Herpes Simplex Virus 1 ICP27 Is Required for Transcription of Two Viral Late (γ2) Genes in Infected Cells

AbstractThe herpes simplex virus infected cell protein 27 (ICP27) is required for the expression of certain early viral proteins and for many late proteins during productive infection. Expression of at least one late (γ2) gene, that encoding glycoprotein C, is severely restricted in the absence of functional ICP27. The exact mode of action by which ICP27 induces late gene expression is not known, but the effect is apparent at the mRNA level as demonstrated by Northern blot analysis. To determine whether ICP27 activates late genes via transcriptional or posttranscriptional mechanisms, we initially used nuclear run-on assays to measure transcription of viral genes in Vero cells infected with wild-type (WT) virus or an ICP27 nonsense mutant virus, n504. We observed a 4-fold reduction in the nuclear run-on signal from the coding strand of the gC gene for n504-infected cells compared to that of WT-infected cells. However, interpretation of the results was complicated by the observation of a significant signal from the noncoding strand in these experiments. To obviate the problem of symmetrical transcription, we utilized in vivo RNA pulse-labeling to measure the amount of transcription of viral genes in cells infected with either WT virus or n504 virus. We found a 5- to 10-fold reduction in the transcription of the gC and UL47 genes, two late genes, in cells infected with n504 compared to that in cells infected with WT virus. In contrast, transcription of the ICP8 gene, an early gene, was similar in WT and n504 virus-infected cells. We also examined the stability of the gC and UL47 gene transcripts in n504-infected cells, and we found it to be comparable to that in WT virus-infected cells, further supporting an effect on transcription. Transcription of the gC and UL47 genes by n504 was normal in a cell line that expresses WT ICP27. From these results we conclude that ICP27 is required for transcription of the late gC and UL47 genes during productive infection

The ability of multimerized cyclophilin A to restrict retrovirus infection

AbstractIn owl monkeys, the typical retroviral restriction factor of primates, TRIM5α, is replaced by TRIMCyp. TRIMCyp consists of the TRIM5 RING, B-box 2 and coiled-coil domains, as well as the intervening linker regions, fused with cyclophilin A. TRIMCyp restricts infection of retroviruses, such as human immunodeficiency virus (HIV-1) and feline immunodeficiency virus (FIV), with capsids that can bind cyclophilin A. The TRIM5 coiled coil promotes the trimerization of TRIMCyp. Here we show that cyclophilin A that is oligomeric as a result of fusion with a heterologous multimer exhibits substantial antiretroviral activity. The addition of the TRIM5 RING, B-box 2 and Linker 2 to oligomeric cyclophilin A generated a protein with antiretroviral activity approaching that of wild-type TRIMCyp. Multimerization increased the binding of cyclophilin A to the HIV-1 capsid, promoting accelerated uncoating of the capsid and restriction of infection

Distinct host cell proteins incorporated by SIV replicating in CD4+ T Cells from natural disease resistant versus non-natural disease susceptible hosts

<p>Abstract</p> <p>Background</p> <p>Enveloped viruses including the simian immunodeficiency virus (SIV) replicating within host cells acquire host proteins upon egress from the host cells. A number of studies have catalogued such host proteins, and a few have documented the potential positive and negative biological functions of such host proteins. The studies conducted herein utilized proteomic analysis to identify differences in the spectrum of host proteins acquired by a single source of SIV replicating within CD4⁺T cells from disease resistant sooty mangabeys and disease susceptible rhesus macaques.</p> <p>Results</p> <p>While a total of 202 host derived proteins were present in viral preparations from CD4⁺T cells from both species, there were 4 host-derived proteins that consistently and uniquely associated with SIV replicating within CD4⁺T cells from rhesus macaques but not sooty mangabeys; and, similarly, 28 host-derived proteins that uniquely associated with SIV replicating within CD4⁺T cells from sooty mangabeys, but not rhesus macaques. Of interest was the finding that of the 4 proteins uniquely present in SIV preparations from rhesus macaques was a 26 S protease subunit 7 (MSS1) that was shown to enhance HIV-1 'tat" mediated transactivation. Among the 28 proteins found in SIV preparations from sooty mangabeys included several molecules associated with immune function such as CD2, CD3ε, TLR4, TLR9 and TNFR and a bioactive form of IL-13.</p> <p>Conclusions</p> <p>The finding of 4 host proteins that are uniquely associated with SIV replicating within CD4⁺T cells from disease susceptible rhesus macaques and 28 host proteins that are uniquely associated with SIV replicating within CD4⁺T cells from disease resistant sooty mangabeys provide the foundation for determining the potential role of each of these unique host-derived proteins in contributing to the polarized clinical outcome in these 2 species of nonhuman primates.</p

