RRE (the 1,042-bp as well as the 272-bp fragment).     

Mutational analysis of the human T-cell leukemia virus type I trans-acting rex gene product.

Expression of the human T-cell leukemia virus type I (HTLV-I) rex gene is a prerequisite for the expression of the retroviral structural proteins. We have generated internal deletion mutants of this 27-kDa nucleolar trans-acting gene product to define functional domains in the Rex protein. The phenotype of the various mutant proteins was tested on the homologous HTLV-I rex response element sequence and the heterologous human immunodeficiency virus type 1 (HIV-1) rev response element sequence. Our results indicate that a region between amino acid residues 55 and 132 in the 189-amino-acid Rex protein is required for Rex-mediated trans activation on both retroviral response element sequences. In addition, substitution of the Rex nuclear localization signal by a sequence of the HIV-1 rev gene product targets the Rex protein to the correct subcellular compartment required for Rex function.     

The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region.

The study of expression of several human immunodeficiency virus type 1 proviral mutants in human cells in the presence or absence of rev (trs/art) protein reveals that rev increases the levels of unspliced and env mRNA and the accumulated structural viral proteins. rev protein produced from appropriate expression vectors fully complements the rev-defective mutants. rev requires the presence of a specific cis-acting sequence for its function. This rev-responsive element sequence has been localized within a 520 base-pair fragment in the env region of human immunodeficiency virus type 1. gag and env expression is coordinately regulated by rev. Two independent cis-acting elements localized in the gag and env regions are responsible for the low levels of gag and env mRNA in the absence of rev. These elements are different than the rev-responsive element and act independent of each other.     

Mutational analysis of simian immunodeficiency virus from African green monkeys and human immunodeficiency virus type 2

We constructed ten mutants of simian immunodeficiency virus isolated from African green monkey (SIVAGM), and nine mutants of human immunodeficiency virus type 2 (HIV-2) in vitro. Their infectivity, cytopathogenicity, transactivation potential, virus RNA, and protein synthesis were examined by transfection and infection experiments. Mutations in three structural (gag, pol, env) and two regulator (tat, rev) genes abolished the infectivity of both viruses, but vpx, vpr (HIV-2), and nef were dispensable and mutant viruses were indistinguishable phenotypically from wild type virus. A vif mutant of HIV-2 showed poor infectivity in cell-free condition, whereas SIVAGM mutants grew equally well with wild type virus. In transient transfection assays, rev mutants derived from both viruses produced mainly small mRNA species and no detectable virus proteins and particles. Transactivation potential of tat mutants originated from both viruses was about three- to ten-fold less than that of respective wild type DNAs, generating small amounts of virus.     

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.     

Mutational analysis of the human immunodeficiency virus type 2 (HIV-2) genome in relation to HIV-1 and simian immunodeficiency virus SIV (AGM).

We constructed an infectious molecular clone of the human immunodeficiency virus type 2 (HIV-2) and generated nine frameshift mutants corresponding to nine open reading frames identified so far. Three structural (gag, pol, env) and two regulative (tat, rev) gene mutants were not infectious, whereas vif, vpx, vpr, and nef genes were dispensable for infectivity. All of the mutants except env and rev were cytopathic in CD4+ human leukemia cells. In transfection assays, the expression of HIV-2 long terminal repeat was activated by infectious clones of HIV-1, HIV-2, and simian immunodeficiency virus from African green monkey but not by the tat mutants. However, an HIV-2 tat mutant could produce small amounts of virus proteins and particles in contrast to a rev mutant, which directed no detectable synthesis of virus proteins and virions.     

Functional similarity of HIV-I rev and HTLV-I rex proteins: identification of a new nucleolar-targeting signal in rev protein

We have tested the functional compatibility between rev protein of human immunodeficiency virus type I (HIV-I) and rex protein of human T-cell lymphotropic virus type I (HTLV-I). Each protein recognized the other's cis-acting sequence, albeit at reduced levels. Both proteins localize predominantly in the nucleolus. We have identified a new nucleolar-targeting signal in rev protein, which was homologous to that of rex protein. The sequence [35-RQARRNRRRRWRERQR-50] in rev protein, when fused to the amino-terminus of beta-galactosidase, directed the hybrid protein to the cell nucleolus. A deletion mutant which lacks several amino acid residues within the signal failed to function in the CAT assay system. These results demonstrate that the nucleolar targeting signals are essential for the functions of Rev and Rex.     

Effects of chimeric mutants of human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex on nucleolar targeting signals.

Two chimeric mutant genes derived from rev of human immunodeficiency virus type 1 and rex of human T-cell leukemia virus type I were constructed to investigate the functions of the nucleolar-targeting signals (NOS) in Rev and Rex proteins. A chimeric Rex protein whose NOS region was substituted with the NOS of Rev was located predominantly in the cell nucleolus and functioned like the wild-type protein in the Rex assay system. However, a chimeric Rev with the NOS of Rex abolished Rev function despite its nucleolar localization. This nonfunctional nucleolar-targeting chimeric protein inhibited the function of both Rex and Rev. In the same experimental conditions, this mutant interfered with the localization of the functional Rex in the nucleolus.     

Identification of feline immunodeficiency virus rev gene activity.

We constructed 16 deletion mutants from an infectious molecular clone of feline immunodeficiency virus (FIV) and a reporter plasmid carrying the bacterial chloramphenicol acetyltransferase (CAT) gene to identify the rev transactivator activity of the virus. Cotransfections of various mutants and the rev reporter clone bearing a portion of FIV env in addition to the CAT gene revealed that the sequence important for the augmentation of CAT production was located in three separate parts of the virus genome. This enhancement was FIV specific in that the human retrovirus rev and rex gene products did not activate the reporter. The phenotypic properties of an FIV proviral mutant containing a small deletion in the genome were similar to those of rev mutants derived from primate immunodeficiency viruses. These results indicate that FIV, like the other lentiviruses, contains the rev gene in its genome.     

