Jonathan Leis

Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55Gag

Ubiquitination appears to be involved in virus particle release from infected cells. Free ubiquitin (Ub), as well as Ub covalently bound to a small fraction of p6 Gag, is detected in mature HIV particles. Here we report that the p6 region in the Pr55Gag structural precursor polyprotein binds to Tsg101, a putative Ub regulator that is involved in trafficking of plasma membrane-associated proteins. Tsg101 was found to interact with Gag in (i) a yeast two-hybrid assay, (ii) in vitro coimmunoprecipitation by using purified Pr55Gag and rabbit reticulocyte lysate-synthesized Tsg101, and (iii) in vivo in the cytoplasm of COS cells transfected with gag. The PTAPP motif [or late (L) domain] within p6, which is required for release of mature virus from the plasma membrane, was the determinant for binding Pr55Gag. The N-terminal region in Tsg101, which is homologous to the Ubc4 class of Ub-conjugating (E2) enzymes, was the determinant of interaction with p6. Mutation of Tyr-110 in Tsg101, present in place of the active-site Cys that binds Ub in E2 enzymes, and other residues unique to Tsg101, impaired p6 interaction, indicating that features that distinguish Tsg101 from active E2 enzymes were important for binding the viral protein. The results link L-domain function in HIV to the Ub machinery and a specific component of the cellular trafficking apparatus.

The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro.

The integration of viral DNA into the host cell chromosome is an essential feature of the retroviral life cycle. The integration reaction requires cis-acting sequences at the ends of linear viral DNA and a trans-acting product of the pol gene, the integration protein (IN). Previously, we demonstrated that avian sarcoma-leukosis virus (ASLV) IN is able to carry out the first step in the integration process in vitro: nicking of the ends of linear viral DNA. In this paper, using two independent assays, we demonstrate that IN, alone, is sufficient to carry out the second step: cleavage and joining to the target DNA. These results demonstrate that the retroviral IN protein is an integrase.

Proteins related to the Nedd4 family of ubiquitin protein ligases interact with the L domain of Rous sarcoma virus and are required for gag budding from cells

The late assembly (L) domain of retrovirus Gag, required in the final steps of budding for efficient exit from the host cell, is thought to mediate its function through interaction with unknown cellular factors. Here, we report the identification of the Nedd4-like family of E3 ubiquitin protein ligases as proteins that specifically interact with the Rous sarcoma virus (RSV) L domain in vitro and in vivo. We screened a chicken embryo cDNA expression library by using a peptide derived from the RSV p2b sequence, isolating two unique partial cDNA clones. Neither clone interacted with a peptide containing mutations known to disrupt in vivo RSV L domain function or with human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) L domain-derived peptides. The WW domain region of one of the clones, late domain-interacting protein 1 (LDI-1), but not the C2 domain, bound RSV Gag and inhibited RSV Gag budding from human 293 cells in a dominant-negative manner, functionally implicating LDI-1 in RSV particle budding from cells. RSV Gag can be coimmune precipitated from cell extracts with an antisera directed at an exogenously expressed hemagglutinin (HA)-tagged LDI-1 or endogenous Nedd4 proteins. These findings mechanistically link the cellular ubiquitination pathway to retrovirus budding.

Site-Directed Mutagenesis Using Overlap Extension PCR

Site-directed mutagenesis and the polymerase chain reaction (PCR) represent two powerful techniques that have led to rapid advances in our understanding of gene expression and function. Early protocols for site-directed mutagenesis depended on the production of single-stranded DNA containing the gene of interest (1), using Ml 3 phage, or phagemids such as pBluescript. One limitation with this method is the presence of inverted repeat sequences and other complementary regions, which form extensive secondary structures within single-stranded DNA, which often severely decrease the ability to extend annealed primers containing the mutation of interest. Recently, several groups have described PCR-based site-directed mutagenesis techniques that avoid this problem by using thermal stable DNA polymerases and high temperatures to melt secondary structure (2— 4). Although several variations of these techniques have been reported, this chapter focuses on a derivative of the Sarkar and Sommer (4) megaprimer method termed Overlap Extension Mutagenesis, which is schematically depicted in Fig. 1. In the first round of PCR, primer A and mutagenic primer B are used to amplify the region of interest as well as introduce the desired mutation. This amplified product is then used as a primer in the second round of PCR and, as such, is referred to as a megaprimer. In the second round of PCR, megaprimer A/B and primer C

The Interferon-Induced Gene ISG15 Blocks Retrovirus Release from Cells Late in the Budding Process