Unique features of TRIM5α among closely related human TRIM family members

AbstractThe tripartite motif (TRIM) protein, TRIM5α, restricts some retroviruses, including human immunodeficiency virus (HIV-1), from infecting the cells of particular species. TRIM proteins contain RING, B-box, coiled-coil and, in some cases, B30.2(SPRY) domains. We investigated the properties of human TRIM family members closely related to TRIM5. These TRIM proteins, like TRIM5α, assembled into homotrimers and co-localized in the cytoplasm with TRIM5α. TRIM5α turned over more rapidly than related TRIM proteins. TRIM5α, TRIM34 and TRIM6 associated with HIV-1 capsid–nucleocapsid complexes assembled in vitro; the TRIM5α and TRIM34 interactions with these complexes were dependent on their B30.2(SPRY) domains. Only TRIM5α potently restricted infection by the retroviruses studied; overexpression of TRIM34 resulted in modest inhibition of simian immunodeficiency virus (SIVmac) infection. In contrast to the other TRIM genes examined, TRIM5 exhibited evidence of positive selection. The unique features of TRIM5α among its TRIM relatives underscore its special status as an antiviral factor

Effects of human TRIM5α polymorphisms on antiretroviral function and susceptibility to human immunodeficiency virus infection

AbstractTRIM5α acts on several retroviruses, including human immunodeficiency virus (HIV-1), to restrict cross-species transmission. Using natural history cohorts and tissue culture systems, we examined the effect of polymorphism in human TRIM5α on HIV-1 infection. In African Americans, the frequencies of two non-coding SNP variant alleles in exon 1 and intron 1 of TRIM5 were elevated in HIV-1-infected persons compared with uninfected subjects. By contrast, the frequency of the variant allele encoding TRIM5α 136Q was relatively elevated in uninfected individuals, suggesting a possible protective effect. TRIM5α 136Q protein exhibited slightly better anti-HIV-1 activity in tissue culture than the TRIM5α R136 protein. The 43Y variant of TRIM5α was less efficient than the H43 variant at restricting HIV-1 and murine leukemia virus infections in cultured cells. The ancestral TRIM5 haplotype specifying no observed variant alleles appeared to be protective against infection, and the corresponding wild-type protein partially restricted HIV-1 replication in vitro. A single logistic regression model with a permutation test indicated the global corrected P value of <0.05 for both SNPs and haplotypes. Thus, polymorphism in human TRIM5 may influence susceptibility to HIV-1 infection, a possibility that merits additional evaluation in independent cohorts

The Cholesterol Transport Inhibitor U18666A Interferes with Pseudorabies Virus Infection

Many viruses require the maintenance of lysosomal cholesterol homeostasis for a successful infection; however, the role of lysosomal cholesterol homeostasis in the alphaherpesvirus life cycle is not clear. Here we show that the lysosomal cholesterol transport inhibitor U18666A interferes with the replication of pseudorabies virus (PRV), a member of the alphaherpesvirus subfamily. The treatment with U18666A caused a significant reduction in the production of infectious virus particles. The U18666A treatment was shown to suppress the release of PRV particles. Pretreating PRV virions with U18666A did not affect virus production, whereas pretreating target cells with U18666A led to a substantial reduction in virus yield. Our previous study showed that two cyclodextrin derivatives, 2-hydroxypropyl-&beta;-cyclodextrin (HP&beta;CD) and 2-hydroxypropyl-&gamma;-cyclodextrin (HP&gamma;CD), can rescue the cholesterol accumulation defect in primary fibroblasts derived from a Niemann&ndash;Pick disease type C (NPC) patient. Here, we demonstrate that treatment with HP&beta;CD or HP&gamma;CD not only rescues the U18666A-induced cholesterol accumulation but also rescues the U18666A-induced inhibition of PRV production. Collectively, our data suggest that U18666A interferes with PRV infection via altering cellular functions that are critical for the viral life cycle and may include lysosomal cholesterol homeostasis

