George Englund

Phosphorylation of Residue 131 of HIV-1 Matrix Is Not Required for Macrophage Infection

infectivity in primary human macrophage cultures even of HIV-1 Matrix Is Not Required in the context of a mutant Vpr. To test the possibility that the apparent discrepancy for Macrophage Infection between our results and those reported by Gallay et al. (1995a) might reflect differences between the virus isolates used in the two studies, the molecular clones It has been suggested that the matrix (MA) domain of used in the Gallay et al. experiments were obtained from HIV-1 Gag plays a central role in translocating the HIV-1 D. Trono (Salk Institute). The ability of these clones to preintegration complex to the nucleus following infec-productively infect MDM was tested in parallel with our tion of nondividing cells. Gallay et al. proposed that wild-type and 131YF mutant clones (Figure 1B). Consis-phosphorylation of 1% of MA on a C-terminal Tyr (resi-tent with the results obtained with our wild-type and due 131) was required to reverse the membrane binding mutant virus stocks, the macrophage-tropic isolate properties of MA and promote an association between R7Bal and the Tyr mutant derivative MAY132F. R7Bal MA and the integrase protein, thus enabling MA to assist replicated with indistinguishable kinetics. We note that in translocating the HIV-1 preintegration complex to the R7Bal and MAY132F.R7Bal contain a truncated vpr gene nucleus (Gallay et al., 1995a,b). The critical piece of (von Schwedler et al., 1994). biological data in support of this model was that muta-It has been suggested that, in some primary macro-tion of MA Tyr 131 to Phe blocked infection of differenti-phage systems, virus infection occurs primarily in a ated primary human monocyte-derived macrophages small subpopulation of cells which are actively prolifer-(MDM) (Gallay et al., 1995a). ating (Schuitemaker et al., 1994). To test whether this To confirm this observation, we introduced the same is the case in our experiments, we determined the frac-single amino acid substitution (Tyr to Phe) at residue tion of cells which are actively proliferating at the time 131 of MA. We initially introduced the 131YF mutation of infection and at two-weeks post-infection. This was into the T-cell line tropic molecular clone pNL4-3, and done by labeling nuclear DNA with propidium iodide and determined that this substitution did not affect Gag pre-using flow cytometry to determine relative DNA content, cursor processing, virion assembly and release, or virus then quantitating the proportion of nuclei in G0/G1, S, spread in a T-cell line (data not shown). To analyze the …

Potent and stable attenuation of live-HIV-1 by gain of a proteolysis-resistant inhibitor of NF-kappaB (IkappaB-alphaS32/36A) and the implications for vaccine development.

Live-attenuated human immunodeficiency viruses (HIVs) are candidates for Acquired Immunodeficiency Syndrome (AIDS) vaccine. Based on the simian immunodeficiency virus (SIV) model for AIDS, loss-of-function (e.g. deletion of accessory genes such as nef) has been forwarded as a primary approach for creating enfeebled, but replication-competent, HIV-1/SIV. Regrettably, recent evidence suggests that loss-of-function alone is not always sufficient to prevent the emergence of virulent mutants. New strategies that attenuate via mechanisms distinct from loss-of-function are needed for enhancing the safety phenotype of viral genome. Here, we propose gain-of-function to be used simultaneously with loss-of-function as a novel approach for attenuating HIV-1. We have constructed an HIV-1 genome carrying the cDNA of a proteolysis-resistant nuclear factor-κB inhibitor (IκB-αS32/36A) in the nef region. HIV-1 expressing IκB-αS32/36A down-regulates viral expression and is highly attenuated in both Jurkat and peripheral blood mononuclear cells. We provide formal proof that the phenotypic and attenuating characteristics of IκB-αS32/36A permit its stable maintenance in a live, replicating HIV-1 despite 180 days of forced ex vivopassaging in tissue culture. As compared with other open-reading frames embedded into HIV/SIV genome, this degree of stability is unprecedented. Thus, IκB-αS32/36A offers proof-of-principle that artifactually gained functions, when used to attenuate the replication of live HIV-1, can be stable. These findings illustrate gain-of-function as a feasible strategy for developing safer live-attenuated HIVs to be tested as candidates for AIDS vaccine.

A novel HIV-1 isolate containing alterations affecting the NF-κB element

Abstract Three molecular clones of HIV-1, derived from a single isolate (Al-1), exhibited distinct replicative and cytopathic properties during propagation in a human T cell line. The phenotypic differences observed were attributable, in large part, to changes affecting the viral LTR. Nucleotide sequence and PCR analyses demonstrated the presence of novel duplications or deletions involving the NF-κB motif. These changes in the enhancer element were identified in the original AL1 virus stock. Subcloning of the variant NF-κB segments into LTR-driven CAT expression vectors confirmed a correlation between promoter activity and replicative/cytopathic capacity.

Evaluation of the structural proteins of the hamster neurotropic strain of measles virus with monoclonal antibodies

The virus-specified proteins produced by cells persistently infected with hamster neurotropic strain (HNT) of measles virus were assessed by a panel of monoclonal antibodies. In comparison to the Edmonston strain a number of differences were detected. The HA protein lacks epitopes identified by certain monoclonal antibodies. Some of the absent epitopes may relate to agglutination, because this biological phenomenon is not produced by HNT virus. Phosphoprotein (P) of the usual size (70 kDa) was not detected. Instead a protein with an approximate MW of 36 kDa which reacted with one monoclonal anti-P was identified. Although matrix protein (M) was not detected, mRNAs for P and M proteins were present. However, these in contrast to the mRNAs from Edmonston strain, did not translate in vitro. These abnormalities may relate to neurotropism and the capacity of HNT to produce persistent CNS infection.