Live, Attenuated Simian Immunodeficiency Virus SIVmac-M4, with Point Mutations in the Env Transmembrane Protein Intracytoplasmic Domain, Provides Partial Protection from Mucosal Challenge with Pathogenic SIVmac251

ABSTRACT Attenuated molecular clones of simian immunodeficiency virus (SIVmac) are important tools for studying the correlates of protective immunity to lentivirus infection in nonhuman primates. The most highly attenuated SIVmac mutants fail to induce disease but also fail to induce immune responses capable of protecting macaques from challenge with pathogenic virus. We recently described a novel attenuated virus, SIVmac-M4, containing multiple mutations in the transmembrane protein (TM) intracytoplasmic domain. This domain has been implicated in viral assembly, infectivity, and cytopathogenicity. Whereas parental SIVmac239-Nef+ induced persistent viremia and simian AIDS in rhesus macaques, SIVmac-M4 induced transient viremia in juvenile and neonatal macaques, with no disease for at least 1 year postinfection. In this vaccine study, 8 macaques that were infected as juveniles (n = 4) or neonates (n = 4) with SIVmac-M4 were challenged with pathogenic SIVmac251 administered through oral mucosa. At 1 year postchallenge, six of the eight macaques had low to undetectable plasma viremia levels. Assays of cell-mediated immune responses to SIVmac Gag, Pol, Env, and Nef revealed that all animals developed strong CD8+ T-cell responses to Gag after challenge but not before. Unvaccinated control animals challenged with SIVmac251 developed persistent viremia, had significantly weaker SIV-specific T-cell responses, and developed AIDS-related symptoms. These findings demonstrate that SIVmac-M4, which contains a full-length Nef coding region and multiple point mutations in the TM, can provide substantial protection from mucosal challenge with pathogenic SIVmac251.

Effects of Truncation of the Carboxy Terminus of Pseudorabies Virus Glycoprotein B on Infectivity

ABSTRACT Glycoproteins homologous to the type I membrane glycoprotein B (gB) of herpes simplex virus 1 (HSV-1) are the most highly conserved glycoproteins within the family Herpesviridae and are present in members of each herpesvirus subfamily. In the alphaherpesvirus pseudorabies virus (PrV), gB is required for entry into target cells and for direct viral cell-to-cell spread. These processes, though related, appear to be distinct, and thus it was interesting to analyze whether they require different functions of gB. To this end, we established cell lines stably expressing different carboxy-terminally truncated versions of PrV gB by deleting either (i) one predicted intracytoplasmic α-helical domain encompassing putative YQRL and dileucine internalization signals, (ii) two predicted intracytoplasmic α-helical domains, (iii) the complete intracytoplasmic domain, or (iv) the intracytoplasmic domain and the transmembrane anchor region. Confocal laser scanning microscopy showed that gB derivatives lacking at least the last 29 amino acids (aa) localize close to the plasma membrane, while the full-length protein accumulates in intracellular aggregations. Trans-complementation studies with a gB-deleted PrV (PrV-gB−) demonstrated that the 29-aa truncated form lacking the putative internalization signals and the C-terminal α-helical domain (gB-008) was efficiently incorporated into PrV-gB− virions and efficiently complemented infectivity and cell-to-cell spread. Moreover, gB-008 exhibited an enhanced fusogenic activity. In contrast, gB proteins lacking both α-helical domains (gB-007), the complete intracytoplasmic domain, or the intracytoplasmic domain and transmembrane anchor were only inefficiently or not at all incorporated into PrV-gB− virions and did not complement infectivity. However, gB-007 was able to mediate cell-to-cell spread of PrV-gB−. Similar phenotypes were observed when virus recombinants expressing gB-008 or gB-007, respectively, instead of wild-type gB were isolated and analyzed. Thus, our data show that internalization of gB is not required for gB incorporation into virions nor for its function in either entry or cell-to-cell spread. Moreover, they indicate different requirements for gB in these membrane fusion processes.

The Intracytoplasmic Domain of the Env Transmembrane Protein Is a Locus for Attenuation of Simian Immunodeficiency Virus SIVmac in Rhesus Macaques

ABSTRACT The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unusually long intracytoplasmic domains (ICD-TM). These domains are suggested to play a role in envelope fusogenicity, interaction with the viral matrix protein during assembly, viral infectivity, binding of intracellular calmodulin, disruption of membranes, and induction of apoptosis. Here we describe a novel mutant virus, SIVmac-M4, containing multiple mutations in the coding region for the ICD-TM of pathogenic molecular clone SIVmac239. Parental SIVmac239-Nef+ produces high-level persistent viremia and simian AIDS in both juvenile and newborn rhesus macaques. The ICD-TM region of SIVmac-M4 contains three stop codons, a +1 frameshift, and mutation of three highly conserved, charged residues in the conserved C-terminal alpha-helix referred to as lentivirus lytic peptide 1 (LLP-1). Overlapping reading frames for tat,rev, and nef are not affected by these changes. In this study, four juvenile macaques received SIVmac-M4 by intravenous injection. Plasma viremia, as measured by branched-DNA (bDNA) assay, reached a peak at 2 weeks postinoculation but dropped to below detectable levels by 12 weeks. At over 1.5 years postinoculation, all four juvenile macaques remain healthy and asymptomatic. In a subsequent experiment, four neonatal rhesus macaques were given SIVmac-M4 intravenously. These animals exhibited high levels of viremia in the acute phase (2 weeks postinoculation) but are showing a relatively low viral load in the chronic phase of infection, with no clinical signs of disease for 1 year. These findings demonstrated that the intracytoplasmic domain of the transmembrane Env (Env-TM) is a locus for attenuation in rhesus macaques.