The HIV-1 Rev response element (RRE) adopts alternative conformations that promote different rates of virus replication

The HIV Rev protein forms a complex with a 351 nucleotide sequence present in unspliced and incompletely spliced human immunodeficiency virus (HIV) mRNAs, the Rev response element (RRE), to recruit the cellular nuclear export receptor Crm1 and Ran-GTP. This complex facilitates nucleo-cytoplasmic export of these mRNAs. The precise secondary structure of the HIV-1 RRE has been controversial, since studies have reported alternative structures comprising either four or five stem-loops. The published structures differ only in regions that lie outside of the primary Rev binding site. Using in-gel SHAPE, we have now determined that the wt NL4-3 RRE exists as a mixture of both structures. To assess functional differences between these RRE ‘conformers’, we created conformationally locked mutants by site-directed mutagenesis. Using subgenomic reporters, as well as HIV replication assays, we demonstrate that the five stem-loop form of the RRE promotes greater functional Rev/RRE activity compared to the four stem-loop counterpart.     

Continuous propagation of RRE(-) and Rev(-)RRE(-) human immunodeficiency virus type 1 molecular clones containing a cis-acting element of simian retrovirus type 1 in human peripheral blood lymphocytes.

Molecular clones of human immunodeficiency virus type 1 that contained either 37 point mutations in the Rev-responsive element (RRE) that did not affect the overlapping env reading frame or both a mutated RRE and two mutations that eliminated Rev were constructed. The mutations in the RRE were shown to remove both negative and Rev-inducible positive effects of the RRE on gene expression (G. Nasioulas, A. S. Zolotukhin, C. Tabernero, L. Solomin, C. P. Cunningham, G. N. Pavlakis, and B. K. Felber, J. Virol. 68:2986-2993, 1994). Upon insertion of a cis-acting element of simian retrovirus type 1 (SRV-1) into these clones, both RRE(-) and Rev(-)RRE(-) clones were expressed efficiently. The element of SRV-1 has properties similar to those of the recently identified element of Mason-Pfizer monkey virus (M. Bray, S. Prasad, J. W. Dubay, E. Hunter, K.-T. Jeang, D. Rekosh, and M.-L. Hammarskjold, Proc. Natl. Acad. Sci. USA 4:1256-1260, 1994). We demonstrated that virus preparations produced after transfections of these SRV-1 element-containing molecular clones in human cells were infectious after cell-free transmission, that they replicated about 5 to 10 times less efficiently than wild-type virus, and that they were propagated continuously for more than 7 months in human peripheral blood mononuclear cells. Growth characteristics and sequence analysis of these viruses after long-term culture demonstrated that no RRE(+)Rev(+) revertants developed. These data demonstrate that human immunodeficiency virus type 1 Rev and RRE can be replaced by heterologous regulatory systems, resulting in efficient virus production. The resulting Rev(-)RRE(-) virus can be prepared and propagated efficiently in tissue culture and can be used for further studies of the life cycle of the virus. The data also suggest that Rev acts exclusively through the RRE interaction and that it does not have any additional essential function in the life cycle of the virus.     

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).     

A human chromosome 12-associated 83-kilodalton cellular protein specifically binds to the loop region of human immunodeficiency virus type 1 trans-activation response element RNA.

trans activation of human immunodeficiency virus type 1 (HIV-1) involves the viral trans-activator protein (Tat) and a cellular factor(s) encoded on human chromosome 12 (HuChr12) that targets the trans-activation response element (TAR) in the viral long terminal repeat. Because nascent TAR RNA is predicted to form a secondary structure that specifically binds cellular proteins, we investigated the composition of the TAR RNA-protein complex for HuChr12-specific proteins. UV cross-linking of TAR RNA-nuclear protein complexes formed in vitro identified an 83-kDa protein in human cells and in a human-hamster hybrid cell containing only HuChr12. The 83-kDa TAR RNA-binding protein was absent in the parental hamster cells. TAR RNA mutations that inhibited binding of the 83-kDa protein in vitro also inhibited HuChr12-dependent Tat trans activation. These TAR mutations changed the native sequence or secondary structure of the TAR loop. The TAR RNA binding activity of the 83-kDa protein also correlated with a HuChr12-dependent increase in steady-state HIV-1 RNA expression during Tat trans activation. Our results suggest that either a species-specific 83-kDa TAR RNA loop-binding protein is directly encoded on HuChr12 or a HuChr12 protein(s) induces the expression of an 83-kDa TAR-binding protein in nonprimate cells.     

Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor.

