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.     

Functional comparison of transactivation by simian immunodeficiency virus from rhesus macaques and human immunodeficiency virus type 1.

Simian immunodeficiency virus from rhesus macaques (SIVmac), like human immunodeficiency virus type 1 (HIV-1), encodes a transactivator (tat) which stimulates long terminal repeat (LTR)-directed gene expression. We performed cotransfection assays of SIVmac and HIV-1 tat constructs with LTR-CAT reporter plasmids. The primary effect of transactivation for both SIVmac and HIV-1 is an increase in LTR-directed mRNA accumulation. The SIVmac tat gene product partially transactivates an HIV-1 LTR, whereas the HIV-1 tat gene product fully transactivates an SIVmac LTR. Significant transactivation is achieved by the product of coding exon 1 of the HIV-1 tat gene; however, inclusion of coding exon 2 results in a further increase in mRNA accumulation. In contrast, coding exon 2 of the SIVmac tat gene is required for significant transactivation. These results imply that the tat proteins of SIVmac and HIV-1 are functionally similar but not interchangeable. In addition, an in vitro-generated mutation in SIVmac tat disrupts splicing at the normal splice acceptor site at the beginning of coding exon 2 and activates a site approximately 15 nucleotides downstream. The product of this splice variant stimulates LTR-directed gene expression. This alternative splice acceptor site is also used by a biologically active provirus with an efficiency of approximately 5% compared with the upstream site. These data suggest that a novel tat protein is encoded during the course of viral infection.     

Wild-type bacteriophage T4 is restricted by the lambda rex genes.

The bacteriophage T4 rII genes and the lambda rex (r exclusion) genes interact; rII mutants are unable to productively infect rex+ lambda lysogens. The relationship between rex and rII has been found to be quantitative, and plasmid clones of rex have excluded not only rII mutants but T4 wild type and most other bacteriophages as well. Mutations in the T4 motA gene substantially reversed exclusion of T4 by rex.     

Persistence of attenuated rev genes in a human immunodeficiency virus type 1-infected asymptomatic individual.

With the goal of examining the functional diversity of human immunodeficiency virus type 1 (HIV-1) env genes within the peripheral blood mononuclear cells of an asymptomatic individual, we substituted four complete env genes into the replication-competent NL4-3 provirus. Despite encoding full-length open reading frames for gp120 and gp41 and the second coding exon of tat and rev, each chimera was replication defective. Site-directed mutagenesis of codon 78 in the Rev activation domain (from a hitherto unique Ile to the subtype B consensus Leu) partially restored infectivity for two of three chimeras tested. Similarly, mutagenesis of rev codon 78 of NL4-3 from Leu to Ile partially attenuated this virus. Ile-78 was found in all 13 clones examined from samples taken from this asymptomatic subject 4.5 years after infection, including 9 from peripheral blood mononuclear cells and 4 from a virus isolate, as well as 4 additional clones each from peripheral blood mononuclear cells sampled 37 and 51 months later. We next examined conservation of the Rev activation domain within and among long-term survivors (LTS) and patients with AIDS, as well as T-cell-line-adapted strains of HIV-1. Putative attenuating mutations were found in a minority of sequences from all five LTS and two of four patients with AIDS. Of the 11 T-cell-line-adapted viruses examined, none had these changes. Among and within LTS virus population had marginally higher levels of diversity in Rev than in Env; patients with AIDS had similar levels of diversity in the two reading frames; and T-cell-line-adapted viruses had higher levels of diversity in Env. These results are consistent with the hypothesis that asymptomatic individuals harbor attenuated variants of HIV-1 which correlate with and contribute to their lack of disease progression.     

Regulation of human immunodeficiency virus env expression by the rev gene product.

A single simian virus 40 late replacement vector which expresses both the rev and envelope (env) genes of human immunodeficiency virus was used to examine the mechanism underlying the dependence of env gene expression on the rev protein. When rev was deleted from the vector, no envelope protein expression could be detected in transfected cells, and the levels of cytoplasmic env mRNA were dramatically reduced. In contrast to this, the levels of env RNA in total cellular RNA preparations were similar with or without rev coexpression, and analysis of nuclear RNA showed that the levels of nuclear env RNA were increased in the absence of rev. These results suggest that rev functions to regulate nuclear export of env mRNA. It was possible to restore env expression from the vector lacking rev by supplying rev in trans, provided that a cis-acting sequence was also present. This sequence was mapped to a 854-base-pair region within the env open reading frame, and it was shown that the sequence could be moved but that it worked only in its original orientation.     