ABSTRACT The release of retroviruses from cells requires ubiquitination of Gag and recruitment of cellular proteins involved in endosome sorting, including the ESCRT-III proteins and the Vps4 ATPase. In response to infection, cells have evolved an interferon-induced mechanism to block virus replication through expression of the interferon-stimulated gene 15 (ISG15), a dimer homologue of ubiquitin, which interferes with ubiquitin pathways in cells. Previously, it has been reported that ISG15 expression inhibited the E3 ubiquitin ligase, Nedd4, and prevented association of the ESCRT-I protein Tsg101 with human immunodeficiency virus type 1 (HIV-1) Gag. The budding of avian sarcoma leukosis virus and HIV-1 Gag virus-like particles containing L-domain mutations can be rescued by fusion to ESCRT proteins, which cause entry into the budding pathway beyond these early steps. The release of these fusions from cells was susceptible to inhibition by ISG15, indicating that there was a block late in the budding process. We now demonstrate that the Vps4 protein does not associate with the avian sarcoma leukosis virus or the HIV-1 budding complexes when ISG15 is expressed. This is caused by a loss in interaction between Vps4 with its coactivator protein LIP5 needed to promote the formation of the ESCRT-III-Vps4 double-hexamer complex required for membrane scission and virus release. The inability of LIP5 to interact with Vps4 is the probable result of ISG15 conjugation to the ESCRT-III protein, CHMP5, which regulates the availability of LIP5. Thus, there appear to be multiple levels of ISG15-induced inhibition acting at different stages of the virus release process.

Modeling the Late Steps in HIV-1 Retroviral Integrase-catalyzed DNA Integration

Model oligodeoxyribonucleotide substrates representing viral DNA integration intermediates with a gap and a two-nucleotide 5′ overhang were used to examine late steps in human immunodeficiency virus, type 1 (HIV-1) retroviral integrase (IN)-catalyzed DNA integration in vitro. HIV-1 or avian myeloblastosis virus reverse transcriptase (RT) were capable of quantitatively filling in the gap to create a nicked substrate but did not remove the 5′ overhang. HIV-1 IN also failed to remove the 5′ overhang with the gapped substrate. However, with a nicked substrate formed by RT, HIV-1 IN removed the overhang and covalently closed the nick in a disintegration-like reaction. The efficiency of this closure reaction was very low. Such closure was not stimulated by the addition of HMG-(I/Y), suggesting that this protein only acts during the early processing and joining reactions. Addition of Flap endonuclease-1, a nuclease known to remove 5′ overhangs, abolished the closure reaction catalyzed by IN. A series of base pair inversions, introduced into the HIV-1 U5 long terminal repeat sequence adjacent to and/or including the conserved CA dinucleotide, produced no or only a small decrease in the HIV-1 IN-dependent strand closure reaction. These same mutations caused a significant decrease in the efficiency of concerted DNA integration by a modified donor DNA in vitro, suggesting that recognition of the ends of the long terminal repeat sequence is required only in the early steps of DNA integration. Finally, a combination of HIV-1 RT, Flap endonuclease-1, and DNA ligase is capable of quantitatively forming covalently closed DNA with these model substrates. These results support the hypothesis that cellular enzyme(s) may catalyze the late steps of retroviral DNA integration.

3 Regulation of Initiation of Reverse Transcription of Retroviruses

Shortly after the discovery of reverse transcriptase in 1970 by Temin (Temin and Mizutani 1970) and Baltimore (1970), its basic enzymatic properties were established (for review, see Weiss et al. 1985; Coffin 1990). Although reverse transcriptase was an unusual DNA polymerase in that it utilized both RNA and DNA as a template, many of its intrinsic properties were found to be similar to those already ascribed to the well-studied bacterial DNA polymerases. These properties included the absolute requirement for a primer to initiate DNA synthesis (Hurwitz and Leis 1972; Leis and Hurwitz 1972; Dahlberg et al. 1974). In contrast to bacterial systems where the primer is synthesized during replication, retroviruses, as well as retroelements, utilize preexisting host-encoded transfer RNAs as primers (Weiss et al. 1985). Depending on the virus, different tRNAs are used (see Table 1). The tRNA primers are encapsidated in virions through interactions with both the viral RNA (Weiss et al. 1985) and reverse transcriptase (RT) (Panet et al. 1975; Barat et al. 1989). Annealing of the primer to viral RNA occurs in the untranslated region close to the 5′ terminus (Weiss et al. 1985) at a site referred to as the primer-binding site (PBS) (see Fig. 1). The acceptor stem of the tRNA is unwound and between 14 and 22 nucleotides (Weiss et al. 1985) form a base-paired duplex with the viral RNA. The RNA 5′ to the PBS is called U5 and the RNA 3′ to the PBS is called the leader. The presence of the...

