Somatic Cell Mutants Resistant to Retrovirus Replication: Intracellular Blocks during the Early Stages of Infection

To identify cellular functions involved in the early phase of the retroviral life cycle, somatic cell mutants were isolated after selection for resistance to infection. Rat2 fibroblasts were treated with chemical mutagens, and individual virus-resistant clones were recovered after selection for resistance to infection. Two clones were characterized in detail. Both mutant lines were resistant to infection by both ecotropic and amphotropic murine viruses, as well as by human immunodeficiency virus type 1 pseudotypes. One clone showed a strong block to reverse transcription of the retroviral RNA, including formation of the earliest DNA products. The second clone showed normal levels of viral DNA synthesis but did not allow formation of the circular DNAs normally found in the nucleus. Cell fractionation showed that the viral preintegration complex was present in a form that could not be extracted under conditions that readily extracted the complex from wild-type cells. The results suggest that the DNA was trapped in a nonproductive state and excluded from the nucleus of the infected cell. The properties of these two mutant lines suggest that host gene products play important roles both before and after reverse transcription

Mutagenesis analysis of the zinc-finger antiviral protein

BACKGROUND: The zinc-finger antiviral protein (ZAP) specifically inhibits the replication of certain viruses, including murine leukemia virus (MLV), by preventing the accumulation of viral mRNA in the cytoplasm. ZAP directly binds to the viral mRNA through the zinc-finger motifs and recruits the RNA exosome to degrade the target RNA. RNA helicase p72 is required for the optimal function of ZAP. In an attempt to understand the structure-function relationship of ZAP, we performed alanine scanning analysis. RESULTS: A series of ZAP mutants was generated, in which three consecutive amino acids were replaced with three alanines. The mutants were analyzed for their antiviral activities against pseudotyped MLV vector. Out of the nineteen mutants analyzed, seven displayed significantly lower antiviral activities. Two mutations were in the very N-terminal domain, and five mutations were within or around the first and second zinc-finger motifs. These mutants were further analyzed for their abilities to bind to the target RNA, the exosome, and the RNA helicase p72. Mutants Nm3 and Nm63 lost the ability to bind to RNA. Mutants Nm 63 and Nm93 displayed compromised interaction with p72, while the binding of Nm133 to p72 was very modest. The interactions of all the mutants with the exosome were comparable to wild type ZAP. CONCLUSIONS: The integrity of the very N-terminal domain and the first and second zinc-finger motifs appear to be required for ZAP's antiviral activity. Analyses of the mutants for their abilities to interact with the target RNA and RNA helicase p72 confirmed our previous results. The mutants that bind normally to the target RNA, the exosome, and the RNA helicase p72 may be useful tools for further understanding the mechanism underlying ZAP's antiviral activity

Isolation of suppressor genes that restore retrovirus susceptibility to a virus-resistant cell line

BACKGROUND: Genetic selections in mammalian cell lines have recently been developed for the isolation of mutant cells that are refractory to infection by retroviruses. These selections have been used to recover lines that block early postentry stages of infection, either before reverse transcription or before nuclear entry. The mechanisms of action of these blocks remain unknown. RESULTS: We have devised a method for the selection of genes from cDNA libraries that suppress the block to virus infection, and so restore virus susceptibility. The protocol involves the transformation of pools of resistant cells by cDNA expression libraries, followed by the selection for rare virus-sensitive cells, using multiple rounds of selection after infection by marked viral vector genomes. The suppressor genes were then recovered from these virus sensitive cells, and their ability to restore virus susceptibility was confirmed by reintroduction of these cDNAs into the resistant line. CONCLUSIONS: The identities of these genes provide insights into the mechanism of virus resistance and will help to define new pathways used during retrovirus infection. The methods for gene isolation developed here will also permit the identification of similar suppressors that modify or override other recently identified virus resistance genes

Hrs inhibits citron kinase-mediated HIV-1 budding via its FYVE domain

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a key component of the endosomal sorting complexes required for transport and has been demonstrated to play a regulatory role in endocytosis/exocytosis and the accumulation of internal vesicles in multivesicular bodies. Citron kinase is a Ser/The kinase that we previously reported to enhance human immunodeficiency virus type 1 (HIV-1) virion production. However, the relationship between Hrs and citron kinase in HIV-1 production remains elusive. Here, we report that Hrs interacts with citron kinase via its FYVE domain. Overexpression of Hrs or the FYVE domain resulted in a significant decrease in HIV-1 virion production. Depletion of Hrs by RNA interference in HEK293T cells increased HIV-1 virion production and enhanced the activity of citron kinase. These data suggest that Hrs inhibits HIV-1 production by inhibiting citron kinase-mediated exocytosis

Energy-efficient optimal power allocation in integrated wireless sensor and cognitive satellite terrestrial networks

