Biomedical subjects

David E Ott

Publications and source records attributed to David E Ott.

7 recordsLinked to original sources

PubMed2003

Elimination of protease activity restores efficient virion production to a human immunodeficiency virus type 1 nucleocapsid deletion mutant.

The nucleocapsid (NC) region of human immunodeficiency virus type 1 (HIV-1) Gag is required for specific genomic RNA packaging. To determine if NC is absolutely required for virion formation, we deleted all but seven amino acids from NC in a full-length NL4-3 proviral clone. This construct, DelNC, produced approximately four- to sixfold fewer virions than did the wild type, and these virions were noninfectious (less than 10(-6) relative to the wild type) and severely genomic RNA deficient. Immunoblot and high-pressure liquid chromatography analyses showed that all of the mature Gag proteins except NC were present in the mutant virion preparations, although there was a modest decrease in Gag processing. DelNC virions had lower densities and were more heterogeneous than wild-type particles, consistent with a defect in the interaction assembly or I domain. Electron microscopy showed that the DelNC virions displayed a variety of aberrant morphological forms. Inactivating the protease activity of DelNC by mutation or protease inhibitor treatment restored virion production to wild-type levels. DelNC-protease mutants formed immature-appearing particles that were as dense as wild-type virions without incorporating genomic RNA. Therefore, protease activity combined with the absence of NC causes the defect in DelNC virion production, suggesting that premature processing of Gag during assembly causes this effect. These results show that HIV-1 can form particles efficiently without NC.

Cell Line↗

PubMed2003

Quantitation of HLA class II protein incorporated into human immunodeficiency type 1 virions purified by anti-CD45 immunoaffinity depletion of microvesicles.

Among the many host cell-derived proteins found in human immunodeficiency virus type 1 (HIV-1), HLA class II (HLA-II) appears to be selectively incorporated onto virions and may contribute to mechanisms of indirect imunopathogenesis in HIV infection and AIDS. However, the amount of HLA-II on the surface of HIV-1 particles has not been reliably determined due to contamination of virus preparations by microvesicles containing host cell proteins, including HLA-II. Even rigorous sucrose density centrifugation is unable to completely separate HIV-1 from microvesicles. CD45, a leukocyte integral membrane protein, is found on microvesicles, yet appears to be excluded from HIV-1 particles. Exploiting this observation, we have developed a CD45-based immunoaffinity depletion method for removing CD45-containing microvesicles that yields highly purified preparations of virions. Examination of CD45-depleted HIV-1(MN) by high-pressure liquid chromatography, protein sequencing, and amino acid analyses determined a molar ratio of HLA-II to Gag of 0.04 to 0.05 in the purified virions, corresponding to an estimated average of 50 to 63 native HLA-II complexes (i.e., a dimer of alpha and beta heterodimers) per virion. These values are approximately 5- to 10-fold lower than those previously determined for other virion preparations that contained microvesicles. Our observations demonstrate the utility of CD45 immunoaffinity-based approaches for producing highly purified retrovirus preparations for applications that would benefit from the use of virus that is essentially free of microvesicles.

Blotting, Western↗

PubMed2003

Retroviruses have differing requirements for proteasome function in the budding process.

Proteasome inhibitors reduce the budding of human immunodeficiency virus types 1 (HIV-1) and 2, simian immunodeficiency virus, and Rous sarcoma virus. To investigate this effect further, we examined the budding of other retroviruses from proteasome inhibitor-treated cells. The viruses tested differed in their Gag organization, late (L) domain usage, or assembly site from those previously examined. We found that proteasome inhibition decreased the budding of murine leukemia virus (plasma membrane assembly, PPPY L domain) and Mason-Pfizer monkey virus (cytoplasmic assembly, PPPY L domain), similar to the reduction observed for HIV-1. Thus, proteasome inhibitors can affect the budding of a virus that assembles within the cytoplasm. However, the budding of mouse mammary tumor virus (MMTV; cytoplasmic assembly, unknown L domain) was unaffected by proteasome inhibitors, similar to the proteasome-independent budding previously observed for equine infectious anemia virus (plasma membrane assembly, YPDL L domain). Examination of MMTV particles detected Gag-ubiquitin conjugates, demonstrating that an interaction with the ubiquitination system occurs during assembly, as previously found for other retroviruses. For all of the cell lines tested, the inhibitor treatment effectively inactivated proteasomes, as measured by the accumulation of polyubiquitinated proteins. The ubiquitination system was also inhibited, as evidenced by the loss of monoubiquitinated histones from treated cells. These results and those from other viruses show that proteasome inhibitors reduce the budding of viruses that utilize either a PPPY- or PTAP-based L domain and that this effect does not depend on the assembly site or the presence of monoubiquitinated Gag in the virion.

Animals↗

PubMed2002

Fusion proteins with COOH-terminal ubiquitin are stable and maintain dual functionality in vivo.

