Xiaojian Yao

Interaction of Human Immunodeficiency Virus Type 1 Integrase with Cellular Nuclear Import Receptor Importin 7 and Its Impact on Viral Replication

Similar to all other viruses, human immunodeficiency virus type 1 (HIV-1) depends heavily on cellular factors for its successful replication. In this study we have investigated the interaction of HIV-1 integrase (IN) with several host nuclear import factors using co-immunoprecipitation assays. Our results indicate that IN interacts specifically with host importin 7 (Imp7) in vivo, but does not interact with importin 8 (Imp8) or importin α (Rch1). In contrast, another HIV-1 karyophilic protein MAp17, which is capable of binding Rch1, fails to interact with Imp7, suggesting that IN and Map17 may interact with different cellular pathways during HIV-1 replication. Genetic analysis revealed that the C-terminal domain of IN is the region responsible for interaction between IN with Imp7, and an IN mutant (K240A,K244A/R263A,K264A) disrupted the Imp7 binding ability of the protein, indicating that both regions (235WKGPAKLLWKG and 262RRKAK) within the C-terminal domain of IN are required for efficient IN/Imp7 interaction. Using a vesicular stomatitis virus G glycoprotein pseudotyped HIV single-cycle replication system, we showed that the IN/Imp7 interaction-deficient mutant was unable to mediate viral replication and displayed impairment at both viral reverse transcription and nuclear import steps. Moreover, transient knockdown of Imp7 in both HIV-1 producing and target cells resulted in a 2.5–3.5-fold inhibition of HIV infection. Altogether, our results indicate that HIV-1 IN specifically interacts with Imp7, and this viral/cellular protein interaction contributes to efficient HIV-1 infection.

Intracellular Transport of Human Immunodeficiency Virus Type 1 Genomic RNA and Viral Production Are Dependent on Dynein Motor Function and Late Endosome Positioning

Our earlier work indicated that the human immunodeficiency virus type 1 (HIV-1) genomic RNA (vRNA) is trafficked to the microtubule-organizing center (MTOC) when heterogeneous nuclear ribonucleoprotein A2/B1 is depleted from cells. Also, Rab7-interacting lysosomal protein promoted dynein motor complex, late endosome and vRNA clustering at the MTOC suggesting that the dynein motor and late endosomes were involved in vRNA trafficking. To investigate the role of the dynein motor in vRNA trafficking, dynein motor function was disrupted by small interference RNA-mediated depletion of the dynein heavy chain or by p50/dynamitin overexpression. These treatments led to a marked relocalization of vRNA and viral structural protein Gag to the cell periphery with late endosomes and a severalfold increase in HIV-1 production. In contrast, rerouting vRNA to the MTOC reduced virus production. vRNA localization depended on Gag membrane association as shown using both myristoylation and Gag nucleocapsid domain proviral mutants. Furthermore, the cytoplasmic localization of vRNA and Gag was not attributable to intracellular or internalized endocytosed virus particles. Our results demonstrate that dynein motor function is important for regulating Gag and vRNA egress on endosomal membranes in the cytoplasm to directly impact on viral production.

