Nuclear Import Of Preintegration Complex Research Articles

73. An inhibitory effect of a sequence just upstream of the central polypurine tract of HIV-1 on RNA production of an HIV-1-based vector

The central polypurine tract (cPPT) with downstream central termination sequence (CTS) (about 100nt apart) is common in all lentivirus genomes. In reverse transcription of human immunodeficiency virus type 1 (HIV-1), plus-strand DNA synthesis is initiated at cPPT and 3'PPT. The latter stops at CTS. Thus, the final product of reverse transcription is a linear DNA molecule with a stable plus strand overlap, the central DNA flap. Reportedly, flap-negative HIV-1 was unable to replicate and defective for nuclear import of preintegration complex. In contrast, many flap-negative HIV-1 vectors could transduce many kind of cells. To confirm these observations and to develop high-titer HIV-1 vectors, we tested an effect of insertion of cPPT/CTS on vector titers. We chose the HXN vector as a parental vector, which carried a TK-promoter-driven neomycin-resistant gene (neor) between the 5'- and 3'-LTRs. We inserted 282nt or 178nt fragment containing cPPT/CTS of the HIV-1LAI just upstream of the TK promoter in the HXN DNA in the sense or anti-sense orientation, generating HXN-282S, HXN-282R, HXN-178S, or HXN-178R DNA, respectively. We produced vector particles by cotransfecting 293T cells with one of the above vector DNAs together with expression plasmids for Gag-Pol (pCMV-GP) and vesicular somatitis viral envelope protein (pMD.G, a gift from Dr. Trono). We obtained about 2 times more G418-resistant colonies using HXN-178S or HXN-178R than the parental HXN vector normalized to one nanogram of p24, after two weeks of G418-selection. In contrast, the number of G418 resistant colonies with HXN-282S or HXN-282R was 5-10 times less than that with HXN. The two cPPT/CTS fragments differed in that the longer had 104 (=282-178) nt additional sequence at 5' end. We named it as the dZ sequence. In order to know whether the dZ sequence had made HXN-282S vector particles less infectious, we constructed pHXN-dZ, pHXN DNA with dZ but without cPPT/CTS by inserting dZ into pHXN. Surprisingly, RT-PCR revealed that little vector RNA was present in the culture supernatant even though abundant p24 was present. Similarly, northern blot analysis showed that little vector RNA was present in transfected Huh7 cells. We concluded that dZ completely decreased an intracellular amount of vector RNA. This conclusion simply predicted that insertion of the longer cPPT/CTS (282nt) — a fusion of the inhibitory dZ and the enhancing shorter cPPT (178nt) — to HXN would have resulted in production of vector particles with a medium amount of vector RNA per unit amount of p24 capsid protein. In fact, RT-PCR revealed that the RNA/p24 ratio of HXN-282S was about 8 times lower than that of HXN-178S and about 100 times higher than that of HXN-dZ as if a tug-of-war took place between the negative and positive sequence elements. dZ was likely to decrease an amount of intracellular RNA molecules by rendering them instable rather than by suppressing transcription per se because insertion of dZ upstream of luciferase gene did not affect luciferase activity very much.

Nuclear import of the pre-integration complex (PIC): the Achilles heel of HIV?

Current treatments against the Aquired immune deficiency syndrome (AIDS) are reasonably effective in reducing the amount of human immunodeficiency virus (HIV) present in infected patients, but their side-effects, and the emergence of drug-resistant HIV strains have intensified the renewed search for novel anti-HIV therapies. An essential step in HIV infection is the integration of the viral genome into the host cell chromosomes within the nucleus. Unlike other retroviruses, HIV can transport its genetic material, in the form of the large nucleoprotein pre-integration complex (PIC), into the nucleus through the intact nuclear envelope (NE). This enables HIV to infect non-dividing cells such as macrophages and microglial cells. Detailed knowledge of the signal-dependent pathways by which cellular proteins and RNAs cross the NE has accumulated in the past decade, but although several different components of the PIC have been implicated in its nuclear import, the mechanism of nuclear entry remains unclear. Since specifically inhibiting PIC nuclear import would undoubtedly block HIV infection in non-dividing cells, this critical step of HIV replication is of great interest as a drug target. This review examines the complex and controversial literature regarding three PIC components--the HIV proteins matrix, integrase and Vpr--proposed to facilitate PIC nuclear import, and existing models of HIV PIC nuclear import. It also suggests approaches to move towards a better understanding of PIC nuclear import, through examining the role of individual PIC components in the context of the intact PIC by direct visualisation, in order to develop new anti-HIV therapeutics.

