Ashok Aiyar

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.

The Amino Terminus of Epstein-Barr Virus (EBV) Nuclear Antigen 1 Contains AT Hooks That Facilitate the Replication and Partitioning of Latent EBV Genomes by Tethering Them to Cellular Chromosomes

ABSTRACT During latency, Epstein-Barr virus (EBV) is stably maintained as a circular plasmid that is replicated once per cell cycle and partitioned at mitosis. Both these processes require a single viral protein, EBV nuclear antigen 1 (EBNA1), which binds two clusters of cognate binding sites within the latent viral origin, oriP. EBNA1 is known to associate with cellular metaphase chromosomes through chromosome-binding domains within its amino terminus, an association that we have determined to be required not only for the partitioning of oriP plasmids but also for their replication. One of the chromosome-binding domains of EBNA1 associates with a cellular nucleolar protein, EBP2, and it has been proposed that this interaction underlies that ability of EBNA1 to bind metaphase chromosomes. Here we demonstrate that EBNA1s chromosome-binding domains are AT hooks, a DNA-binding motif found in a family of proteins that bind the scaffold-associated regions on metaphase chromosomes. Further, we demonstrate that the ability of EBNA1 to stably replicate and partition oriP plasmids correlates with its AT hook activity and not its association with EBP2. Finally, we examine the contributions of EBP2 toward the ability of EBNA1 to associate with metaphase chromosomes in human cells, as well as support the replication and partitioning of oriP plasmids in human cells. Our results indicate that it is unlikely that EBP2 directly mediates these activities of EBNA1 in human cells.

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

Metaphase Chromosome Tethering Is Necessary for the DNA Synthesis and Maintenance of oriP Plasmids but Is Insufficient for Transcription Activation by Epstein-Barr Nuclear Antigen 1

ABSTRACT Epstein-Barr Virus (EBV) infects resting B cells, within which it establishes latency as a stable, circular episome with only two EBV components, the cis element oriP and the latently expressed protein EBNA1. It is believed that EBNA1s ability to tether oriP episomes to metaphase chromosomes is required for its stable replication. We created fusions between the DNA-binding domain (DBD) of EBNA1 and the cellular chromatin-binding proteins HMGA1a and HMG1 to determine the minimal requirements for stable maintenance of an oriP-based episome. These two proteins differ in that HMGA1a can associate with metaphase chromosomes but HMG1 cannot. Interestingly, coinciding with metaphase chromosome association, HMGA1a-DBD but not HMG1-DBD supported both the transient replication and stable maintenance of oriP plasmids, with efficiencies quantitatively similar to that of EBNA1. However, HMGA1a-DBD activated transcription from EBNA1-dependent episomal reporter to only 20% of the level of EBNA1. Furthermore, EBNA1 but not HMGA1a-DBD activated transcription from a chromosomally integrated EBNA1-dependent transcription reporter. This indicates that EBNA1 possesses functional domains that support transcription activation independent of its ability to tether episomal oriP plasmids to cellular chromosomes. We provide evidence that metaphase chromosome tethering is a fundamental requirement for maintenance of an oriP plasmid but is insufficient for EBNA1 to activate transcription.

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...

Establishment of Latent Epstein-Barr Virus Infection and Stable Episomal Maintenance in Murine B-Cell Lines

ABSTRACT Epstein-Barr virus (EBV) is a strict human pathogen for which no small animal models exist. Plasmids that contain the EBV plasmid origin of replication, oriP, and express EBV nuclear antigen 1 (EBNA1) are stably maintained extrachromosomally in human cells, whereas these plasmids replicate poorly in rodent cells. However, the ability of oriP and EBNA1 to maintain the entire EBV episome in proliferating rodent cells has not been determined. Expression of the two human B-cell receptors for EBV on the surfaces of murine B cells allows efficient viral entry that leads to the establishment of latent EBV infection and long-term persistence of the viral genome. Latent gene expression in these cells resembles the latency II profile in that EBNA1 and LMP1 can be detected whereas EBNA2 and the EBNA3s are not expressed.

The spacing between adjacent binding sites in the family of repeats affects the functions of Epstein-Barr nuclear antigen 1 in transcription activation and stable plasmid maintenance.

Epstein-Barr virus (EBV) and the closely related Herpesvirus papio (HVP) are stably replicated as episomes in proliferating latently infected cells. Maintenance and partitioning of these viral plasmids requires a viral sequence in cis, termed the family of repeats (FR), that is bound by a viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). Upon binding FR, EBNA1 maintains viral genomes in proliferating cells and activates transcription from viral promoters required for immortalization. FR from either virus encodes multiple binding sites for the viral maintenance protein, EBNA1, with the FR from the prototypic B95-8 strain of EBV containing 20 binding sites, and FR from HVP containing 8 binding sites. In addition to differences in the number of EBNA1-binding sites, adjacent binding sites in the EBV FR are typically separated by 14 base pairs (bp), but are separated by 10 bp in HVP. We tested whether the number of binding sites, as well as the distance between adjacent binding sites, affects the function of EBNA1 in transcription activation or plasmid maintenance. Our results indicate that EBNA1 activates transcription more efficiently when adjacent binding sites are separated by 10 bp, the spacing observed in HVP. In contrast, using two separate assays, we demonstrate that plasmid maintenance is greatly augmented when adjacent EBNA1-binding sites are separated by 14 bp, and therefore, presumably lie on the same face of the DNA double helix. These results provide indication that the functions of EBNA1 in transcription activation and plasmid maintenance are separable.

