Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV). HIV protease, reverse transcriptase, and integrase are targets of current drugs to treat the disease. However, anti-viral drug-resistant strains have emerged quickly due to the high mutation rate of the virus, leading to the demand for the development of new drugs. One attractive target is Gag-Pol polyprotein, which plays a key role in the life cycle of HIV. Recently, we found that a combination of M50I and V151I mutations in HIV-1 integrase can suppress virus release and inhibit the initiation of Gag-Pol autoprocessing and maturation without interfering with the dimerization of Gag-Pol. Additional mutations in integrase or RNase H domain in reverse transcriptase can compensate for the defect. However, the molecular mechanism is unknown. There is no tertiary structure of the full-length HIV-1 Pol protein available for further study. Therefore, we developed a workflow to predict the tertiary structure of HIV-1 NL4.3 Pol polyprotein. The modeled structure has comparable quality compared with the recently published partial HIV-1 Pol structure (PDB ID: 7SJX). Our HIV-1 NL4.3 Pol dimer model is the first full-length Pol tertiary structure. It can provide a structural platform for studying the autoprocessing mechanism of HIV-1 Pol and for developing new potent drugs. Moreover, the workflow can be used to predict other large protein structures that cannot be resolved via conventional experimental methods.

Nuclear inclusion a protease (NIaPro), a major protease of potyvirids, processes its cognate viral polyprotein at distinct cleavage sites. Although Potyviridae is the largest family of the realm Riboviria, the individual NIaPro enzymes and their cleavage sites are believed to be species-specific. In the present study, the NIaPro amino acid sequences of 165 potyvirids of 10 genera and their 1154 cleavage sites were compared to understand their genus/species-specificity and functional regulation. Of these, the NIaPro of macluraviruses, maintains a constant length of 217 amino acids, while those of other genera allow variation. In particular, poaceviruses exhibited a broad range of NIaPro amino acid sequence lengths. Alignment of 162 NIaPro amino acid sequences showed that the N- and C-terminal regions allow variations, while the central region, with the catalytic triad and S1 subsite, are highly conserved. NIaPro cleavage sites are composed of seven amino acids (heptapeptide) denoted as P6-P5-P4-P3-P2-P1/P1\'. A survey of 1154 cleavage sites showed that the P1 position is predominantly occupied by Gln/Glu, as is seen in picornaviruses. The P6 (Glu), P4 (Val/Cys/Gln), P2 (His/Tyr/Leu), and P1\' (Ser/Ala/Gly/Met) positions are predominantly occupied by genus-specific residues, while P5 and P3 are not genus-specific. The 6K2-VPg and VPg-NIaPro junctions possess Glu at the P1 position in order to maintain latency.

One of the most exciting developments in the field of biological physics in recent years is the ability to manipulate single molecules and probe their properties and function. Since its emergence over two decades ago, single molecule force spectroscopy has become a powerful tool to explore the response of biological molecules, including proteins, DNA, RNA and their complexes, to the application of an applied force. The force versus extension response of molecules can provide valuable insight into its mechanical stability, as well as details of the underlying energy landscape. In this review we will introduce the technique of single molecule force spectroscopy using the atomic force microscope (AFM), with particular focus on its application to study proteins. We will review the models which have been developed and employed to extract information from single molecule force spectroscopy experiments. Finally, we will end with a discussion of future directions in this field.

The cell line IPLB-LD-652Y, derived from the gypsy moth (Lymantria dispar L.), is routinely used to study interactions between viruses and insect hosts. Here we report the full genome sequence and biological characteristics of a small RNA virus, designated Lymantria dispar iflavirus 1 (LdIV1), that was discovered to persistently infect IPLB-LD-652Y. LdIV1 belongs to the genus Iflavirus. LdIV1 formed icosahedral particles of approx. 30 nm in diameter and contained a 10, 044 nt polyadenylated, positive-sense RNA genome encoding a predicted polyprotein of 2980 aa. LdIV1 was induced by a viral suppressor of RNA silencing, suggesting that acute infection is restricted by RNA interference (RNAi). We detected LdIV1 in all tested tissues of gypsy-moth larvae and adults, but the virus was absent from other L. dispar-derived cell lines. We confirmed LdIV1 infectivity in two of these cell lines (IPLB-LD-652 and IPLB-LdFB). Our results provide a novel system to explore persistent infections in lepidopterans and a new model for the study of iflaviruses, a rapidly expanding group of viruses, many of which covertly infect their hosts.