SIVagm: genetic and biological features associated with replication.

African green monkeys (AGMs) belong to a superspecies that include the following four species: vervet (Chlorocebus pygerythrus), grivet (C. aethiops), sabaeus (C. sabaeus) and tantalus monkeys (C. tantalus). Each species carries a distinct SIVagm subtype, named SIVagm.ver, SIVagm.gri, SIVagm.sab and SIVagm.tan but remain clinically asymtomatic throughout their life. SIVagm needs the CD4 molecule and a chemokine-receptor, usually CCR5, Bonzo and/or Bob, for cell entry. Molecular and functional analyses of AGM CD4 and CCR5 revealed evidence of a true co-evolution between SIVagm and their natural hosts. The V3 loop of the SIVagm external glycoprotein is relatively conserved compared to that of HIV-1, but equally a determinant for coreceptor usage. SIVagm are able to replicate in stimulated peripheral blood mononuclear cells (PBMC) and macrophages. Efficient replication of SIVagm in vitro is associated with a cytopathic effect. SIVagm regulatory proteins show low amino acid identities with that of HIV-1, but many functions are conserved. The Tat protein of SIVagm is a transactivator of SIVagm LTR, albeit to a relatively lesser extent than Tat of HIV-1. Vif of SIVagm increases virus infectivity in AGM PBMC, demonstrating that SIVagm Vif is a positive regulator of virion infectivity. Vpr of SIVagm shares with HIV-1 Vpr its ability to transactivate the viral LTR, causes cell cycle arrest in AGM cells and facilitates nuclear import of pre-integration complexes and infection of non-dividing cells. AGM Vpr has been reported to also induce apoptosis, but the pathways involved in the mechanisms leading to apoptosis seem to be divergent compared to HIV-1 Vpr. SIVagm.ver3delta nef shows limited replicative capacity in vitro and in vivo, which suggests that Nef of SIVagm provides activation signals to the cells that facilitate SIV replication. The levels of plasma viral RNA in naturally infected AGMs are in a wide range (<10(3) to >6.10(6) RNA copies/ml), with many animals displaying steady-state levels of >10(6) RNA copies/ml levels, which in humans are associated with progression towards AIDS. Viral load per se therefore is not directly linked with pathogenicity. SIVagm-infections differ, however, from pathogenic HIV/SIVmac infections by a low viral load in peripheral lymph nodes. These relatively low localized tissue viral load levels are associated with lack of signs of detectable immunopathology. More detailed studies on the precise immunological environment during natural SIV infections, especially during primary infection, and on the interactions between SIVagm and host-specific cellular proteins are needed to better understand the mechanisms by which these naturally SIV infected nonhuman primate species are protected against progression towards AIDS.

Wild-type levels of nuclear localization and human immunodeficiency virus type 1 replication in the absence of the central DNA flap.

Numerous factors have been implicated in the nuclear localization of retroviral preintegration complexes. Whereas sequences in human immunodeficiency virus type 1 (HIV-1) matrix, Vpr, and integrase proteins were initially reported to function specifically in nondividing cells, other recently identified sequences apparently function in dividing cells as well. One of these, the central DNA flap formed during reverse transcription, is specific to lentiviruses. It was previously reported that flap-negative (F(-)) HIV-1(LAI) was completely defective for viral spread in the MT-4 T-cell line, yet F(-) HIV-1 vectors were only 2- to 10-fold defective in various single-round transduction assays. To address these different findings, we analyzed the infectivity and nuclear localization phenotypes of two highly related T-cell-tropic strains, HIV-1(NL4-3) and a derivative of HIV-1(HXBc2) deficient for both Vpr and Nef. In stark contrast to the previous report, F(-) derivatives of both strains replicated efficiently in MT-4 cells. F(-) HIV-1(NL4-3) also spread like wild-type HIV-1(NL4-3) in infected Jurkat and primary T-cell cultures. In contrast, F(-) HIV-1(HXBc2) was replication defective in primary T cells. Results of real-time quantitative PCR assays, however, indicated that F(-) HIV-1(HXBc2) entered primary T-cell nuclei as efficiently as its wild-type counterpart. Thus, the F(-) HIV-1(HXBc2) growth defect did not appear to correlate with defective nuclear import. Consistent with this observation, wild-type nef restored replication to F(-) HIV-1(HXBc2) in primary T cells. Our results indicate that the central DNA flap does not play a major role in either preintegration complex nuclear import or HIV-1 replication in a variety of cell types.