Cholesterol Supplementation During Production Increases the Infectivity of Retroviral and Lentiviral Vectors Pseudotyped with the Vesicular Stomatitis Virus Glycoprotein (VSV-G).

Abstract Cholesterol, a major component of plasma membrane lipid rafts, is important for assembly and budding of enveloped viruses, including influenza and HIV-1. Cholesterol depletion impairs virus assembly and infectivity. This study examined the effects of exogenous cholesterol addition (delivered as a complex with methyl-beta-cyclodextrin (MbCD)) on the production of Molony murine leukemia virus (MoMuLV) retroviral vector and HIV-1-based lentiviral vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Cholesterol supplementation before and during vector production enhanced the infectivity of retroviral and lentiviral vectors up to 4-fold and 6-fold, respectively. In contrast, the amount of retroviral vector produced was unchanged, and that of lentiviral vector was increased less than 2-fold. Both free cholesterol and cholesterol ester content in 293-gag-pol producer cells increased with cholesterol addition. In contrast, the phospholipids headgroup composition was essentially unchanged by cholesterol supplementation in 293-gag-pol packaging cells. Based on these results, it is proposed that cholesterol supplementation increases the infectivity of VSV-G-pseudotyped retroviral and lentiviral vectors, possibly by altering the composition of the producer cell membrane where the viral vectors are assembled and bud, and/or by changing the lipid composition of the viral vectors.

Transduction efficiency of pantropic retroviral vectors is controlled by the envelope plasmid to vector plasmid ratio.

Pantropic retroviral vectors pseudotyped with vesicular stomatitis virus envelope G protein (VSV‐G) are typically produced by transient transfection of the VSV‐G expression plasmid because constitutive expression of VSV‐G is cytotoxic. To produce pantropic vectors, the VSV‐G expression plasmid and the vector plasmid are cotransfected into a packaging cell line, such as 293‐gag‐pol. Typically, the ratio of VSV‐G plasmid to the vector plasmid ranges from 0.33 to 1.0. However, it is not clear that this range is optimal for vector production. In this study we have systematically examined the effect of the ratio of VSV‐G plasmid (pVSV‐G) to vector plasmid on vector production. For this, 293‐gag‐pol stable packaging cells were cotransfected with pVSV‐G and an enhanced green fluorescent protein‐ (EGFP‐) expressing retroviral vector plasmid (pLTR‐EGFP) by use of lipofectamine. Vector was collected following transfection and used to transduce three target cell lines, namely, 3T3 fibroblasts, telomerase‐immortalized human diploid fibroblasts (HDF), and the human hepatoma cell line HuH7. Transduction efficiency was evaluated for vectors produced at different pVSV‐G:pLTR‐EGFP ratios such that the total amount of plasmid transfected into 293‐gag‐pol cells was kept constant. Our results indicate that transduction efficiency is greatest when the pVSV‐G:pLTR‐EGFP ratio is substantially below 1.0. For 3T3 and HDF cells, the maximum transduction efficiency was obtained when a ratio of pVSV‐G:pLTR‐EGFP ranging from 0.053 to 0.2 was used for transfection. The relative magnitude of this effect was greater for lower transduction efficiencies in control cultures. For HuH7 cells, the beneficial effects were smaller than those observed when HDF or 3T3 cells were used. The difference in transduction efficiency for vector produced under various pVSV‐G:pLTR‐EGFP ratios was not due to differences in the proliferation of packaging cells or target cells. Further characterization showed that the amount of vector RNA relative to p30gag decreased as the ratio of pVSV‐G:pLTR‐EGFP increased. These results indicate that transduction efficiency increases with increasing levels of vector RNA as long as a minimally sufficient level of pantropic envelope protein is expressed.

[16] Retrovirus reverse transcription and integration

Publisher Summary Retroviruses contain an RNA genome that is replicated through a double-stranded DNA intermediate. DNA synthesis is initiated from a tRNA primer annealed to the 5’ untranslated region of the viral RNA catalyzed by the virus-encoded reverse transcriptase (RT). During the reverse transcription process, the 5’ unique (U5) and 3’ unique (U3) regions of the viral RNA are duplicated and juxtaposed to form the ends of the linear DNA. Each U3–R–U5 end is referred to as a “long terminal repeat” (LTR). The double-stranded DNA, flanked by the LTRs, is then integrated into the host chromosome through the action of a second virus-encoded enzyme, integrase (IN). Understanding of the mechanistic roles of these two enzymes in viral replication has vastly increased with the cloning of the retrovirus genome and its subsequent molecular genetic analysis. The chapter discusses methods both to introduce mutations into the retrovirus genome and to reconstitute in vitro the activities of RT and IN in the initiation of reverse transcription and integration of viral DNA, respectively. The efficiency of Rous sarcoma virus (RSV) RT to initiate DNA synthesis from the tRNA primer is dependent not only on the primer but also on secondary structures near the primer binding site (PBS) in the viral RNA.