The ubiquitin (Ub) fusion degradation pathway functions to degrade fusion proteins containing a nonremovable Ub moiety at their NH(2) terminus (Johnson, E. S., Ma, P. C., Ota, I. M., and Varshavsky, A. (1995) J. Biol. Chem. 270, 17442-17456). Here we show that ubiquitin fusion degradation also targets proteins for proteasomal degradation when Ub is present in the middle of fusion proteins (X-Ub-Y), in a process that entails polyubiquitylation of Ub Lys(48). By contrast, fusion proteins bearing COOH-terminal Ub (X-Ub) are metabolically stable. Such fusion proteins, either newly biosynthesized or generated by Ub hydrolases, are reversibly conjugated to heterogeneous target proteins in a manner similar to wild-type Ub. Most importantly, the NH(2)-terminal fusion partner (X) can maintain its structure and function in the formed X-Ub conjugates as inferred from the fluorescence of green fluorescent protein-Ub conjugates and the incorporation of human immunodeficiency virus type 1 Gag-Ub into viral particles. These findings strongly suggest that 26S proteasomes exhibit spatial discrimination of Ub-conjugated proteins, sparing domains extended from the NH(2) terminus of Ub from unfolding and degradation. The multifunctionality of X-Ub fusion proteins opens the possibility for a number of novel practical applications, including the imaging of Ub conjugate formation in living cells.

Amino Acid Sequence↗

PubMed2002

Human immunodeficiency virus type 1 preferentially encapsidates genomic RNAs that encode Pr55(Gag): functional linkage between translation and RNA packaging.

Full-length retroviral RNA serves as both messenger and genomic RNA. Therefore, an unspliced RNA could play both roles: viral mRNA could be bound in cis by the same Gag polyprotein that it produced, becoming a packaged genomic RNA. To test this possibility, we used in vivo packaging experiments which coexpressed wild-type NL4-3 RNA and NL4-3-based mutant RNA that, ideally, could not translate Gag. However, mutating the gag initiator produced a mutant (pNLX) that expressed a truncated Gag, Gag*, initiated at methionine 10 in the CA region (142 of Pr55(Gag)). Gag* can be rescued into virions by Gag and, as it contains the NC domain, could package RNA in cis. To eliminate NC and the CA dimerization domain, a nonsense mutation in CA at residue 99 was introduced into pNLX to produce pNLXX, which expresses an RNA that should only be packaged in trans. Cotransfection packaging experiments revealed that wild-type genomic RNA was packaged at an 8-fold greater level than NLXX RNA given equal expression of both RNAs. Experiments that varied the relative amounts of these RNAs in the cell found that the wild-type RNA was encapsidated with a packaging preference (i.e., the relative amount of this RNA in virions versus cells) of 6- to 13-fold over the NLXX RNA, showing that the NLXX RNA did not efficiently compete with NL4-3 RNA. These data suggest that the wild-type RNA's ability to express Pr55(Gag) and, by inference, actively translate Gag confers an advantage in packaging over the nearly identical NLXX RNA. In contrast, the NLX RNA competed with wild-type RNA at a 1-to-3 preference. This ratio is similar to the amounts of Gag* rescued by Gag, suggesting that the presence of Gag* assists in the encapsidation of NLX RNA. Together, our data link translation and particle formation to the packaging of viral RNA and support a model of cis packaging where nascent Gag proteins encapsidate their cognate RNA.

Cell Line↗

PubMed2002

Equine infectious anemia virus and the ubiquitin-proteasome system.

Some retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9(Gag) proteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells. Pulse-chase immunoprecipitation and time course immunoblot analyses showed that proteasome inactivation slightly decreased virus release (at most a twofold effect), while it did not affect Gag processing. These results contrast with those obtained with other viruses which are sensitive to these inhibitors. This suggests that, although its Gag is monoubiquitinated, the requirements for EIAV release are somewhat different from those for retroviruses that are sensitive to proteasome inhibitors.

Animals↗

PubMedDate not supplied

Potential roles of cellular proteins in HIV-1.

Human immunodeficiency virus type-1 particles contain host proteins, both on their surface and interior. This review summarises the cellular proteins found in these virions and covers some of their potential roles in the viral life cycle and pathogenesis. For most proteins studied, their role and function are either unknown or in the hypothesis stage. This reflects the relatively recent emphasis given to these proteins by the HIV-1 field as well as the incomplete understanding of their function in the cell. The study of cellular proteins in HIV-1 promises to help us better understand the interaction of this virus with the cell, the immune system, and the whole human host as well as to shed light on the nature of AIDS and suggest more targets for therapeutic intervention. Finally, many of the cell systems themselves are still poorly understood. The extensive study of HIV-1 has already brought increased attention to the fields of immunology and vaccine science and, in the same way, might assist our understanding of the cellular pathways themselves.

Antigens, Surface↗