Encapsidation of host restriction factor APOBEC3G (A3G) into vif-deficient human immunodeficiency virus type 1 (HIV-1) blocks virus replication at least partly by C-to-U deamination of viral minus-strand DNA, resulting in G-to-A hypermutation. A3G may also inhibit HIV-1 replication by reducing viral DNA synthesis and inducing viral DNA degradation. To gain further insight into the mechanisms of viral inhibition, we examined the metabolism of A3G-exposed viral DNA. We observed that an overall 35-fold decrease in viral infectivity was accompanied by a five-to sevenfold reduction in viral DNA synthesis. Wild-type A3G induced an additional fivefold decrease in the amount of viral DNA that was integrated into the host cell genome and similarly reduced the efficiency with which HIV-1 preintegration complexes (PICs) integrated into a target DNA in vitro. The A3G C-terminal catalytic domain was required for both of these antiviral activities. Southern blotting analysis of PICs showed that A3G reduced the efficiency and specificity of primer tRNA processing and removal, resulting in viral DNA ends that are inefficient substrates for integration and plus-strand DNA transfer. However, the decrease in plus-strand DNA transfer did not account for all of the observed decrease in viral DNA synthesis associated with A3G. These novel observations suggest that HIV-1 cDNA produced in the presence of A3G exhibits defects in primer tRNA processing, plus-strand DNA transfer, and integration.

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is a host cytidine deaminase that is packaged into virions and confers resistance to retroviral infection. APOBEC3G deaminates deoxycytidines in minus strand DNA to deoxyuridines, resulting in G to A hypermutation and viral inactivation. Human immunodeficiency virus type 1 (HIV-1) virion infectivity factor counteracts the antiviral activity of APOBEC3G by inducing its proteosomal degradation and preventing virion incorporation. To elucidate the mechanism of viral suppression by APOBEC3G, we developed a sensitive cytidine deamination assay and analyzed APOBEC3G virion incorporation in a series of HIV-1 deletion mutants. Virus-like particles derived from constructs in which pol, env, and most of gag were deleted still contained high levels of cytidine deaminase activity; in addition, coimmunoprecipitation of APOBEC3G and HIV-1 Gag in the presence and absence of RNase A indicated that the two proteins do not interact directly but form an RNase-sensitive complex. Viral particles lacking HIV-1 genomic RNA which were generated from the gag-pol expression constructs pCHelp and pSYNGP packaged APOBEC3G at 30 -40% of the wild-type level, indicating that interactions with viral RNA are not necessary for incorporation. In addition, viral particles produced from an nucleocapsid zinc finger mutant contained ϳ1% of the viral genomic RNA but ϳ30% of the cytidine deaminase activity. The reduction in APOBEC3G incorporation was equivalent to the reduction in the total RNA present in the nucleocapsid mutant virions. These results indicate that interactions with viral proteins or viral genomic RNA are not essential for APOBEC3G incorporation and suggest that APOBEC3G interactions with viral and nonviral RNAs that are packaged into viral particles are sufficient for APOBEC3G virion incorporation.

Here, we investigated whether A3F similarly interferes with HIV-1 provirus formation. We observed that both A3F and A3G inhibit viral DNA synthesis and integration, but A3F is more potent than A3G in preventing viral DNA integration. We further investigated the mechanisms by which A3F and A3G block viral DNA integration by analyzing their effects on viral cDNA processing using Southern blot analysis. A3G generates a 6-bp extension at the viral U5 end of the 3 long terminal repeat (3-LTR), which is a poor substrate for integration; in contrast, A3F inhibits viral DNA integration by reducing the 3 processing of viral DNA at both the U5 and U3 ends. Furthermore, we demonstrated that a functional C-terminal catalytic domain is more critical for A3G than A3F function in blocking HIV-1 provirus formation. Finally, we showed that A3F has a greater binding affinity for a viral 3-LTR double-stranded DNA (dsDNA) oligonucleotide template than A3G. Taking these results together, we demonstrated that mechanisms utilized by A3F to prevent HIV-1 viral DNA integration were different from those of A3G, and that their target specificities and/or their affinities for dsDNA may contribute to their distinct mechanisms.

Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.

The reverse transcriptase enzyme plays an essential role in the HIV-1 life cycle by converting a single-stranded viral RNA genome into a double-stranded viral DNA through a complex process known as reverse transcription. The resulting double-stranded DNA is integrated into the host chromosome to form a provirus. A small proportion of the viral DNAs form dead-end circular products, which nevertheless can serve as useful surrogate markers for monitoring viral replication. Utilizing real-time PCR technology, it is possible to track and quantify different stages of the reverse transcription process, the proviruses, and the nonintegrated dead-end reverse transcription products.

