Vesicular stomatitis virus-simian retrovirus type 2 vaccine protects macaques from detectable infection and B-cell destruction.

Natural infection with simian retrovirus (SRV) has long been recognized in rhesus macaques (RMs) and may result in an AIDS-like disease. Importantly, SRV infections persist as a problem in recently imported macaques. Therefore, there is a clear need to control SRV spread in macaque colonies. We developed a recombinant vesicular stomatitis virus (VSV)-SRV vaccine consisting of replication-competent hybrid VSVs that express SRV gag and env in separate vectors. The goal of this study was to assess the immunogenicity and protective efficacy of the VSV-SRV serotype 2 vaccine prime-boost approach in RMs. The VSV-SRV vector (expressing either SRV gag or env) vaccines were intranasally administered in 4 RMs, followed by a boost 1 month after the first vaccination. Four RMs served as controls and received the VSV vector alone. Two months after the boost, all animals were intravenously challenged with SRV-2 and monitored for 90 days. After the SRV-2 challenge, all four controls became infected, and viral loads (VLs) ranged from 10(6) to 10(8) SRV RNA copies/ml of plasma. Two animals in the control group developed simian AIDS within 7 to 8 weeks postinfection and were euthanized. Anemia and weight loss were observed in the remaining controls. During acute infection, severe B-cell depletion and no significant changes in T-cell population were observed in the control group. Control RMs with greater preservation of B cells and lower VLs survived longer. SRV-2 was undetectable in vaccinated animals, which remained healthy, with no clinical or biological signs of infection and preservation of B cells. Our study showed that the VSV-SRV vaccine is a strong approach for preventing clinically relevant type D retrovirus infection and disease in RMs, with protection of 4/4 RMs from SRV infection and prevention of B-cell destruction. B-cell protection was the strongest correlate of the long-term survival of all vaccinated and control RMs.

High-resolution structure of a retroviral protease folded as a monomer.

Mason-Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C(α) deviations are large and the active-site 'DTG' loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections, including AIDS.

A virus causes cancer by inducing massive chromosomal instability through cell fusion.

Chromosomal instability (CIN) underlies malignant properties of many solid cancers and their ability to escape therapy, and it might itself cause cancer [1, 2]. CIN is sustained by deficiencies in proteins, such as the tumor suppressor p53 [3-5], that police genome integrity, but the primary cause of CIN in sporadic cancers remains uncertain [6, 7]. The primary suspects are mutations that deregulate telomere maintenance, or mitosis, yet such mutations have not been identified in the majority of sporadic cancers [6]. Alternatively, CIN could be caused by a transient event that destabilizes the genome without permanently affecting mechanisms of mitosis or proliferation [5, 8]. Here, we show that an otherwise harmless virus rapidly causes massive chromosomal instability by fusing cells whose cell cycle is deregulated by oncogenes. This synergy between fusion and oncogenes "randomizes" normal diploid human fibroblasts so extensively that each analyzed cell has a unique karyotype, and some produce aggressive, highly aneuploid, heterogeneous, and transplantable epithelial cancers in mice. Because many viruses are fusogenic, this study suggests that viruses, including those that have not been linked to carcinogenesis, can cause chromosomal instability and, consequently, cancer by fusing cells.

The impact of altered polyprotein ratios on the assembly and infectivity of Mason-Pfizer monkey virus.

Most retroviruses employ a frameshift mechanism during polyprotein synthesis to balance appropriate ratios of structural proteins and enzymes. To investigate the requirements for individual precursors in retrovirus assembly, we modified the polyprotein repertoire of Mason-Pfizer monkey virus (M-PMV) by mutating the frameshift sites to imitate the polyprotein organization of Rous sarcoma virus (Gag-Pro and Gag-Pro-Pol) or Human immunodeficiency virus (Gag and Gag-Pro-Pol). For the "Rous-like" virus, assembly was impaired with no incorporation of Gag-Pro-Pol into particles and for the "HIV-like" virus an altered morphogenesis was observed. A mutant expressing Gag and Gag-Pro polyproteins and lacking Gag-Pro-Pol assembled intracellular particles at a level similar to the wild-type. Gag-Pro-Pol polyprotein alone neither formed immature particles nor processed the precursor. All the mutants were non-infectious except the "HIV-like", which retained fractional infectivity.

