Comparing cytosolic expression to peroxisomal targeting of the chimeric L1/L2 (ChiΔH-L2) gene from human papillomavirus type 16 in the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha.

The chimeric ChiΔH-L2 gene from human papillomavirus type 16, consisting of structural proteins L1 and L2, was successfully expressed in the cytosol of both Pichia pastoris and Hansenula polymorpha during methanol induction. In addition, a novel approach was employed whereby ChiΔH-L2 was targeted to the peroxisome using peroxisomal targeting sequence 1 (PTS1) to compare ChiΔH-L2 yields in the peroxisome vs the cytosol. The ChiΔH-L2 gene was yeast-optimized and cloned into plasmids aimed at genomic integration. Levels of intracellular ChiΔH-L2 accumulation in the cytosol were highest in P. pastoris KM71 strain KMChiΔH-L2 (1.43 mg/l), compared to the maximum production level of 0.72 mg/l obtained with H. polymorpha. ChiΔH-L2 targeting to the peroxisome was successful; however, it appeared to negatively affect ChiΔH-L2 production in both P. pastoris and H. polymorpha.

Prevalence of genotypic resistance to antiretroviral drugs in treatment-naive youths infected with diverse HIV type 1 subtypes and recombinant forms in Dar es Salaam, Tanzania.

As human immunodeficiency virus (HIV) diversity may have an impact on both vaccine efficacy and drug resistance, it is important to have knowledge of circulating genetic variants. With widespread use of antiretroviral (ARV) drugs in Africa, one of the major potential challenges is the risk of emergence of ARV drug-resistant HIV strains. This study aimed to determine the circulating HIV subtypes and recombinant forms, as well as the prevalence of ARV drug resistance mutations, among 75 treatment-naive HIV-infected youths in Dar es Salaam, Tanzania. Gag (n = 48), partial pol (n = 44), and partial env (n = 35) sequencing was performed; all three regions were sequenced in 26 samples. Evidence of infection with recombinant viruses was found in 12 (46%) participants; AC recombinants were the most commonly detected and they were identified in six (23%) participants. Of individuals infected with nonrecombinant strains, subtype A was most commonly detected in seven (27%) participants, followed by subtype C detected in six (23%) participants and subtype D detected in one (4%) participant. Among the pol sequences from 44 individuals, three (7%) had resistance to nucleoside reverse transcriptase (RT) inhibitors and four (9%) had nonnucleoside RT inhibitor resistance mutations. Of these, three (7%) individuals were infected with viruses with cross-resistance mutations to both classes of RT inhibitors. These resistant mutations were all associated with drugs currently used in first-line therapy and in the prevention of vertical transmission. This high prevalence of resistance mutations is of considerable concern in apparently drug-naive populations as it may result in treatment failure and the spread of ARV-resistant strains.

Adaptive changes in HIV-1 subtype C proteins during early infection are driven by changes in HLA-associated immune pressure.

It is unresolved whether recently transmitted human immunodeficiency viruses (HIV) have genetic features that specifically favour their transmissibility. To identify potential "transmission signatures", we compared 20 full-length HIV-1 subtype C genomes from primary infections, with 66 sampled from ethnically and geographically matched individuals with chronic infections. Controlling for recombination and phylogenetic relatedness, we identified 39 sites at which amino acid frequency spectra differed significantly between groups. These sites were predominantly located within Env, Pol and Gag (14/39, 9/39 and 6/39 respectively) and were significantly clustered (33/39) within known immunoreactive peptides. Within 6 months of infection, we detected reversion-to-consensus mutations at 14 sites and potential CTL escape mutations at seven. Here we provide evidence that frequent reversion mutations probably allows the virus to recover replicative fitness which, together with immune escape driven by the HLA alleles of the new hosts, differentiate sequences from chronic infections from those sampled shortly after transmission.

Molecular investigation of two possible cases of accidental HIV-1 transmission in South Africa.