Nonprimate lentiviral vectors.

Advances in the nascent field of gene therapy have created a demand for gene transfer vehicles that are capable of safely and stably delivering genes to both dividing and nondividing cells. Retroviral vectors, by virtue of their ability to permanently integrate genes of interest into the target cell genome, have played a prominent role in much of the gene transfer research to date. However, their inability to transduce quiescent cells has rendered them largely ineffective for clinical applications. In contrast, adenoviral vectors are capable of infecting nondividing cells, but attempts to move these vectors into the clinic have been limited at times by their lack of stable expression and high immunogenicity. Lentiviral vectors offer a unique combination of the advantages of these two systems without many of the pitfalls. Lentiviruses can stably transduce both dividing and nondividing cells, and, unlike adenoviral vectors, are not immunogenic in vivo. The most established and tested lentiviral vectors are those based on the human immunodeficiency virus (HIV-1). As a full-length virus, HIV is naturally optimized for production in and transduction of human cells and, as such, has proved an ideal candidate for development into a human gene transfer vector. Concern over the consequences of using vectors based on a pathogenic virus with such an optimized tropism for human cells has, however, limited the clinical applications of HIV vectors. For these reasons, vector development has begun to shift to other members of the lentivirus family that do not infect primates as part of their normal host range. In addition to the potential biosafety benefits, it is possible that some nonprimate lentiviral vectors will show an increased ability to infect some useful cell types relative to HIV-based vectors. As many of these viruses have not been well characterized biologically, an interesting cross-talk has developed where advances in lentiviral biology impact vector development and vice versa. The recent discovery of the central DNA flap in lentiviral genomes and its role in nuclear import of preintegration complexes, for example, may prove critical in bringing the efficiency of nonprimate lentiviral vectors up to the level of HIV vectors. As it appears that these nonprimate vectors might reach parity with their primate counterparts in terms of efficiency and tropism, it must now be considered whether they are truly safer for use in the clinic. This review will focus on those aspects of nonprimate lentiviral biology which make such vectors potentially useful in clinical settings.

Characterization of a late entry event in the replication cycle of human immunodeficiency virus type 2.

Certain human cell lines and primary macrophage cultures are restricted to infection by some primary isolates of human immunodeficiency virus type 2 (HIV-2), although early steps of the viral life cycle such as fusion at the plasma membrane and reverse transcription are fully supported. The late postintegration events, transcription, translation, assembly, budding, and maturation into infectious virions are functional in restrictive cells. Apart from primary macrophages, the restrictive cell types are actively dividing, and nuclear import of preintegration complexes (PICs) is not required for infection. We therefore postulate that the PICs are trapped in a cellular compartment, preventing subsequent steps in the replication cycle that lead to integration of the provirus. To test this we showed that HIV-2 particles pseudotyped with vesicular stomatitis virus envelope G protein, which delivers HIV into an endocytic compartment, could overcome the block to infection. We suggest that delivery of the viral core into an appropriate cellular compartment is a critical step during the entry process of HIV.

Vpr Is Required for Efficient Replication of Human Immunodeficiency Virus Type-1 in Mononuclear Phagocytes

HIV-1 vpr encodes a 96-amino acid, nuclear protein whose function is not well understood. Unlike the other lentivirus regulatory proteins, Vpr is present in virions at relatively high copy number. In cells, Vpr is localized to the nucleus. Possible functions for vpr consistent with these findings include the nuclear import of preintegration complexes, transactivation of cellular genes, or induction of cellular differentiation. We show here, using both replication competent, macrophage-tropic virus and a sensitive, single-cycle luciferase HIV-1 reporter vector, that vpr is important for efficient viral replication in primary monocyte/macrophages, but appears to play no role in activated or resting T cell infection. The block to infection in monocytes was localized by PCR analysis of newly synthesized viral DNA and with the luciferase reporter vector to a stage in the viral life cycle after entry and reverse transcription, yet prior to, or at the time of, proviral transcription. In addition, infection of mononuclear phagocytes with virions that had been loaded with Vpr molecules in the producer cells by trans-complementation still showed a vpr-phenotype. These data suggest a role for vpr molecules produced in newly infected cells, in addition to its presumed function in the virion.