The RNA binding G-patch domain in retroviral protease is important for infectivity and D-type morphogenesis of Mason-Pfizer monkey virus.

Retroviral proteases (PRs) cleave the viral polyprotein precursors into functional mature proteins late during particle release and are essential for viral replication. Unlike most retroviruses, beta-retroviruses, including Mason-Pfizer monkey virus (M-PMV), assemble immature capsids within the cytoplasm of the cell. The activation of beta-retroviral proteases must be highly regulated, because processing of the Gag-related polyprotein precursors occurs only after transport of immature capsids to the plasma membrane and budding. Several beta-retroviral proteases have unique C-terminal extension sequences, containing a glycine-rich motif (G-patch), which specifically binds in vitro to single-stranded nucleic acids. In M-PMV PR the G-patch is removed in vitro as well as in vivo by autoproteolytic processing to yield truncated active forms of PR. To investigate the role of the G-patch domain on the virus life cycle, we introduced mutations within the C-terminal domain of protease. We found that the G-patch domain of M-PMV PR is not required for the processing of viral polyproteins, but it significantly influences the infectivity of M-PMV, the activity of reverse transcriptase, and assembly of immature capsid within the cells. These results demonstrate for the first time that the G-patch domain of M-PMV PR is critical for the life cycle of beta-retroviruses, and its evolutionary conservation within members of this genus suggests its importance for retroviruses that display D-type morphology.

Amino acid residues in the cytoplasmic domain of the Mason-Pfizer monkey virus glycoprotein critical for its incorporation into virions.

Assembly of an infectious retrovirus requires the incorporation of the envelope glycoprotein complex during the process of particle budding. We have recently demonstrated that amino acid substitutions of a tyrosine residue in the cytoplasmic domain block glycoprotein incorporation into budding Mason-Pfizer monkey virus (M-PMV) particles and abrogate infectivity (C. Song, S. R. Dubay, and E. Hunter, J. Virol. 77:5192-5200, 2003). To investigate the contribution of other amino acids in the cytoplasmic domain to the process of glycoprotein incorporation, we introduced alanine-scanning mutations into this region of the transmembrane protein. The effects of the mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of two cytoplasmic residues, valine 20 and histidine 21, inhibits viral protease-mediated cleavage of the cytoplasmic domain that is observed during virion maturation, but the mutant virions show only moderately reduced infectivity. We also demonstrate that the cytoplasmic domain of the M-PMV contains three amino acid residues that are absolutely essential for incorporation of glycoprotein into virions. In addition to the previously identified tyrosine at residue 22, an isoleucine at position 18 and a leucine at position 25 each mediate the process of incorporation and efficient release of virions. While isoleucine 18 may be involved in direct interactions with immature capsids, antibody uptake studies showed that leucine 25 and tyrosine 22 are part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein. These results demonstrate that the cytoplasmic domain of M-PMV Env, in part through its YXXL-mediated endocytosis and intracellular trafficking signals, plays a critical role in the incorporation of glycoprotein into virions.

Mutations within the transmembrane glycoprotein of Mason-Pfizer monkey virus: loss of SU-TM association and effects on infectivity.

We have previously reported that an 11-amino-acid deletion (D33) within the immunosuppressive peptide (ISP) region of the Mason-Pfizer monkey virus transmembrane (TM) glycoprotein, gp22, caused the loss of interaction between TM and the surface (SU) glycoprotein, gp70. This resulted in the secretion of large amounts of biologically active SU glycoprotein, and we postulated that the ISP might represent a point of contact between the two glycoproteins. To further define the amino acids that might be involved in this proposed region of interaction, we have made two neighboring 4-amino-acid deletions within the area defined by the D33 mutation and have carried out saturation mutagenesis on this 8-amino-acid region. We found that one of the smaller deletions (delta D), and two single point mutations (R68S and L72P), gave the same phenotype as the original D33 mutant. These results provide additional support for the hypothesis that this region of the TM glycoprotein is in contact with the SU glycoprotein and is important in maintaining the noncovalent interactions of the glycoproteins that function to hold the complex together.