This paper proposes novel satellite-based wireless sensor networks (WSNs), which integrate the WSN with the cognitive satellite terrestrial network. Having the ability to provide seamless network access and alleviate the spectrum scarcity, cognitive satellite terrestrial networks are considered as a promising candidate for future wireless networks with emerging requirements of ubiquitous broadband applications and increasing demand for spectral resources. With the emerging environmental and energy cost concerns in communication systems, explicit concerns on energy efficient resource allocation in satellite networks have also recently received considerable attention. In this regard, this paper proposes energy-efficient optimal power allocation schemes in the cognitive satellite terrestrial networks for non-real-time and real-time applications, respectively, which maximize the energy efficiency (EE) of the cognitive satellite user while guaranteeing the interference at the primary terrestrial user below an acceptable level. Specifically, average interference power (AIP) constraint is employed to protect the communication quality of the primary terrestrial user while average transmit power (ATP) or peak transmit power (PTP) constraint is adopted to regulate the transmit power of the satellite user. Since the energy-efficient power allocation optimization problem belongs to the nonlinear concave fractional programming problem, we solve it by combining Dinkelbach’s method with Lagrange duality method. Simulation results demonstrate that the fading severity of the terrestrial interference link is favorable to the satellite user who can achieve EE gain under the ATP constraint comparing to the PTP constraint

Endophilins interact with Moloney murine leukemia virus Gag and modulate virion production

BACKGROUND: The retroviral Gag protein is the central player in the process of virion assembly at the plasma membrane, and is sufficient to induce the formation and release of virus-like particles. Recent evidence suggests that Gag may co-opt the host cell's endocytic machinery to facilitate retroviral assembly and release. RESULTS: A search for novel partners interacting with the Gag protein of the Moloney murine leukemia virus (Mo-MuLV) via the yeast two-hybrid protein-protein interaction assay resulted in the identification of endophilin 2, a component of the machinery involved in clathrin-mediated endocytosis. We demonstrate that endophilin interacts with the matrix or MA domain of the Gag protein of Mo-MuLV, but not of human immunodeficiency virus, HIV. Both exogenously expressed and endogenous endophilin are incorporated into Mo-MuLV viral particles. Titration experiments suggest that the binding sites for inclusion of endophilin into viral particles are limited and saturable. Knock-down of endophilin with small interfering RNA (siRNA) had no effect on virion production, but overexpression of endophilin and, to a lesser extent, of several fragments of the protein, result in inhibition of Mo-MuLV virion production, but not of HIV virion production. CONCLUSIONS: This study shows that endophilins interact with Mo-MuLV Gag and affect virion production. The findings imply that endophilin is another component of the large complex that is hijacked by retroviruses to promote virion production

Genotyping of TRIM5 locus in northern pig-tailed macaques (Macaca leonina), a primate species susceptible to Human Immunodeficiency Virus type 1 infection

<p>Abstract</p> <p>Background</p> <p>The pig-tailed macaques are the only Old World monkeys known to be susceptible to human immunodeficiency virus type 1 (HIV-1) infection. We have previously reported that the <it>TRIM5-Cyclophilin A </it>(<it>TRIMCyp</it>) fusion in pig-tailed macaques (<it>Macaca nemestrina</it>) is dysfunctional in restricting HIV-1, which may explain why pig-tailed macaques are susceptible to HIV-1 infection. Similar results have also been reported by other groups. However, according to the current primate taxonomy, the previously reported <it>M. nemestrina </it>are further classified into three species, which all belong to the <it>Macaca spp</it>. This calls for the need to look into the previous studies in more details.</p> <p>Results</p> <p>The local species Northern pig-tailed macaque (<it>M. leonina</it>) was analyzed for the correlation of <it>TRIM5 </it>structure and HIV-1 infection. Eleven <it>M. leonina </it>animals were analyzed, and all of them were found to possess <it>TRIM5-CypA </it>fusion at the <it>TRIM5 </it>locus. The transcripts encoding the dysfunctional <it>TRIM5-CypA </it>should result from the G-to-T mutation in the 3'-splicing site of intron 6. Polymorphism in the putative TRIMCyp recognition domain was observed. The peripheral blood mononuclear cells (PBMCs) of <it>M. leonina </it>were susceptible to HIV-1 infection. Consistent with the previous results, expression of the <it>M. leonina </it>TRIMCyp in HeLa-T4 cells rendered the cells resistant to HIV-2_RODbut not to SIVmac239 infection.</p> <p>Conclusion</p> <p>The susceptibility of <it>M. leonina </it>to HIV-1 infection is due to the dysfunctional <it>TRIM5-CypA </it>fusion in the <it>TRIM5 </it>locus. This finding should broaden our perspective in developing better HIV/AIDS non-human primate animal models.</p

HIV-1 can infect northern pig-tailed macaques (Macaca leonina) and form viral reservoirs in vivo

Viral reservoirs of HIV-1 are a major obstacle for curing AIDS. The novel animal models that can be directly infected with HIV-1 will contribute to develop effective strategies for eradicating infections. Here, we inoculated 4 northern pig-tailed macaques (NPM) with the HIV-1 strain HIV-1NL4.3 and monitored the infection for approximately 3 years (150 weeks). The HIV-1-infected NPMs showed transient viremia for about 10 weeks after infection. However, cell-associated proviral DNA and viral RNA persisted in the peripheral blood and lymphoid organs for about 3 years. Moreover, replication-competent HIV-1 could be successfully recovered from peripheral blood mononuclear cells (PBMCs) during long-term infection. The numbers of resting CD4+ T cells in HIV-1 infected NPMs harboring proviruses fell within a range of 2- to 3-log10 per million cells, and these proviruses could be reactivated both ex vivo and in vivo in response to co-stimulation with the latency-reversing agents JQ1 and prostratin. Our results suggested that NPMs can be infected with HIV-1 and a long-term viral reservoir was formed in NPMs, which might serve as a potential model for HIV-1 reservoir research