A synthetic peptide, RPI 312, that specifically inhibits the protease of the human immunodeficiency virus type 1 (HIV-1) showed a potent inhibition on virus production, maturation, and infectivity. Treatment with this agent prevented the cleavage of Gag protein at the site between p17 and p24 in HIV-1 chronically infected MOLT-4 cells as well as in the released virus. Passage of HIV-1 in the presence of gradually increasing concentrations of this protease inhibitor resulted in emergence of a variant that could evade the drug effects. In the resistant variant the maturation of Gag proteins appeared normal, but its infectivity was reduced compared with that of the parent virus. The nucleotides coding the amino acids at and around the cleavage site between Gag proteins p17 and p24 were not changed. One point mutation (A-->G) at site 2082 of the pol gene that resulted in one amino acid change at site 84 of the protease from isoleucine to valine (I-84-->V) could be detected in the resistant variant. An HIV-1 infectious DNA clone with the I-84-->V mutation also showed reduced sensitivity to this protease inhibitor. The findings that the resistant variant had lower infectivity and was still affected by higher doses of the drug support the speculation that resistance to protease inhibitors may not be as problematic as other drug resistance.     

A human immunodeficiency virus type 1 (HIV-1) infection-enhancing factor in seropositive sera

While testing sera for Human Immunodeficiency Virus neutralizing antibody titers, three sera were identified which had the ability to enhance infectivity of the virus. The sera were from three different individuals residing in Nashville, TN. The enhancing factor was not removed by either filtration through 0.05 micron filters or by incubation for one hour with a stoichiometric amount of protein A sepharose. Two of the sera were able to enhance infection by two divergent isolates (HTLV-IIIB and HTLV-IIIRF) while one was only capable of enhancing infection of target cells by HTLV-IIIB. None of the sera induced syncytium formation in chronic HIV-infected cells. The findings suggest that the substance is neither a virus nor an IgG class 1 or 2.     

A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene

The human immunodeficiency virus (HIV) genome is AU rich, and this imparts a codon bias that is quite different from the one used by human genes. The codon usage is particularly marked for the gag, pol, and env genes. Interestingly, the expression of these genes is dependent on the presence of the Rev/Rev-responsive element (RRE) regulatory system, even in contexts other than the HIV genome. The Rev dependency has been explained in part by the presence of RNA instability sequences residing in these coding regions. The requirement for Rev also places a limitation on the development of HIV-based vectors, because of the requirement to provide an accessory factor. We have now synthesized a complete codon-optimized HIV-1 gag-pol gene. We show that expression levels are high and that expression is Rev independent. This effect is due to an increase in the amount of gag-pol mRNA. Provision of the RRE in cis did not lower protein or RNA levels or stimulate a Rev response. Furthermore we have used this synthetic gag-pol gene to produce HIV vectors that now lack all of the accessory proteins. These vectors should now be safer than murine leukemia virus-based vectors.     

Robust growth of human immunodeficiency virus type 1 (HIV-1)

The persistence of human immunodeficiency virus type-1 (HIV-1) has long been attributed to its high mutation rate and the capacity of its resulting heterogeneous virus populations to evade host immune responses and antiviral drugs. However, this view is incomplete because it does not explain how the virus persists in light of the adverse effects mutations in the viral genome and variations in host functions can potentially have on viral functions and growth. Here we show that the resilience of HIV-1 can be credited, at least in part, to a robust response to perturbations that emerges as an intrinsic property of its intracellular development. Specifically, robustness in HIV-1 arises through the coupling of two feedback loops: a Rev-mediated negative feedback and a Tat-mediated positive feedback. By employing a mechanistic kinetic model for its growth we found that HIV-1 buffers the effects of many potentially detrimental variations in essential viral and cellular functions, including the binding of Rev to mRNA; the level of rev mRNA in the pool of fully spliced mRNA; the splicing of mRNA; the Rev-mediated nuclear export of incompletely-spliced mRNAs; and the nuclear import of Tat and Rev. The virus did not, however, perform robustly to perturbations in all functions. Notably, HIV-1 tended to amplify rather than buffer adverse effects of variations in the interaction of Tat with viral mRNA. This result shows how targeting therapeutics against molecular components of the viral positive-feedback loop open new possibilities and potential in the effective treatment of HIV-1.     