Identification of Amino Acids in HIV-1 and Avian Sarcoma Virus Integrase Subsites Required for Specific Recognition of the Long Terminal Repeat Ends

A tetramer model for HIV-1 integrase (IN) with DNA representing 20 bp of the U3 and U5 long terminal repeats (LTR) termini was assembled using structural and biochemical data and molecular dynamics simulations. It predicted amino acid residues on the enzyme surface that can interact with the LTR termini. A separate structural alignment of HIV-1, simian sarcoma virus (SIV), and avian sarcoma virus (ASV) INs predicted which of these residues were unique. To determine whether these residues were responsible for specific recognition of the LTR termini, the amino acids from ASV IN were substituted into the structurally equivalent positions of HIV-1 IN, and the ability of the chimeras to 3 ′ process U5 HIV-1 or ASV duplex oligos was determined. This analysis demonstrated that there are multiple amino acid contacts with the LTRs and that substitution of ASV IN amino acids at many of the analogous positions in HIV-1 IN conferred partial ability to cleave ASV substrates with a concomitant loss in the ability to cleave the homologous HIV-1 substrate. HIV-1 IN residues that changed specificity include Val72, Ser153, Lys160–Ile161, Gly163–Val165, and His171–Leu172. Because a chimera that combines several of these substitutions showed a specificity of cleavage of the U5 ASV substrate closer to wild type ASV IN compared with chimeras with individual amino acid substitutions, it appears that the sum of the IN interactions with the LTRs determines the specificity. Finally, residues Ser153 and Val72 in HIV-1 IN are among those that change in enzymes that develop resistance to naphthyridine carboxamide- and diketo acid-related inhibitors in cells. Thus, amino acid residues involved in recognition of the LTRs are among these positions that change in development of drug resistance.

Mechanisms for Priming DNA Synthesis

DNA replication is a semiconservative process in which a DNA polymerase uses one DNA strand as a template for the synthesis of a second, complementary, DNA strand. However, in contrast to RNA polymerases, which can initiate RNA synthesis on a DNA template de novo, all DNA polymerases require a preexisting primer on which to initiate DNA synthesis (Kornberg and Baker 1992). One apparent exception to this rule is a mitochondrial DNA (mtDNA)-encoded reverse transcriptase (RT) in Neurospora (Wang and Lambowitz 1993). Preexisting primers can be classified into four groups. The simplest primer consists of the 3′-hydroxyl (3′-OH) termini of DNA chains that are complementary to the DNA template and thereby form a stable duplex structure at the site where DNA synthesis begins. This primer is used for DNA repair (Friedberg and Wood, this volume), parvovirus DNA replication (Brush and Kelly; Cotmore and Tattersall; both this volume), some RTs. The second type of primer consists of a deoxyribonucleoside monophosphate that is covalently attached to a specific serine, threonine, or tyrosine residue of a protein. Examples are bacteriophage, plasmids, and animal viruses that replicate as a linear DNA genome, and animal viruses such as hepadnaviruses whose genome is partially double-stranded and partially single-stranded. The third type of primer consists of tRNA molecules that anneal to specific sequences in the RNA genomes of retroviruses where their 3′-OH termini are utilized by RT. The fourth class of primers consists of nascent RNA chains. These comprise nascent RNA transcripts that are processed to create a...

The Functionally Exchangeable L Domains in RSV and HIV-1 Gag Direct Particle Release Through Pathways Linked by Tsg101

The functionally exchangeable L domains of HIV‐1 and Rous sarcoma virus (RSV) Gag bind Tsg101 and Nedd4, respectively. Tsg101 and Nedd4 function in endocytic trafficking, and studies show that expression of Tsg101 or Nedd4 fragments interfere with release of HIV‐1 or RSV Gag, respectively, as virus‐like particles (VLPs). To determine whether functional exchangeability reflects use of the same trafficking pathway, we tested the effect on RSV Gag release of co‐expression with mutated forms of Vps4, Nedd4 and Tsg101. A dominant‐negative mutant of Vps4A, an AAA ATPase required for utilization of endosomal sorting proteins that was shown previously to interfere with HIV‐1 budding, also inhibited RSV Gag release, indicating that RSV uses the endocytic trafficking machinery, as does HIV. Nedd4 and Tsg101 interacted in the presence or absence of Gag and, through its binding of Nedd4, RSV Gag interacted with Tsg101. Deletion of the N‐terminal region of Tsg101 or the HECT domain of Nedd4 did not prevent interaction; however, three‐dimensional spatial imaging suggested that the interaction of RSV Gag with full‐length Tsg101 and N‐terminally truncated Tsg101 was not the same. Co‐expression of RSV Gag with the Tsg101 C‐terminal fragment interfered with VLP release minimally; however, a significant fraction of the released VLPs was tethered to each other. The results suggest that, while Tsg101 is not required for RSV VLP release, alterations in the protein interfere with VLP budding/fission events. We conclude that RSV and HIV‐1 Gag direct particle release through independent ESCRT‐mediated pathways that are linked through Tsg101–Nedd4 interaction.