Novel human endogenous sequences related to human immunodeficiency virus type 1.

Endogenous retrovirus-related sequences exist within the normal genomic DNA of all eukaryotes, and these endogenous sequences have been shown to be important to the nature and biology of related exogenous retroviruses and may also play a role in cellular functions. To date, no endogenous sequences related to human immunodeficiency virus type 1 (HIV-1) have been reported. Herein we describe the first report of the presence of nucleotide sequences related to HIV-1 in human, chimpanzee, and rhesus monkey DNAs from normal uninfected individuals. We also present the isolation and characterization of two of these endogenous HIV-1-related sequences, EHS-1 and EHS-2. With use of low-stringency Southern blot hybridization, complex banding patterns were detected in human DNA with 5' and 3' HIV-1-derived probes. When an HIV-1 env region probe was used, we detected a less complex, conserved banding pattern in human DNA as well as a related but distinct banding pattern in chimpanzee and rhesus monkey DNAs. EHS-1 and -2 were cloned from normal human genomic DNA libraries by using the env region probe. Clone EHS-1 shows sequence similarity with the domain of the envelope cellular protease cleavage site of HIV-1, while EHS-2 has sequence similarity to the overlapping reading frame for Rev and gp41. Stringent hybridization of EHS-1 back to primate genomic DNA indicates two distinct EHS-1 loci in normal human DNA, an identical band pattern in chimpanzee DNA, and a single locus in rhesus monkey DNA. Likewise, EHS-2 is present as a single highly conserved locus in all three species. An oligonucleotide derived from EHS-2 across a region of near identity to HIV-1 detects a complex banding pattern in all primates tested similar to that seen with the 3' HIV-1 probe. These data suggest that most of the HIV-1-related sequences identified in primate DNA share a common core of nucleic acid sequence found in both EHS-2 and rev and that some of these HIV-1-related sequences have additional larger regions of sequence similarity to HIV-1.     

HIV-1 ancestry primordial expansions of RRE and RRE related sequences.

The human T-lymphotropic retroviruses HTLV-I and HIV-1/-2 share a complementary patchwork homology in which the RRE regions of HIV-1 and -2 cover 78.1% of a 169 nucleotide (nt) region (PX1,2) positioned precisely between open reading frames PX I and PX II of ATLV (HTLV-I). The sequence character of the PX1,2 region is shown to be influenced by a primordial expansion, CTC2T, originating several hundred nucleotides upstream. A second primordial expansion, AGCU(U/C), is identified and shown to represent 52.1% of the HIV-1 RRE region. It is argued that prior to the present AIDS pandemic the efficiency of the rev receptor was enhanced by an ancestral recombination event.     

Retroviral infections transmitted by blood transfusion

Modifications in donor screening and the introduction of laboratory testing of donated blood for anti-HIV-1 and anti-HTLV-I have resulted in a significant reduction in the risks of retroviral infections from blood transfusion. Presently, the American Red Cross detects an average of eight carriers of human immunodeficiency virus, type 1 (HIV-1) per 100,000 otherwise acceptable blood donors (0.008 percent), compared with an average of 35 per 100,000 (0.035 percent) when testing for HIV-1 antibodies began in 1985. Surveillance studies in the United States indicate a small likelihood that HIV-2 carriers will pass current screening procedures and be accepted as blood donors. Even if an HIV-2-infected person were to be accepted as a blood donor, there is a 42-92 percent likelihood that this person's blood would be detected as infective for HIV-2 and excluded because of serological cross-reactions that occur in the EIA for HIV-1 antibodies. During 1989, which was the first year that donated blood was routinely tested for antibodies to human T-lymphotropic virus, type I (HTLV-I) in the United States, approximately nine in 100,000 donors (0.009 percent) were confirmed positive for antibodies to HTLV-I, and their donated blood was excluded. Subsequent testing has revealed that a significant number of these persons whose sera was reactive by the HTLV-I EIA were, in fact, infected by HTLV-II. Epidemiological studies of human retroviral infections (HIV-1, HIV-2, HTLV-I, and HTLV-II) continue to provide important data and direction for improving criteria for qualifying blood donors.     

Analysis of rev gene function on human immunodeficiency virus type 1 replication in lymphoid cells by using a quantitative polymerase chain reaction method.

Most detailed analyses of the human immunodeficiency virus type 1 (HIV-1) rev gene product have relied on transfection of subgenomic env constructs into cells in which amplification of the transfected DNA occurs. This was necessitated by difficulties in quantitating low-abundance HIV-1 mRNA species and in distinguishing different RNAs of similar sizes. We have modified the conventional polymerase chain reaction method for general use as an extremely sensitive procedure for quantitative analysis of RNA species. Using this method, we assessed the role of the HIV-1 rev gene in viral replication following mutagenesis of an infectious molecular clone, HIV-1JR-CSF. Following transfection of wild-type and mutant proviral constructs, we can specifically detect unspliced RNA and distinguish between the spliced tat-rev and nef mRNAs, which are not resolved by standard RNA analyses. Our results show that the rev protein of HIV-1JR-CSF simultaneously down regulates the expression of tat-rev and nef RNAs and up regulates the level of unspliced full-length HIV-1 RNA. A cis-acting element(s), located exclusively within the env sequences, is essential to exhibit this regulation. Fractionation of cells shows that the ultimate effect of Rev is to direct the appearance of unspliced or singly spliced RNAs in the cytoplasm. Models are discussed for possible mechanisms of Rev action.