Rev binds specifically to a purine loop in the SL1 region of the HIV-1 leader RNA

The leader RNA sequence of human immunodeficiency virus type 1 (HIV-1) consists of a complex series of stem loop structures that are critical for viral replication. Three-dimensional structural analysis by NMR of one of these structures, the SL1 stem loop of the packaging signal region, revealed a highly conserved purine rich loop with a structure nearly identical to the Rev-binding loop of the Rev response element. Using band-shift assays, surface plasmon resonance, and further NMR analysis, we demonstrate that this loop binds Rev. HIV-1 appears to have a second Rev-binding site close to the major splice donor site that may have an additional role in the viral life cycle.     

Human T-cell leukemia virus (HTLV) type II Rex protein binds specifically to RNA sequences of the HTLV long terminal repeat but poorly to the human immunodeficiency virus type 1 Rev-responsive element.

The human T-cell leukemia viruses (HTLVs) encode a trans-regulatory protein, Rex, which differentially regulates viral gene expression by controlling the cytoplasmic accumulation of viral mRNAs. Because of insufficient amounts of purified protein, biochemical characterization of Rex activity has not previously been performed. Here, utilizing the baculovirus expression system, we purified HTLV type II (HTLV-II) Rex from the cytoplasmic fraction of recombinant baculovirus-infected insect cells by heparin-agarose chromatography. We directly demonstrated that Rex specifically bound HTLV-II 5' long terminal repeat RNA in both gel mobility shift and immunobinding assays. Sequences sufficient for Rex binding were localized to the R-U5 region of the HTLV-II 5' long terminal repeat and correlate with the region required for Rex function. The human immunodeficiency virus type 1 (HIV-1), has an analogous regulatory protein, Rev, which directly binds to and mediates its action through the Rev-responsive element located within the HIV-1 env gene. We demonstrated that HTLV-II Rex rescued an HIV-1JR-CSF Rev-deficient mutant, although inefficiently. This result is consistent with a weak binding activity to the HIV-1 Rev-responsive element under conditions in which it efficiently bound the HTLV-II long terminal repeat RNA.     

Extensive sequence-specific information throughout the CAR/RRE, the target sequence of the human immunodeficiency virus type 1 Rev protein.

The significance and location of sequence-specific information in the CAR/RRE, the target sequence for the Rev protein of the human immunodeficiency virus type 1 (HIV-1), have been controversial. We present here a comprehensive experimental and computational approach combining mutational analysis, phylogenetic comparison, and thermodynamic structure calculations with a systematic strategy for distinguishing sequence-specific information from secondary structural information. A target sequence analog was designed to have a secondary structure identical to that of the wild type but a sequence that differs from that of the wild type at every position. This analog was inactive. By exchanging fragments between the wild-type sequence and the inactive analog, we were able to detect an unexpectedly extensive distribution of sequence specificity throughout the CAR/RRE. The analysis enabled us to identify a critically important sequence-specific region, region IIb in the Rev-binding domain, strongly supports a proposed base-pairing interaction in this location, and places forceful constraints on mechanisms of Rev action. The generalized approach presented can be applied to other systems.     

Primary human immunodeficiency virus type 1 (HIV-1) infection during HIV-1 Gag vaccination

Vaccination for human immunodeficiency virus type 1 (HIV-1) remains an elusive goal. Whether an unsuccessful vaccine might not only fail to provoke detectable immune responses but also could actually interfere with subsequent natural immunity upon HIV-1 infection is unknown. We performed detailed assessment of an HIV-1 gag DNA vaccine recipient (subject 00015) who was previously uninfected but sustained HIV-1 infection before completing a vaccination trial and another contemporaneously acutely infected individual (subject 00016) with the same strain of HIV-1. Subject 00015 received the vaccine at weeks 0, 4, and 8 and was found to have been acutely HIV-1 infected around the time of the third vaccination. Subject 00016 was a previously HIV-1-seronegative sexual contact who had symptoms of acute HIV-1 infection approximately 2 weeks earlier than subject 00015 and demonstrated subsequent seroconversion. Both individuals reached an unusually low level of chronic viremia (<1,000 copies/ml) without treatment. Subject 00015 had no detectable HIV-1-specific cytotoxic T-lymphocyte (CTL) responses until a borderline response was noted at the time of the third vaccination. The magnitude and breadth of Gag-specific CTL responses in subject 00015 were similar to those of subject 00016 during early chronic infection. Viral sequences from gag, pol, and nef confirmed the common source of HIV-1 between these individuals. The diversity and divergence of sequences in subjects 00015 and 00016 were similar, indicating similar immune pressure on these proteins (including Gag). As a whole, the data suggested that while the gag DNA vaccine did not prime detectable early CTL responses in subject 00015, vaccination did not appreciably impair his ability to contain viremia at levels similar to those in subject 00016.     

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.