Functional Interplay Between the B-box 2 and the B30.2(SPRY) Domains of TRIM5α

The retroviral restriction factors, TRIM5α and TRIMCyp, consist of RING and B-box 2 domains separated by a coiled coil from carboxy-terminal domains. These carboxy-terminal domains (the B30.2(SPRY) domain in TRIM5α and the cyclophilin A domain in TRIMCyp) recognize the retroviral capsid. Here we show that some B-box 2 changes in TRIM5α, but not in TRIMCyp, resulted in decreased human immunodeficiency virus (HIV-1) capsid binding. The phenotypic effects of these B-box 2 changes on the restriction of retroviral infection depended on the potency of restriction and the affinity of the TRIM5α interaction with the viral capsid, two properties specified by the B30.2 (SPRY) domain. Thus, some alterations in the TRIM5α B-box 2 domain apparently affect the orientation or conformation of the B30.2(SPRY) domain, influencing capsid recognition

Cyclodextrins: Assessing the Impact of Cavity Size, Occupancy, and Substitutions on Cytotoxicity and Cholesterol Homeostasis

Cyclodextrins (CDs) are cyclic oligosaccharides; the most common CDs contain six, seven, or eight glucose units called &alpha;-CDs, &beta;-CDs, and &gamma;-CDs, respectively. The use of CDs in biomedical research is increasing due to their ability to interact with membrane lipids as well as a wide variety of poorly water-soluble molecules. We assessed the impact of CD cavity size, occupancy, and substitutions on cytotoxicity and cholesterol homeostasis. The potency of CD-mediated cytotoxicity was in the order of &beta;-CDs, &alpha;-CDs, and &gamma;-CDs. Substitutions with hydroxypropyl or carboxymethyl group attenuated cytotoxicity compared with the native CDs, whereas CDs substituted with methyl groups exhibited cytotoxicity that was similar to that of the native CDs. The lipid components in blood exerted remarkable hemolysis-alleviating effects in methyl-&beta;-CD-induced hemolysis. Occupancy of the CD cavity with cholesterol or a structurally related lipid molecule abrogated the cytotoxic capacity of the CDs. Interestingly, hydroxypropyl-&gamma;-CD (HP&gamma;CD) was able to reduce intracellular cholesterol accumulation in Niemann&ndash;Pick disease type C (NPC) patient-derived fibroblasts as efficiently as HP&beta;CD. Proteomic study indicated that HP&beta;CD and HP&gamma;CD treatments altered the expression pattern of cellular proteins, suggesting that some of the CD-induced cellular proteins may play an important function in modulating intracellular cholesterol homeostasis

Virus-Specific effects of TRIM5αrh RING domain functions on restriction of retroviruses

The tripartite motif protein TRIM5α restricts particular retrovirus infections by binding to the incoming capsid and inhibiting the early stage of virus infection. The TRIM5α RING domain exhibits E3 ubiquitin ligase activity and assists the higher-order association of TRIM5α dimers, which promotes capsid binding. We characterized a panel of RING domain mutants of the rhesus monkey TRIM5α (TRIM5αrh) protein. The RING domain function that significantly contributed to retroviral restriction depended upon the restricted virus. The E3 ubiquitin ligase activity of the RING domain contributes to the potency of HIV-1 restriction. Nonetheless, TRIM5αrh mutants without detectable E3 ubiquitin ligase activity still blocked reverse transcription and inhibited HIV-1 infection at a moderate level. When TRIM5αrh capsid binding was weakened by substitution with a less efficient B30.2/SPRY domain, the promotion of higher-order association by the RING domain was more important to HIV-1 restriction than its E3 ubiquitin ligase activity. For the restriction of N-tropic murine leukemia virus (N-MLV) and equine infectious anemia virus (EIAV) infection, promotion of higher-order association represented the major contribution of the RING domain. Thus, both identity of the target virus and the B30.2/SPRY domain-mediated affinity for the viral capsid determine the relative contribution of the two known RING domain functions to TRIM5α restriction of retrovirus infection. © 2013, American Society for Microbiology.Peer Reviewe