Indeterminate Human Immunodeficiency Virus Type 1 Western Blots: Seroconversion Risk, Specificity of Supplemental Tests, and an Algorithm for Evaluation

1991 ◽  
Vol 164 (4) ◽  
pp. 656-664 ◽  
Author(s):  
C. L. Celum ◽  
R. W. Coombs ◽  
W. Lafferty ◽  
T. S. Inui ◽  
P. H. Louie ◽  
...  
2009 ◽  
Vol 83 (23) ◽  
pp. 12336-12344 ◽  
Author(s):  
Linda L. Dunn ◽  
Mary Jane McWilliams ◽  
Kalyan Das ◽  
Eddy Arnold ◽  
Stephen H. Hughes

ABSTRACT Although human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has been extensively studied, there are still significant questions about the effects of mutations on the maturation and stability of RT. We show here that a significant fraction (>80%) of the single point mutations we generated in the thumb subdomain of HIV-1 (RT) affect the stability of RT in virions. Fragments of the unstable mutant RTs can be detected in Western blots of virion proteins; however, the degree of degradation varies. The titers of the mutants whose virions contain degraded RTs are reduced. Some, but not all, of the unstable RT thumb subdomain mutants we analyzed have a temperature-sensitive phenotype. A preliminary survey of mutations in other subdomains of RT shows that some of these mutations also destabilize RT. The stability of the RT mutants is enhanced by the addition of a protease inhibitor, suggesting that the viral protease plays an important role in the degradation of the mutant RTs. These results confirm and extend earlier reports of mutations that affect the stability of RT in virions. The data suggest that the stability of a mutant RT in virions could be a major factor in determining the virus titer and, by extension, viral fitness, which could affect whether a mutation in RT is acceptable to the virus.


1998 ◽  
Vol 5 (5) ◽  
pp. 636-644 ◽  
Author(s):  
Hans J. W. de Haard ◽  
Bert Kazemier ◽  
Marck J. M. Koolen ◽  
Liekle J. Nijholt ◽  
Rob H. Meloen ◽  
...  

ABSTRACT By application of combinatorial library technology, we generated the first recombinant antibody fragments directed against the major capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A library of single-chain Fv fragments (scFvs) was constructed by using the antibody variable-region (V) genes of B cells derived from the spleen of a viral lysate-immunized mouse. Antibodies were selected by panning or by enrichment with biotinylated antigen, yielding four different families of antibody fragments. The different types of scFvs were characterized by affinity measurements, by antigen recognition on Western blots, and by pepscan analysis. The epitope of one of the scFvs is located near the residues involved in CypA binding, thereby making it an attractive candidate for therapeutic applications. Comparison of the V gene sequence of this scFV with that of a previously described monoclonal antibody reactive against this immunodominant epitope revealed the usage of the identical combination of VH and Vκ regions. Thus, this is one of the rare examples in which the original combination in a library-derived antibody fragment was retrieved. After appropriate affinity and format improvements, the best of our recombinant scFvs may form the basis for a sensitive p24 assay as a measure of viral load. In addition, anti-p24 scFvs could be expressed as intracellular antibodies (intrabodies) to aid in the treatment of HIV infections.


2004 ◽  
Vol 78 (20) ◽  
pp. 11130-11141 ◽  
Author(s):  
Andre J. Marozsan ◽  
Erika Fraundorf ◽  
Awet Abraha ◽  
Heather Baird ◽  
Dawn Moore ◽  
...  

ABSTRACT Most studies on human immunodeficiency virus type 1 (HIV-1) replication kinetics or fitness must rely on a particular assay to initially standardize inocula from virus stocks. The most accurate measure of infectious HIV-1 titers involves a limiting dilution-infection assay and a calculation of the dose required for 50% infectivity of susceptible cells in tissue culture (TCID50). Surrogate assays are now commonly used to measure the amount of p24 capsid, the endogenous reverse transcriptase (RT) activity, or the amount of viral genomic RNA in virus particles. However, a direct comparison of these surrogate assays and actual infectious HIV-1 titers from TCID50 assays has not been performed with even the most conserved laboratory strains, let alone the highly divergent primary HIV-1 isolates of different subtypes. This study indicates that endogenous RT activity, not p24 content or viral RNA load, is the best surrogate measure of infectious HIV-1 titer in both cell-free supernatants and viruses purified on sucrose cushions. Sequence variation between HIV-1 subtypes did not appear to affect the function or activity of the RT enzyme in this endogenous assay but did affect the detection of p24 capsid by both enzyme immunoassays and Western blots. Clear groupings of non-syncytium-inducing (NSI), CCR5-tropic (R5), and SI/CXCR4-tropic (X4) HIV-1 isolates were observed when we compared the slopes derived from correlations of RT activity with infectious titers. Finally, the replication efficiency or fitness of both the NSI/R5 and SI/X4 HIV-1 isolates was not linked to the titers of the virus stocks.


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