HIV-1 sequences from two possible transmission cases in South Africa were examined for evidence of genetic linkage. HIV-1-seropositive blood samples were obtained from a donor and recipient within 8 months following a blood transfusion and from a healthcare worker and her patient within 10 months following a needle-stick injury. A 700-bp region in env and 550-bp region in gag were analyzed. All sequences were phylogenetically associated with HIV-1 subtype C, the predominant HIV-1 subtype in South Africa. The nucleotide sequences from the blood transfusion case grouped together significantly with a bootstrap value of 100%. These samples were 98% and 100% identical in the predicted amino acid sequences of env and gag, respectively. In contrast, sequences from the needle-stick case showed only 67% and 80% amino acid identity in env and gag, respectively, and were separated on a phylogenetic tree. Molecular analysis suggested that HIV transmission occurred in the blood transfusion case but not in the case of the needle-stick injury. These data emphasize the need for molecular investigation of epidemiologically linked cases of HIV transmission.

Human herpesvirus-8 antibodies and DNA in HIV-1 infected patients in South Africa.

HIV-infected individuals with high levels of IgG antibodies against human herpesvirus-8 (HHV-8) are at increased risk of developing Kaposi's sarcoma. The aim of this study was to measure the association between HHV-8 viraemia and IgG antibody responses (by immunofluorescence) in a group of 201 HIV-infected individuals attending outpatient clinics, 91 in-patients with AIDS and 87 HIV-infected patients admitted with Kaposi's sarcoma. Compared to HIV-infected outpatients, the adjusted odds ratio in relation to Kaposi's sarcoma was 15.4 (95% CI 4.4-54.2) in those with viraemia, 25.1 (95% CI 6.6-95.6) in those with a positive immunofluorescent signal and infinity (lower exact CI 33.6) in those with a high immunofluorescent signal (all P trend < 0.001). Among those without HHV-8 viraemia, 23% were IgG-positive, but only 5.5% had a high immunofluorescent signal. In those who were viraemic, 89.1% were IgG-positive, and 28.2% had a high immunofluorescent signal, suggesting viraemia is associated with high HHV-8 immunofluorescence IgG signal.

CCR5 is the major coreceptor used by HIV-1 subtype C isolates from patients with active tuberculosis.

Tuberculosis (TB) is the major opportunistic infection of HIV-infected patients in developing countries and is associated with activation of the immune system and increased HIV-1 expression. The aim of this study was to explore the biological properties of HIV-1 isolates from patients with active TB. Ten HIV-1 subtype C isolates were analyzed for biological phenotypes, using MT-2 cells, and for coreceptor usage, using coreceptor-transfected cell lines. All isolates were nonsyncytium inducing (NSI) in the MT-2 assay and replicated in CCR5-expressing cells. None of the isolates used CXCR4 or any of the minor coreceptors (CCR1, CCR2b, or CCR3) efficiently. Analysis of the V3 region showed that all isolates contained the GPGQ motif characteristic of subtype C and also had a sequence profile typical of NSI viruses. These data indicate that despite their advanced disease state, patients with TB harbor viruses that use the CCR5 coreceptor. It is possible that activation of monocytes and macrophages during TB infection results in the expansion of macrophage-tropic isolates that preferentially use CCR5.

Genetic characterization of HIV type 1 from migrant workers in three South African gold mines.

The phylogenetic relationships between 44 HIV-1 isolates from 43 infected subjects employed by three adjacent South African gold mines were investigated. The patients were migrant workers originating from rural areas of South Africa and the neighboring countries of Lesotho, Botswana, Swaziland, and Mozambique. Proviral HIV-1 DNA was subtyped using a heteroduplex mobility assay (HMA) based on the 700-bp V3-V5 region of the env gene. DNA sequence analysis was used to confirm the subtype designation and to determine phylogenetic relationships between isolates. All 44 HIV-1 isolates were identified as env subtype C using both HMA and phylogenetic analysis. These isolates did not show a distinct phylogenetic relatedness based on the geographic origins of the migrant workers or show close homology to other subtype C sequences from southern Africa or India. However, five clusters of closely related sequences were identified, mainly involving miners of disparate geographic origins, suggesting possible epidemiological linkage in these few cases. The characteristic tetrapeptide sequence, GPGQ, at the tip of the V3 loop of subtype C viruses was conserved in the predicted amino acid sequences of most isolates. The heterogeneity of HIV-1 sequences among migrant workers in a mining cohort suggests multiple introductions of HIV-1 subtype C into this population that are not apparently linked to the geographic origins of the patients.