Importin α3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication

ABSTRACT HIV-1 employs the cellular nuclear import machinery to actively transport its preintegration complex (PIC) into the nucleus for integration of the viral DNA. Several viral karyophilic proteins and cellular import factors have been suggested to contribute to HIV-1 PIC nuclear import and replication. However, how HIV interacts with different cellular machineries to ensure efficient nuclear import of its preintegration complex in dividing and nondividing cells is still not fully understood. In this study, we have investigated different importin α (Impα) family members for their impacts on HIV-1 replication, and we demonstrate that short hairpin RNA (shRNA)-mediated Impα3 knockdown (KD) significantly impaired HIV infection in HeLa cells, CD4+ C8166 T cells, and primary macrophages. Moreover, quantitative real-time PCR analysis revealed that Impα3-KD resulted in significantly reduced levels of viral 2-long-terminal repeat (2-LTR) circles but had no effect on HIV reverse transcription. All of these data indicate an important role for Impα3 in HIV nuclear import. In an attempt to understand how Impα3 participates in HIV nuclear import and replication, we first demonstrated that the HIV-1 karyophilic protein integrase (IN) was able to interact with Impα3 both in a 293T cell expression system and in HIV-infected CD4+ C8166 T cells. Deletion analysis suggested that a region (amino acids [aa] 250 to 270) in the C-terminal domain of IN is involved in this viral-cellular protein interaction. Overall, this study demonstrates for the first time that Impα3 is an HIV integrase-interacting cofactor that is required for efficient HIV-1 nuclear import and replication in both dividing and nondividing cells.

Assessment of the role of the central DNA flap in human immunodeficiency virus type 1 replication by using a single-cycle replication system

ABSTRACT In this study, reverse transcriptase (RT)- and integrase (IN)-defective human immunodeficiency virus type 1 (HIV-1) was transcomplemented with Vpr-RT-IN fusion proteins to delineate pol sequences important for HIV-1 replication. Our results reveal that a 194-bp sequence encompassing the 3′end of the IN gene and containing the central DNA flap is necessary and sufficient for efficient HIV-1 single-cycle replication in dividing and nondividing cells. Furthermore, we show that the central DNA flap enhances HIV-1 single-round replication by five- to sevenfold, primarily by facilitating nuclear import of proviral DNA. In agreement with previous reports, our data support a functional role of the central DNA flap during the early stages of HIV-1 infection.

The Cellular Antiviral Protein APOBEC3G Interacts with HIV-1 Reverse Transcriptase and Inhibits Its Function during Viral Replication

ABSTRACT The cytidine deaminase APOBEC3G (A3G) exerts a multifaceted antiviral effect against HIV-1 infection. First, A3G was shown to be able to terminate HIV infection by deaminating the cytosine residues to uracil in the minus strand of the viral DNA during reverse transcription. Also, a number of studies have indicated that A3G inhibits HIV-1 reverse transcription by a non-editing-mediated mechanism. However, the mechanism by which A3G directly disrupts HIV-1 reverse transcription is not fully understood. In the present study, by using a cell-based coimmunoprecipitation (Co-IP) assay, we detected the direct interaction between A3G and HIV-1 reverse transcriptase (RT) in produced viruses and in the cotransfected cells. The data also suggested that their interaction did not require viral genomic RNA bridging or other viral proteins. Additionally, a deletion analysis showed that the RT-binding region in A3G was located between amino acids 65 and 132. Overexpression of the RT-binding polypeptide A3G65-132 was able to disrupt the interaction between wild-type A3G and RT, which consequently attenuated the anti-HIV effect of A3G on reverse transcription. Overall, this paper provides evidence for the physical and functional interaction between A3G and HIV-1 RT and demonstrates that this interaction plays an important role in the action of A3G against HIV-1 reverse transcription.

Inactivation of microbial infectiousness by silver nanoparticles-coated condom: a new approach to inhibit HIV- and HSV-transmitted infection.