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).     

Functional role of VNTR polymorphism of human genes

A brief overview of the current views on the functional role of genetic VNTR polymorphism in humans is given. Data on the involvement of VNTRs in the regulation of gene expression and in the formation of complex phenotypes are presented. According to these data, the effects of VNTRs are determined by the number of repeats, the structure of their monomers and flanking haplotypes, epigenetic mechanisms (in the case of localization in imprinted regions) and can be modified by environmental factors. Some possible mechanisms of the influence of VNTRs on the level of expression are considered.     

Ancestry of a human endogenous retrovirus family.

The human endogenous retrovirus type II (HERVII) family of HERV genomes has been found by Southern blot analysis to be characteristic of humans, apes, and Old World monkeys. New World monkeys and prosimians lack HERVII proviral genomes. Cellular DNAs of humans, common chimpanzees, gorillas, and orangutans, but not lesser ape lar gibbons, appear to contain the HERVII-related HLM-2 proviral genome integrated at the same site (HLM-2 maps to human chromosome 1). This suggests that the ancestral HERVII retrovirus(es) entered the genomes of Old World anthropoids by infection after the divergence of New World monkeys (platyrrhines) but before the evolutionary radiation of large hominoids.     

Functional and nonfunctional measles virus matrix genes from lethal human brain infections.

Subacute sclerosing panencephalitis (SSPE) is a lethal disease induced by the persistence of measles virus in the human brain. In many SSPE cases, the viral matrix (M) protein cannot be detected; in others, M proteins of the expected size are found and sequence analysis of M cDNAs has confirmed that the reading frames are intact, showing only several missense mutations. To determine whether these alterations result in nonfunctional proteins, we have replaced the M gene of an infectious full-length genomic cDNA (from vaccine strain Edmonston) with different M genes derived from four patients with SSPE. One of the SSPE M genes tested proved to be functionally competent, giving rise to a virus yielding titers similar to those of viruses containing the M gene from control lytic strains. The other three SSPE M genes were not functionally competent in the same test. In all three cases, the inactivating changes resided in the carboxyl-terminal half of the M protein, as shown by the exchange of either of the two genes halves. In summary, mutational M gene alterations, which either prevent synthesis of M protein altogether or only allow synthesis of nonfunctional M protein, have been detected by us and by others in 9 of 10 SSPE cases. The one functional M gene appears to be an exception to the rule, indicating that M gene alteration might not be an absolute requirement for disease development.     

Transcellular transactivation by the human immunodeficiency virus type 1 tat protein.

The human immunodeficiency virus type 1 (HIV-1) transactivator (tat) protein produced in one cell activated HIV-1 promoter-directed gene expression in a second cell, provided the cells were in direct contact with one another. This observation suggests that the tat protein produced in HIV-1-infected cells has a physiological effect on neighboring cells.     

Epstein-Barr virus latent membrane protein LMP-2A is sufficient for transactivation of the human endogenous retrovirus HERV-K18 superantigen

Superantigens are microbial proteins that strongly stimulate T cells. We described previously that the Epstein-Barr virus (EBV) transactivates a superantigen encoded by the human endogenous retrovirus, HERV-K18. We now report that the transactivation is dependent upon the EBV latent cycle proteins. Moreover, LMP-2A is sufficient for induction of HERV-K18 superantigen activity.     

Suppression of simian immunodeficiency virus replication by human immunodeficiency virus type 1 trans-dominant negative rev mutants.

We demonstrate that trans-dominant negative rev mutants are able to suppress simian immunodeficiency virus provirus replication in both transient cotransfection assays and stably transduced HUT 78 cells. These studies suggest that the efficacy of trans-dominant rev strategies in reducing viral burden may be evaluated in a simian immunodeficiency virus-rhesus macaque animal model.