Ukraine has one of the largest HIV epidemics in Europe, historically driven by people who inject drugs (PWID). The epidemic showed signs of stabilization in 2012, but the recent war in eastern Ukraine may be reigniting virus spread. We investigated the movement of HIV-infected people within Ukraine before and during the conflict. We analyzed HIV-1 subtype-A nucleotide sequences sampled during 2012-2015 from 427 patients of 24 regional AIDS centers and used phylogeographic analysis to reconstruct virus movement among different locations in Ukraine. We then tested for correlations between reported PWID behaviors and reconstructed patterns of virus spread. Our analyses suggest that Donetsk and Lugansk, two cities not controlled by the Ukrainian government in eastern Ukraine, were significant exporters of the virus to the rest of the country. Additional analyses showed that viral dissemination within the country changed after 2013. Spearman correlation analysis showed that incoming virus flow was correlated with the number of HIV-infected internally displaced people. Additionally, there was a correlation between more intensive virus movement and locations with a higher proportion of PWID practicing risky sexual behaviors. Our findings suggest that effective prevention responses should involve internally displaced people and people who frequently travel to war-affected regions. Scale-up of harm reduction services for PWID will be an important factor in preventing new local HIV outbreaks in Ukraine.

Reovirus infection induces the formation of large cytoplasmic inclusions that serve as the major site of viral assembly. Reovirus strains type 3 Dearing (T3D) and type 1 Lang (T1L) differ in the rate of inclusion formation in L929 cells. The median time of inclusion formation is 18 h in cells infected with T3D and 39 h in cells infected with T1L. Using reassortant viruses that contain combinations of gene segments derived from T1L and T3D, we found that the M1 gene, which encodes the mu2 protein, is the primary determinant of the rate of inclusion formation. The S3 gene, which encodes the nonstructural protein sigmaNS, plays a secondary role in this process. The subcellular location of the mu2 protein was determined by confocal laser scanning microscopy using dual-fluorescence labeling of mu2 and the outer-capsid protein mu1/mu1C. In virus-infected cells, mu2 protein colocalized with other viral proteins in inclusions and was also distributed diffusely in the cytoplasm and nucleus. Expression of recombinant T1L and T3D mu2 proteins resulted in the formation of protein complexes resembling inclusions in both the cytoplasm and the nucleus with kinetics that reflected the strain of origin. The median time of mu2 protein complex formation was 22 h in cells transfected with the T3D M1 gene and 43 h in cells transfected with the T1L M1 gene. These findings suggest that the mu2 protein influences the rate of inclusion formation and contributes to inclusion morphogenesis. The requirement of mu2 protein in inclusion formation was tested by determining the subcellular localization of mu2 in cells infected with temperature-sensitive (ts) mutants that are defective in viral assembly. In contrast to infection with wild-type virus, mu2 did not colocalize with mu1/mu1C protein in subcellular structures that formed in cells infected at nonpermissive temperature with ts mutants tsH11.2, tsC447, and tsG453 with mutations in the M1, S2, and S4 genes, respectively. These results suggest that despite the role of the mu2 protein in controlling the rate of inclusion formation, this process is a concerted function of several reovirus proteins.

Sustained viral response (SVR) rates for direct‐acting antiviral (DAA) therapy for hepatitis C virus (HCV) infection routinely exceed 95%. However, a small number of patients require retreatment. Sofosbuvir, velpatasvir and voxilaprevir (SOF/VEL/VOX) is a potent DAA combination primarily used for the retreatment of patients who failed by DAA therapies. Here we evaluate retreatment outcomes and the effects of resistance‐associated substitutions (RAS) in a real‐world cohort, including a large number of genotype (GT)3 infected patients. 144 patients from the UK were retreated with SOF/VEL/VOX following virologic failure with first‐line DAA treatment regimens. Full‐length HCV genome sequencing was performed prior to retreatment with SOF/VEL/VOX. HCV subtypes were assigned and RAS relevant to each genotype were identified. GT1a and GT3a each made up 38% (GT1a n = 55, GT3a n = 54) of the cohort. 40% (n = 58) of patients had liver cirrhosis of whom 7% (n = 4) were decompensated, 10% (n = 14) had hepatocellular carcinoma (HCC) and 8% (n = 12) had received a liver transplant prior to retreatment. The overall retreatment SVR12 rate was 90% (129/144). On univariate analysis, GT3 infection (50/62; SVR = 81%, p = .009), cirrhosis (47/58; SVR = 81%, p = .01) and prior treatment with SOF/VEL (12/17; SVR = 71%, p = .02) or SOF+DCV (14/19; SVR = 74%, p = .012) were significantly associated with retreatment failure, but existence of pre‐retreatment RAS was not when viral genotype was taken into account. Retreatment with SOF/VEL/VOX is very successful for non‐GT3‐infected patients. However, for GT3‐infected patients, particularly those with cirrhosis and failed by initial SOF/VEL treatment, SVR rates were significantly lower and alternative retreatment regimens should be considered.