Recent research suggests that today’s condoms are only 85% effective in preventing human immunodeficiency virus (HIV) and other sexually transmitted diseases. In response, there has been a push to develop more effective ways of decreasing the spread of the disease. The new nanotechnology-based condom holds the promise of being more potent than the first-generation products. The preliminary goal of this study was to develop a silver nanoparticles (Ag-NPs)-coated polyurethane condom (PUC) and to investigate its antimicrobial potential including the inactivation of HIV and herpes simplex virus (HSV) infectiousness. The Ag-NPs-coated PUC was characterized by using ultraviolet-visible spectrophotometry, Fourier transform-infrared spectroscopy, high-resolution scanning electron microscopy, and energy-dispersive analysis of X-ray spectroscopy. Nanoparticles were stable on the PUC and not washed away by water. Morphology of the PUC was retained after coating. The NP binding is due to its interaction with the nitrogen atom of the PUC. No significant toxic effects was observed when human HeLa cells, 293T and C8166 T cells were contacted to Ag-NPs-coated PUC for three hours. Interestingly, our results demonstrated that the contact of the Ag-NPs-coated PUC with HIV-1 and HSV-1/2 was able to efficiently inactivate their infectiousness. In an attempt to elucidate the antiviral action of the Ag-NPs, we have demonstrated that the anti-HIV activity was primarily mediated by the Ag-NPs, which are associated with the PUC. In addition, the data showed that both macrophage (M)-tropic and T lymphocyte (T)-tropic strains of HIV-1 were highly sensitive to the Ag-NPs-coated PUC. Furthermore, we also showed that the Ag-NPs-coated PUC was able to inhibit the growth of bacteria and fungi. These results demonstrated that the Ag-NPs-coated PUC is able to directly inactivate the microbe’s infectious ability and provides another defense line against these sexually transmitted microbial infections.

Evaluation of conserved and variable influenza antigens for immunization against different isolates of H5N1 viruses

The combination of rapid evolution and high mortality in human cases of infections has raised concerns that the H5N1 avian influenza virus may become a new, possibly severe, pandemic virus. Vaccination is likely to be the most efficient strategy to mitigate the impact of the next influenza pandemic. The present study evaluates B and T cell immune responses generated by the H5N1 viral antigens, hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), or the M2 ion channel in parallel, expressed from a DNA vaccine vehicle. Protection studies of immunized mice challenged with 100 LD50 of homologous or heterologous H5N1 viruses indicate that HA afforded better protection than the NA, NP or M2 DNA vaccines. The antibody response was also higher in HA-vaccinated mice as determined by hemagglutination inhibition (HI) and neutralizing antibodies (NAB) assays. Interestingly, the T cell response was higher against HA than against NA, NP or M2 and was detectable at low doses of the DNA-HA vaccine capable of inducing complete protection, despite the absence of a detectable B cell response. This study emphasizes the need to evaluate the relationship between both arms of the adaptive immune responses in regards to protective efficacy against influenza virus.

Contribution of the C-terminal tri-lysine regions of human immunodeficiency virus type 1 integrase for efficient reverse transcription and viral DNA nuclear import

BackgroundIn addition to mediating the integration process, HIV-1 integrase (IN) has also been implicated in different steps during viral life cycle including reverse transcription and viral DNA nuclear import. Although the karyophilic property of HIV-1 IN has been well demonstrated using a variety of experimental approaches, the definition of domain(s) and/or motif(s) within the protein that mediate viral DNA nuclear import and its mechanism are still disputed and controversial. In this study, we performed mutagenic analyses to investigate the contribution of different regions in the C-terminal domain of HIV-1 IN to protein nuclear localization as well as their effects on virus infection.ResultsOur analysis showed that replacing lysine residues in two highly conserved tri-lysine regions, which are located within previously described Region C (235WKGPAKLLWKGEGAVV) and sequence Q (211KELQKQITK) in the C-terminal domain of HIV-1 IN, impaired protein nuclear accumulation, while mutations for RK263,4 had no significant effect. Analysis of their effects on viral infection in a VSV-G pseudotyped RT/IN trans-complemented HIV-1 single cycle replication system revealed that all three C-terminal mutant viruses (KK215,9AA, KK240,4AE and RK263,4AA) exhibited more severe defect of induction of β-Gal positive cells and luciferase activity than an IN class 1 mutant D64E in HeLa-CD4-CCR5-β-Gal cells, and in dividing as well as non-dividing C8166 T cells, suggesting that some viral defects are occurring prior to viral integration. Furthermore, by analyzing viral DNA synthesis and the nucleus-associated viral DNA level, the results clearly showed that, although all three C-terminal mutants inhibited viral reverse transcription to different extents, the KK240,4AE mutant exhibited most profound effect on this step, whereas KK215,9AA significantly impaired viral DNA nuclear import. In addition, our analysis could not detect viral DNA integration in each C-terminal mutant infection, even though they displayed various low levels of nucleus-associated viral DNA, suggesting that these C-terminal mutants also impaired viral DNA integration ability.ConclusionAll of these results indicate that, in addition to being involved in HIV-1 reverse transcription and integration, the C-terminal tri-lysine regions of IN also contribute to efficient viral DNA nuclear import during the early stage of HIV-1 replication.