BACKGROUND: A tool for stoichiometric co-expression of effector and target proteins to study intracellular protein trafficking processes has been provided by the so called 2A peptide technology. In this system, the 16-20 amino acid 2A peptide from RNA viruses allows synthesis of multiple gene products from single transcripts. However, so far the use of the 2A technology in plant systems has been limited.
RESULTS: The aim of this work was to assess the suitability of the 2A peptide technology to study the effects exerted by dominant mutant forms of three small GTPase proteins, RABD2a, SAR1, and ARF1 on intracellular protein trafficking in plant cells. Special emphasis was given to CAH1 protein from Arabidopsis, which is trafficking to the chloroplast via a poorly characterized endoplasmic reticulum-to-Golgi pathway. Dominant negative mutants for these GTPases were co-expressed with fluorescent marker proteins as polyproteins separated by a 20 residue self-cleaving 2A peptide. Cleavage efficiency analysis of the generated polyproteins showed that functionality of the 2A peptide was influenced by several factors. This enabled us to design constructs with greatly increased cleavage efficiency compared to previous studies. The dominant negative GTPase variants resulting from cleavage of these 2A peptide constructs were found to be stable and active, and were successfully used to study the inhibitory effect on trafficking of the N-glycosylated CAH1 protein through the endomembrane system.
CONCLUSIONS: We demonstrate that the 2A peptide is a suitable tool when studying plant intracellular protein trafficking and that transient protoplast and in planta expression of mutant forms of SAR1 and RABD2a disrupts CAH1 trafficking. Similarly, expression of dominant ARF1 mutants also caused inhibition of CAH1 trafficking to a different extent. These results indicate that early trafficking of the plastid glycoprotein CAH1 depends on canonical vesicular transport mechanisms operating between the endoplasmic reticulum and Golgi apparatus.

We examined the safety, immunogenicity and efficacy of a prime-boost vaccination regime involving two poxvirus malaria subunit vaccines, FP9-PP and MVA-PP, expressing the same polyprotein consisting of six pre-erythrocytic antigens from Plasmodium falciparum. Following safety assessment of single doses, 15 volunteers received a heterologous prime-boost vaccination regime and underwent malaria sporozoite challenge. The vaccines were safe but interferon-γ ELISPOT responses were low compared to other poxvirus vectors, despite targeting multiple antigens. There was no vaccine efficacy as measured by delay in time to parasitaemia. A number of possible explanations are discussed, including the very large insert size of the polyprotein transgene.

Forty-four adult patients with cutaneous leishmaniasis (CL) were enrolled in a randomized, double-blind, controlled, dose-escalating clinical trial and were randomly assigned to receive three injections of either the LEISH-F1+MPL-SE vaccine (consisting of 5, 10, or 20 μg recombinant Leishmania polyprotein LEISH-F1 antigen+25 μg MPL-SE adjuvant) (n=27), adjuvant alone (n=8), or saline placebo (n=9). The study injections were given subcutaneously on Days 0, 28, and 56, and the patients were followed through Day 336 for safety, immunological, and clinical evolution endpoints. All patients received chemotherapy with meglumine antimoniate starting on Day 0. The vaccine was safe and well tolerated. Nearly all vaccine recipients and no adjuvant-alone or placebo recipients demonstrated an IgG antibody response to LEISH-F1 at Day 84. Also at Day 84, 80% of vaccine recipients were clinically cured, compared to 50% and 38% of adjuvant-alone and placebo recipients. The LEISH-F1+MPL-SE vaccine was safe and immunogenic in CL patients and appeared to shorten their time to cure when used in combination with meglumine antimoniate chemotherapy.