Variable sensitivity to substitutions in the N-terminal heptad repeat of Mason-Pfizer monkey virus transmembrane protein.

The transmembrane protein of Mason-Pfizer monkey virus contains two heptad repeats that are predicted to form amphipathic alpha-helices that mediate the conformational change necessary for membrane fusion. To analyze the relative sensitivity of the predicted hydrophobic face of the N-terminal heptad repeat to the insertion of uncharged, polar, and charged substitutions, mutations that introduced alanine, serine, or glutamic acid into positions 436, 443, 450, and 457 of the envelope protein were examined. Novel systems using Tat protein and the GHOST cell line were developed to test and quantitate the effects of the mutations on Env-mediated fusion and infectivity of the virus. While no single amino acid change at any of the positions interfered significantly with the synthesis, processing, or transport to the plasma membrane of glycoprotein complexes, 9 of the 12 nonconservative mutations in these residues completely abolished fusion activity and virus infectivity. Mutations in the central positions (443 and 450) of the heptad repeat region were the most detrimental to Env function, and even single alanine substitutions in these positions dramatically altered the fusogenicity of the protein. These results demonstrate that this N-terminal heptad repeat plays a critical role in Env-mediated membrane fusion and highlight the key function of central hydrophobic residues in this process and the sensitivity of all positions to charge substitutions.

Postassembly cleavage of a retroviral glycoprotein cytoplasmic domain removes a necessary incorporation signal and activates fusion activity.

Viral protease-mediated cleavage within the cytoplasmic domain of the transmembrane (TM) glycoprotein of the type D retrovirus, Mason-Pfizer monkey virus, removes approximately 16 amino acids from the carboxy terminus of the protein. To determine the functional significance of this cleavage in the virus life cycle, we introduced premature stop codons into the TM coding domain, resulting in the production of truncated glycoproteins. Progressive truncated of the cytoplasmic domain identified the carboxy-terminal third as being required for efficient incorporation of the glycoprotein complex into budding virions and profoundly increased the fusogenic capability of the TM glycoprotein. These results, together with the ability of matrix protein mutations to suppress TM cleavage, imply that this portion of the glycoprotein interacts specifically with the capsid proteins during budding, suppressing glycoprotein fusion function until virus maturation has occurred.

A tyrosine motif in the cytoplasmic domain of mason-pfizer monkey virus is essential for the incorporation of glycoprotein into virions.

Mason-Pfizer monkey virus (M-PMV) encodes a transmembrane (TM) glycoprotein with a 38-amino-acid-long cytoplasmic domain. After the release of the immature virus, a viral protease-mediated cleavage occurs within the cytoplasmic domain, resulting in the loss of 17 amino acids from the carboxy terminus. This maturational cleavage occurs between a histidine at position 21 and a tyrosine at position 22 in the cytoplasmic domain of the TM protein. We have demonstrated previously that a truncated TM glycoprotein with a 21-amino-acid-long cytoplasmic tail showed enhanced fusogenicity but could not be incorporated into virions. These results suggest that postassembly cleavage of the cytoplasmic domain removes a necessary incorporation signal and activates fusion activity. To investigate the contribution of tyrosine residues to the function of the glycoprotein complex and virus replication, we have introduced amino acid substitutions into two tyrosine residues found in the cytoplasmic domain. The effects of these mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of tyrosine 34 to alanine had little effect on glycoprotein function. In contrast, substitutions at tyrosine 22 modulated fusion activity in either a positive or negative manner, depending on the substituting amino acid. Moreover, any nonaromatic substitution at this position blocked glycoprotein incorporation into virions and abolished infectivity. These results demonstrate that M-PMV employs a tyrosine signal for the selective incorporation of glycoprotein into budding virions. Antibody uptake studies show that tyrosine 22 is part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein that can also be positively and negatively influenced by changes at this site.