Host protein Ku70 binds and protects HIV-1 integrase from proteasomal degradation and is required for HIV replication

HIV-1 integrase (IN) is a key viral enzymatic protein acting in several viral replication steps, including integration. IN has been shown to be an unstable protein degraded by the N-end rule pathway through the host ubiquitin-proteasome machinery. However, it is still not fully understood how this viral protein is protected from the host ubiquitin-proteasome system within cells during HIV replication. In the present study, we provide evidence that the host protein Ku70 interacts with HIV-1 IN and protects it from the Lys48-linked polyubiquitination proteasomal pathway. Moreover, Ku70 is able to down-regulate the overall protein polyubiquitination level within the host cells and to specifically deubiquitinate IN through their interaction. Mutagenic studies revealed that the C terminus of IN (residues 230–288) is required for IN binding to the N-terminal part of Ku70 (Ku70(1–430)), and their interaction is independent of Ku70/80 heterodimerization. Finally, knockdown of Ku70 expression in both virus-producing and target CD4+ T cells significantly disrupted HIV-1 replication and rendered two-long terminal repeat circles and integration undetectable, indicating that Ku70 is required for both the early and the late stages of the HIV-1 life cycle. Interestingly, Ku70 was incorporated into the progeny virus in an IN-dependent way. We proposed that Ku70 may interact with IN during viral assembly and accompany HIV-1 IN upon entry into the new target cells, acting to 1) protect IN from the host defense system and 2) assist IN integration activity. Overall, this report provides another example of how HIV-1 hijacks host cellular machinery to protect the virus itself and to facilitate its replication.

Incorporation of Vpr into human immunodeficiency virus type 1: role of conserved regions within the P6 domain of Pr55gag.

The 96-amino acid Vpr protein is the only virion-associated regulatory protein encoded by the human immunodeficiency virus type 1 (HIV-1). Vpr incorporation into the viral particle is most likely due to an interaction with a viral structural protein. Recent data have shown that DNA encoding for the p55 Gag precursor protein (Pr55gag) is the minimal viral genetic information necessary for Vpr incorporation. Other studies have suggested that the p6 portion of Pr55gag, which is unique to lentiviruses, is involved in Vpr incorporation. To investigate the mechanism of incorporation of Vpr into HIV-1 virions, COS-7 cells were cotransfected with ptrENV, an expression vector that encodes all of the HIV-1 regulatory proteins including Rev and Vpr, and different constructs of pIIIgagCAR, a rev-dependent Gag expression plasmid that encodes Pr55gag and the viral protease. Virions produced from gag constructs containing a premature p6 termination codon at positions Leu-1, Ser-17, Tyr-36, or Leu-44 lacked detectable Vpr. In contrast, gag constructs with double Pro-10-Pro-11 substitutions for Leu-10-Leu-11 or a premature termination codon at position Pro-49 of p6 were still able to incorporate Vpr, however, with lower efficiency than wild type. The mutations described in this study affected directly two short regions within the p6 domain, which are highly conserved among primate immunodeficiency viruses. Our results suggest that the conserved (P-T/S-A-P-P) and (L-X-S-L-F-G) motifs located near the N-terminus and C-terminus, respectively, of the p6 domain of Gag are critical for Vpr incorporation into HIV-1 virions.